摘要:

Prof. Helliwellopened the discussion of the Introductory Lecture: Your conversion efficiency in the Pt–Pt complex experiment was only 2%; have you explored,e.g.by simulation or calculation, the limits of applicability to lighter,i.e.weaker scattering, elements than Pt due to this?Prof. Coppensresponded: We believe that we can reach higher conversion percentages by working with smaller crystals at more intense sources, or by using more complex solids in which the active species are diluted. The ‘limiting percentage’ depends on the distortion, for example, for the large change in dihedral angle predicted to occur (for the isolated molecule) on excitation of benzophenone, the limiting percentage will be lower than for small changes. We have done some simulations; you are right that in general the conversion percentage must be higher when the scattering is not dominated by one or a few strongly scattering atoms.Prof. Bürgiasked: An illuminated crystal of TEA3HPt2(H2P2O4)4is composed of two species, 98% ground state molecules , 2% excited state molecules. Does the difference in Pt–Pt distances give rise to a diffuse scattering signal?Prof. Coppensanswered: We have assumed here and in our work on transition metal linkage isomers that the distribution of the excited molecules in the crystal is random. This assumption is supported by the lack of additional diffraction spots and the results of the structural analyses. So there should be a diffuse scattering contribution, but we have not looked for it.Dr Schmidtasked: (i) Did you see changes in the crystal parameters, since your observed atomic displacements are significant relative to the unit cell parameters? (ii) If yes, how did you correct for these?Prof. Coppensreplied: Yes. In the case of the binuclear Pt complex the excited state population was too small to observe an effect on the cell dimensions. However in our work on photoinduced metastable transition-metal nitrosyl and sulfur dioxide complexes, the conversion percentages were much higher (sometimes close to 50%), and significant cell dimension changes were observed. The sign of such changes correlates with the change in the molecular shape. For example, we observed the largest effect in (pentamethylcyclopentadienyl)nitrosyl-nickel, [Ni(NO)(η5-Cp*)], in which the NO groups are closely aligned with theb-axis. This axis shortens by 0.28 Å on photoinduced conversion of 47% of the molecules to the metastable state, while thec-axis shows a smaller decrease. In the combined analysis of the light-on and dark data it is necessary to take such changes into account. For the photodifference maps in which the ground state density is subtracted, the fractional coordinates must be converted to the new cell in order to prevent a bond length change on transfer to the new cell. In least squares refinement of the response ratios we use two sets of cell dimensions, in the metastable state work we use a slightly different procedure in which the ground state molecule is allowed to undergo rigid body motions after it has been included with corrected fractional coordinates to preserve the intramolecular distances.Dr Chensaid: I am not sure about the Pt–Pt distance in the compound, but we have encountered difficulties in determining the Rh–Rh distance in another similar compound. This distance was too long for EXAFS in solution at room temperature. (i) What were your excited state structure calculation results on the nearest neighbour to Cu distance? (ii) Would you please comment on any problem in making comparisons between your calculations that were carried out on a single molecule and your experimental results that were obtained in single crystals where the effect of crystal packing was significant?Prof. Coppensreplied: (i) We find that the Cu–N distance decreases by about 0.04 Å upon metal-to-ligand charge transfer. The shortening is as expected from the change in oxidation state of copper. We are working on the calculation of the five-coordinate exciplex, and hope to have results shortly. (ii) There are crystal structures for three different salts of the binuclear Pt complex. They are very different in terms of intermolecular interactions, two are acid salts with infinite hydrogen bonded networks, while the third, the potassium salt, is held together by cation–anion contacts. The variation in ground state distances between the three structures is about 0.01 Å at most, which makes it seem unlikely that the excited state distances would be very much affected by the crystal packing, in this case at least. The variation between distances from the various theoretical calculations for either the ground or excited state is about ten times larger.Prof. Helliwellopened the discussion of Prof. Wulff's paper: Your beamline at ESRF encompasses the spectrum of chemistry fields from biochemistry through to “pure” chemistry what features are common to all the chemical experiments in terms of structure to function rather than ‘simple’ characterization?Prof. Wulffresponded: The main focus of our work has been to follow structural changes following breakage or formation of a chemical bond in condensed systems. For systems such as myoglobin and iodine, the cage around the photo-activated molecule determines the reaction pathways and to some extent their time course.Dr Nibberingsaid: Most of the I2recombines very fast because of the cage effect, and what you observe is only the minor fraction that can escape the cage, diffuse away and finally recombine again. Would it not be beneficial if one were to persue an experiment where I2is inside a nanocavity, so that all I2molecules can dissociate to larger internuclear distances, and the expected signals in X-ray diffraction will be more pronounced.Prof. Wulffreplied: The photo-induced signal indicates that 15–50% of the photolysed iodine molecules dissociate (solvent dependent),i.e.break through the solvent cage. The signal from atomic iodine is therefore already quite strong.Dr Techertsaid: One advantage of time-resolved X-ray scattering is the extendedqrange, which can be scanned in one measurement. Therefore, it is possible to determine intra- as well as inter-molecular distances of a system within one experiment, in contrast to spectroscopic methods. This is summarised in Figs. 12 and 13 of your paper. In the reported experiment a ns laser was used as excitation source. In Fig. 13, the experimental difference maps at lowqrange are interpreted as thermal heating effects. How can the found thermal effects be affected by energy flow? Can, in general, energy flow be studied by time-resolved scattering techniques? How does the thermally induced change in the Boltzmann distribution function (see the paper) influence the difference map at highq?Prof. Wulffanswered: When two iodine atoms recombine to form a molecule, the binding energy is transferred to the solvent,i.e.the solvent cools the nascent molecule. The flow of energy is slaved to the recombination. It proceeds though at three stages: direct recombinationviavibrational cooling on the X-state potential (100 ps), intersystem crossing from the A-state (500–2700 ns) and atomic recombinationviadiffuse motion (μs). The result is that the solvent starts to expand. If the expansion was instantaneous, the distance between nearest neighbours, as measured by the angular position of the liquid peak, would be a direct measure of the energy flow. In a real liquid, the expansive motion and its time scale have to be included. The expansion is seen starting from 25–50 ns onwards. After about 1 μs, the expansion stops,i.e.the system reaches thermal equilibrium. We are currently working with Savo Bratos, University of Paris, to separate the thermal expansion from the energy flow. Concerning your second question, if an iodine molecule is (thermally) excited to a vibrational level near the ground state, its average bond length will increase. A distribution of bond lengths will shift and broaden the difference oscillations and the effect is largest at highq. However this broadening does not prevent us from seeing oscillations up to aQ = 9.0 Å−1in iodine and we have the impression that the effect is small in iodine. The effect should be treated as a Debye–Waller factor as in crystallography.Dr Mayasked: Is there any chance of activating the C–Cl bond and what would be the consequences?Prof. Wulffreplied: From recent studies of CH2I2in the solvent CH2Cl2, we know that this solvent can be activatedviatwo-photon absorption. It is our impression that the solvent CCl4is completely transparent to the (softer) 515 nm laser pulse used here. We are planning an experiment to check this point.Prof. Greavessaid: You use the molecular potential for iodine to interpret your laser excited X-ray scattering data. Do these new experiments enable you to refine the potential in any way or do they simply demonstrate its correctness?Prof. Wulffreplied: Our initial fit functions, based on simple molecular form factors with the solvent cage, are not very sensitive to the bond length of the A/A-state. We will soon include cage effects and hope that that will enhance our sensitivity. In addition, new multi-layer optics will reduce the polychromatic bandwidth from 3–4% to about 1%. Combined, we hope to enhance our model selectivity.Prof. Coppensasked: Is your spatial resolution affected by the use of a 3% band width. As the lineshape of your undulator harmonic is asymmetric does it introduce a bias?Prof. Wulffanswered: The polychromatic X-ray spectrum from a single-harmonic undulator leads to a slight increase in the background of the radial intensity on a CCD detector and that dampens the oscillation amplitude in the difference pattern. Simulations show that the damping is small and greatly compensated by the 250–500 fold gain in flux over conventional monochromatic methods.Dr Techertopened the discussion of Dr Nibbering's paper: In the reported work the out-of-plane-twisting mode was found to be 60 cm−1, which is surprisingly high if it is compared with the twist motion of stilbene (ca.11 cm−1). Is this high value due to the rigidity of the system, which enhances the harmonic contribution (in contrast to the rotational contribution in stilbene) to this vibrational mode?Dr Nibberingresponded: The high value is indeed due to the strong intramolecular hydrogen bond, making the molecular structure more rigid. We compared the out-of-plane mode of 2-(2′-hydroxyphenyl)benzothiazole (HBT) with that of its anion (where the proton is removed by a strong base) in quantum chemical calculations. In the case of the anion, where no hydrogen bond exists, the frequency of the out-of-plane mode is very close to that of stilbene.Prof. Moffatasked: Have similar ultra-fast spectroscopic measurements been made either in single crystals or in HBT bound to a small protein? In other words, how does the “solvent” environment affect the mechanism and rates of reaction?Dr Nibberinganswered: The observed H-transfer reaction in HBT in tetrachloroethene represents a clear case of intramolecular H-transfer with minor solvent contributions. When dissolving HBT in polar solvents, like DMSO, alcohols or water, the efficiency of intramolecular hydrogen bond formation will be affected and compete with intermolecular hydrogen bonds with solvent molecules. Since these changes in the hydrogen bond have pronounced effects on the optical spectra, HBT in polar solvents has been studied in optical pump–probe experiments, where different proton transfer times have been deduced from the observed transients. We have not performed femtosecond IR experiments on HBT in polar solvents yet. We expect to see different dynamics dependent on the hydrogen bond configurations.Dr Techertasked: How isexcitonic couplingdefined in the liquid phase (or does this only refer to IR studies on peptides)?Dr Nibberingreplied: Excitonic coupling refers to coupling of vibrational modes within a larger molecular system, that may itself be in the condensed phase (liquids, proteins, solids) or even in the gas phase. These vibrational modes have a mutual coupling when their vibrational bands spectrally overlap or are at very small detuning. Coupling might take place by a through space transition dipole–dipole coupling mechanism with angle and distance dependences. Another mechanism might involve a through bond coupling scheme. Examples include the amide I band in amino acid subunits in peptides, or hydrogen bonded OH-groups in dimeric systems, such as the acetic acid dimer or the DNA base pairs in the double helix.Dr Chenasked: How do you distinguish anharmonic coupling in the proton transfer processes that you are probing from heterogeneity of H-bonding distribution that was disturbed by the laser excitation, which may also result in the spectral shift of the O–H shielding band?Dr Nibberinganswered: The theory of O–H stretching vibration line shapes shows that anharmonic coupling of the high-frequency O–H stretching mode to other vibrational modes leads to (i) a red-shifting, a consequence of the weakening of the O–H stretching vibration; (ii) a Franck–Condon progression due to coupling with underdamped low-frequency modes; (iii) a broadening, due to coupling to overdamped low-frequency modes; (iv) level splitting due to Fermi resonances with vibrational overtone or combination modes. We probe here the carbonyl stretching mode marking the formation of the keto*-product state. We observe in a clear way the effects of anharmonic coupling of coherently excited underdamped intramolecular low-frequency modes. We observe these coherently excited low-frequency modes also in the O–H/N–H stretching region. Interpretation is more problematic though due to the above mentioned other contributions to the overall line shape of O–H and N–H stretching bands. Since the O–H and N–H stretching bands spectrally overlap to a significant extent, it is difficult to distinguish the coherent motions in the keto*-product and enol-ground states.Prof. Coppenssaid: Is the time scale of H-transfer distance dependent? Would it be slower for intermolecular transfers?Dr Nibberingreplied: The time scale of H-transfer is first and foremost determined by the shape of the potential energy surfaces. If barriers exist, reaction times may be longer, and even the surrounding solvent may play a decisive role. For the photoacid molecules we currently study the dynamics appear to take place on picosecond time scales. However, even in these cases recent findings suggest that the H-transfer distance is not the most important parameter, but a more complex relaxation scenario through different electronic states may be the rate-limiting step.Prof. Wilsonsaid: You are correct that determination of hydrogen atoms is difficult with X-ray diffraction but the accurate “end-point” structures can be examined by a “slow technique”—neutron diffraction. Such precise/accurate structural techniques provide the ideal basis for setting upe.g.theoretical chemical modelling as identified by Prof. Coppens in his Introductory Lecture. In addition, such static methods can show “precursor” effects which set up conditions for processes such as proton transfer. For example, variable temperature or variable pressure studies can begin to probe the average shapes of potentials, for example in low barrier potentials.Dr Nibberingresponded: I agree that for a full understanding of the dynamics one needs to determine the structural information in real time. While awaiting the development of time-resolved structure resolving techniques with femtosecond time resolution, such as ultrafast X-ray spectroscopy, X-ray diffraction, electron diffraction or even neutron diffraction, we pursue femtosecond vibrational spectroscopy. However, for a full interpretation of our studies the understanding of the potential energy surfaces is a necessary requirement. Here the mentioned “slow techniques”, or I would rather say “steady-state techniques with averaging over a broad range of time scales” may be helpful in giving part of that information on the steady-state configurations, with high accuracy as well as the option to study temperature or pressure dependences. Nevertheless, for the study of transient states time resolved techniques are the only experimental approaches that potentially reveal the structural dynamical information.Dr Coleasked: If you were to isotopically enrich the subject sample and perform an otherwise identical experiment to that described, could you use the comparison of the data,viaexploitation of the isotope effect, to draw out more quantitative information from the results,e.g.with regard to force constants, and the alike? If experimental complications could be overcome, I wondered if it would prove very useful, not only for this quantification of the results, but also to help highlight the harmonic frequencies which could be used to give further consistency to the results in hand.Dr Nibberinganswered: It is known that in numerous H-transfer studies a kinetic isotope effect exists. Isotopic labelling will then give information on the potentials (i.ethe force constants). For the case of HBT the currently adopted model ascribes the finite H-transfer time to the motion of a low-frequency mode modulating the hydrogen bond distance. Since in this case the dynamics involve relative motions of the O and N atoms constituting the hydrogen bond, no significant H/D-exchange effect on the reaction time scale can be expected. This is reflected by the fact that the low-frequency modes do not alter signifcantly their frequencies upon H/D exchange.Prof. Helliwellsaid: You are advocating in your conclusions the femtosecond IR spectroscopy method but what systems do you have in prospect to apply it to? (Finding systems that are appropriate can be difficult and of course it is the proposition of this meeting that such research can be scientifically rewarding at the “Smoking Gun” level.)Dr Nibberingreplied: We are currently studying the dynamics of intermolecular proton transfer of photoacids in liquid solution, and we aim to extend these studies to photoacids in protein surroundings. In a different line of experiments we now also use femtosecond IR spectroscopy in the study of the photoinduced dynamics of photochromic switches. In principle the method can be extended to any chemical process induced by an ultrafast trigger, that may be an optical light pulse (for dynamics in electronic excited states), but could also be an IR pump pulse, initializing reactions in the electronic ground state.Prof. Helliwellopened the discussion of Prof. Bürgi's paper: Central to your approach is that the structure database scatter plot reveals sampling of metastable intermediate structures but, if I may use the analogy of jumping off a cliff, at the cliff and on the beach you have a larger population of structures than you do falling to the beach. How do you really reveal snapshots between the top and the bottom? (After all to isolate structural intermediatesviatime-resolved experiments requires considerable ingenuity to trap them in time.)Prof. Bürgiresponded: Structural databases sample stable structures only. For a set of structures with a particular fragment in common, the structures of the common fragment—the only part comparable among the members of the set—may vary considerably. The variation in bond lengths and angles reflects the influence of those parts of the structures, which are not common to the set,i.e.the influence of the environments. Our approach does not presume one cliff and one beach, but rather a series of closely related energy surfaces whose minima delineate a (more or less well defined) region of parameter space such as a reaction path (see Fig. 6 of our paper, for example).Dr Sagiasked: How can a dynamic process be elucidated from a collection of data points that were taken in different crystallization conditions. For example, ionic strength, pH, solventetc.Prof. Bürgireplied: Techniques in time resolved chemistry localize chemical processes along the arrow of time. In our approach we attempt localization in the structural domain. The approach is based on the hypothesis that a given class of ground state dynamic processes is associated with a common reaction path on a generic energy surface. The details of the path and the surface for a specific process,i.e.its minima and transition states, depend somewhat on the specific molecule undergoing this process. Our hypothesis implies that the stationary points characterizing the various molecules tend to congregate in the low-lying parts of the generic surface, which—by definition—includes the reaction path of the process. They can be said to ‘map the reaction path’ along the structural coordinate (principles of structure–structure and structure–energy correlation, see section 3.1). Empirically the hypothesis has been found to hold for many different chemical systems (see ref. 11 of the paper). (My answer to Prof. Helliwell's question is also relevant.)Dr Techertasked: How would the presented simulation change if the assumptions of the Eyring theory/transition state theory (equilibrium reactant ↔ TS) were to break down?Prof. Bürgianswered: I assume that you refer to the empirical correlations between ground state structures obtained from crystal structure analyses and activation energies derived from reaction rate constants measured in solution (see section 5 of the paper). These correlations apply to thermally activated processes and illustrate the Hammond principle from a structural point of view. They are similar in a way to correlations that can be understood in terms of a Marcus relationship, which emphasises the energy point of view. The reactions you are interested in are initiated by a photo-excitation and followed by vibrational relaxation. These processes lead to a more or less long lived intermediate associated with a structure on an excited-state surface that usually differs significantly from the original ground state structure. Therefore the simple correlations described in section 5 will not apply.Dr Hirstsaid: One section of your paper mentions the non-planarity of amides, referring to deviations of up to 10° in proteins. Do you believe that protein crystallography can resolve such deviations reliably? And if so would you like to expand on your speculation that this extra degree of freedom is important in protein folding?Prof. Bürgireplied: To measure the non-planarity of amides accurate positions of the atoms Ci−1′, Ni, αCiand HNiare needed. For a number of high-resolution X-ray structures several torsion anglesω(αCi−1–Ci−1′–Ni–αCi) have been reported to deviate from 180° by more than 10° (for a recent example see ref. 1). The position of HNicannot be determined accurately from even the highest-resolution X-ray diffraction experiments, but could be determined by high-resolution neutron diffraction (<1 Å). The hinge-like flexibility associated with this degree of freedom could help to minimize strain energy of protein folds, especially with respect to N–H⋯X hydrogen bonds. It could also facilitate domain motions associated with protein function. Even though the energy increment associated with the flexibility of a single N–H⋯X hydrogen bond may be small, a substantial contribution to the total energy may result, because of the large number of such hydrogen bonds. However, these are speculations and require experimental verification.1 J. Symersky, Y. Devedjiev, K. Moore, C. Brouillette and L. DeLucas,Acta Crystallogr., Sect. D, 2002,58, 1138–1146.Prof. Sir John Meurig Thomascommented: Prof. Burgi's persuasive arguments about how much we can learn about fast chemical processes from slow diffraction experiments prompts me to draw to the Discussion's attention to how much it becomes possible to gain quite useful insights into the process of catalytic turnover at a well defined active site from steady-state measurements of X-ray absorption fine structure coupled with density functional theory computations. In papers that my colleagues and I have published elsewhere1–3we showed that the commercial Ti–SiO2catalyst for the epoxidation of olefins has an active site which is a Tiivion tripodally attached ,viaoxygens, to the underlying silica support. This ion has also attached to it an OH group. In the presence of hydroperoxide oxidant and the alkene, the XANES fingerprint of the Tiivunmistakably shows it to be 6-coordinated,3and EXAFS analysis yields precise bond distances and quite good bond angles in which the Tiivis involved (see the figures in the paper). These tally well with the corresponding values deduced from DFT. Taking all experimental factors into consideration we may plausibly portray the key catalytic act as the “plucking away” of one of the oxygens of the bound peroxide, which leaves the active site in its original 4-coordinated state, ready for further catalytic turnover. Fuller details are described in a recent paper.4(A fictional animation of the conversion of cyclohexene to its epoxide was shown during this contribution.)1 T. Maschmeyer, G. Sankar, F. Ray and J. M. Thomas,Nature, 1995,378, 159.2 J. M. Thomas, G. Sankar and C. R. A. Catlow,Top. Catal., 2000,10, 225.3 J. M. Thomas and G. Sankar,Acc. Chem. Res., 2001,34, 571.4 G. Sankar, J. M. Thomas C. R. A. Catlow, C. M. Baker, D. Gleeson and N. Kaltsoyannis.Prof. Greavessaid: In your intriguing video which models the structural changes taking place during the catalytic event around Tiiv, the start and end configurations come directly from static experiments.Prof. Wilsoncommented: To emphasise the point ofin situversusquenching experiments, oftenin situexperiments with extrapolation can give more accurate (and less misleading) information than more traditional methods which often involved quenching and the implicit assumption that the state frozen-in by the quenching process is actually of relevance to the reaction under study.Prof. Helliwellopened the discussion of Prof. Moffat's paper: (i) Re your target proteins, how many are membrane bound (i.e. which would then be very difficult to crystallise)? (ii) Re Fig. 1 of your paper. A monochromatic chirping approach will involve a profile method. What accuracy of intensity measurements are to be expected?Prof. Moffatresponded: (i) The target proteins are identified only by sequence homology. Some such as the photosynthetic reaction center and light-harvesting complexes are indeed integral membrane proteins; others such as phototropin are loosely membrane-associated and can readily be solubilized, yet others are probably cytoplasmic and soluble in aqueous media. The experimental distribution into these three subsets, to my knowledge, has not been established. My point is that experimental organisms such asArabidopsisoffer several light-sensitive target proteins (and even more targets if domains of the full-length proteins are considered, or of the light-insensitive components that lie further downstream in the overall, light-driven signal transduction pathways). (ii) This has not yet been explored. The accuracy will depend on, among other factors, the nature of the spatial chirp, the mosaicity of the crystal and the resolution of the spot being considered. One way to approach this problem is to introduce time-dependent Ewald spheres into the static treatment for oscillation spot size and shape developed 20 years ago by, for example, Greenhoughet al.1–31 T. J. Greenhough, J. R. Helliwell and S. A. Rule,J. Appl. Crystallogr., 1983,16, 242–250.2 T. J. Greenhough and J. R. Helliwell,J. Appl. Crystallogr., 1982,15, 493–508.3 T. J. Greenhough and J. R. Helliwell,Prog. Biophys. Mol. Biol., 1983,41, 67–123.Dr Mayasked: (i) You mentioned that there are 58 photosensitive proteins inArabidopsis. How many proteins are not photosensitive? Only direct photoreactions are very rapid. (ii) You may not be able to observe all reactions (and their consequences) that are going on in the protein within a crystal, because the movement may not be possible in the crystal.Prof. Moffatanswered: (i) The exact number of proteins inArabidopsisthat are not photosensitive is now known; but it is certainly orders of magnitude larger than 58. That is, only a small fraction of proteins in an organism are naturally photosensitive. Yes, only direct photoreactions are very rapid, but this is characteristic of all naturally light-sensitive proteins. Attempts to confer light sensitivity on otherwise light-inert reactions by preparing so-called “caged” compounds, such as caged ATP, suffer at present from the fact that the initial, direct photoreactions are followed by much slower, dark reactions that ultimately liberate the desired product, such as ATP. It may be possible to develop other forms of caged compounds that do not suffer from this limitation.(ii) Yes, but the intermolecular forces that stabilize the crystal lattice in biological macromolecules are weak, roughly 1 kcal mol−1per interface. I find it worrying that activity in the crystalline state is seldom directly examined, let alone quantitated; we blithely assume that crystal structures always provide an accurate foundation for assessing mechanism. In any case, it is highly desirable to check by optical means that the reaction in the crystal firstly can proceed at all, secondly that it does so by the same reaction mechanism as in dilute solution, and thirdly that the rate coefficients associated with each step in this mechanism are not significantly different from those in dilute solution. (“Significant” here must be thought of in energetic terms; by how much is the free energy of activation for the particular step affected?)Prof. Sir John Meurig Thomassaid: Do you feel that, in using Laue time-resolved and other X-ray crystallographic techniques on enzyme crystals, one is addressing the active sites and other regions of the enzyme under realistic (pseudo-physiological) conditions?Prof. Moffatreplied: Yes—provided always that one can demonstrate that chemical or biochemical activity is quantitatively retained in the crystalline state. If this is NOT the case then time-resolved experiments are of no relevance and indeed, the static enzyme structure itself comes into question.A historical note on this point—I was first stimulated to consider time-resolved crystallography in 1969 by a key paper by Parkhurst and Gibson1who examined quantitatively the reactivity of haemoglobin towards carbon monoxide in the physiological environment of the erythrocyte, in the biochemist's environment of dilute solution, and in the crystallographer's environment of the intact crystal. They concluded that, at least for the reaction studied, the kinetic behaviour of haemoglobin was very closely similar in these three, very different environments. This provided strong supporting evidence for the physiological relevance of the crystal structures of haemoglobin then being determined by Perutz and colleagues. It took a further 25 years before it became clear that the kinetic properties of haemoglobin are in fact significantly different, both qualitatively and quantitatively, in the crystal compared with dilute solution.21 L. J. Parkhurst and Q. H. Gibson,J. Biol. Chem.,1967,242(24), 5762.2 C. Rivetti, A. Mozzarelli, G. L. Rossi, E. R. Henry and W. A. Eaton,Biochemistry, 1993,32(11), 2888–2906.Prof. Greavescommented: If you can bring the time scale of diffraction experiments into the regime of ps or shorter, will this possibly open the door to penetrating the co-operative elements of photostructural phenomena in macromolecules.Prof. Moffatadded: It might bring coherent structural transitions into view—an exciting challenge.Dr Chensaid: I am always very impressed by the movies of protein movement from your studies. However, I would like to understand what happens in the later time after the initial coherent atomic movement triggered by the laser pulse are lost in the protein molecules. One can envision by the potential energy landscape in the protein that multiple conformations exist after the initial protein quake?Prof. Moffatreplied: Coherence is presumably maintained only for a very short period of time after the laser pulse, perhaps a few hundred fs but in any case, a time much shorter than the present time resolution of our experiments. Thereafter, we believe that structural processes evolve purely stochastically; coherence is lost. That is, the subsequent time evolution arises from the build-up and decay of the populations of intermediate structures, exactly as in dilute solution. A consequence is that at all time points, there is indeed a mixture of multiple conformations present. Our challenge is to unscramble this mixture (for example, by singular value decomposition and associated techniques) and to recover the time-independent structures of all intermediates. This is in progress, as illustrated by the poster by Schmidt, Rajagopal, Ren and Moffat presented at this meeting.Prof. Bürgiasked: How does the information from a chirped pulse in a time slice of 1 ps, say, compare with that of a typical Laue experiment?Prof. Moffatanswered: In a time slice of 1 ps, say at a timetafter the laser pulse, only a subset of the Laue spots on a “normal” Laue image would be stimulated. This subset corresponds to those spots stimulated between an X-ray energyEandE + dE, where the values ofEand dEdepend ont, dt(here, 1 ps) and the nature of the chirp. Thus the information content would be a subset of that present in a “normal” Laue image. However, I emphasize that each “chirped” Laue image contains all the spots that would be present in the corresponding “normal” Laue image.Prof. Greavessaid: You say that the time resolution for Laue diffraction from a chirped source in principle is greater than that from an unchirped source even though the former is far longer than the latter. Is this because you have created a more manageable instrument function?Prof. Moffatreplied: The time resolution from an unchirped pulse is today set by the total duration of the X-ray pulse, say 100 ps. If such a pulse is manipulated to produce a (much longer) chirped pulse, the time resolution can never be better than 100 ps since phase space volume must be conserved. Equally, if a 100 fs unchirped pulse from the proposed SPPS or LCLS (Linac Coherent Light Source) is stretched to provide a 1000 times longer, chirped pulse of 100 ps total duration, it may be possible to retain near-100 fs time resolution by the strategies outlined in the main paper. This is not really due to an “instrument function”; rather, it results from the experimental design that maps time into X-ray energy and X-ray energy into space, on the detector.Prof. Finneyasked: Do you have any good physical evidence to justify a statement that, as you improve the resolution towards ∼100 fs, you will have a coherent target system? Light takes 1 ps to cross a 0.3 mm crystal. Does this not raise a basic problem with approaching 100 fs time resolution?Prof. Moffatanswered: Yes it does; the physical dimensions of the crystal (likely to be somewhat smaller than 0.3 mm, say 0.075 mm in typical dimension) does enter into the time resolution. Rather than a disadvantage this fact may be exploited as proposed by Neutze and Hajdu;1but this will be very challenging experimentally.1 R. Neutze and J. Hajdu,Proc. Natl. Acad. Sci. USA,1997,94, 5651–5655.Dr Nibberingasked: Did you consider the effects of group velocity mismatch between the optical pump pulse and the X-ray probe pulse? How thin do your crystals have to be to maintain the anticipated time resolution of about 100 fs (100 μm or less)?What do you expect will happen when probing a molecular wave packet motion that will spread out in the course of time? Will the diffraction signal initially be strong and wash out with the dephasing time (typically 0.5–2 ps)?Prof. Moffatreplied: As in my reply to Prof. Finney, crystal dimensions do inescapably enter into the time resolution.The initial diffraction signal will presumably arise from coherent motion of the excited species; and then evolve (but not “wash out”) as coherence is lost over the few ps time frame when stochastically independent structural processes begin to come into play. (My replies to Prof. Greaves and to Dr Chen are also relevant here.)Prof. Greavessaid: If the pump pulse occurs during the chirped pulse and the X-ray detector has no time resolution, how are the ensuing changes in diffraction differentiated from those that are unperturbed if neither are known beforehand—or is this simply a question of normalising against a pulse-free pattern?Prof. Moffatresponded: Laser-pulse-free patterns will normally be acquired, interleaved in time with the laser-pulse-present patterns. Comparison of the structure amplitudes derived from the latter (which correspond to time zero) with those of the former (which correspond to different times after the laser pulse depending on their X-ray energy) will yield the desired, time-dependent differences in structure amplitudes.Prof. Wilsonsaid: As I understand it, there is no time (energy) or space resolution on the detector. Also, the method does not improve the overall data collection time resolution, but instead is improving the intrinsic time resolution of the Laue sections taken in each shot.Prof. Moffatreplied: There is indeed no time resolution in the detector but it certainly has excellent spatial resolution; and energy resolution is afforded by indexing the Laue pattern and hence assigning an energy (or more accurately stated, a small energy range) to each spot. In conventional Laue experiments, the time resolution is at present limited by the total duration of the X-ray pulse from the synchrotron, typically 100 ps. The time resolution of a Laue experiment conducted with a chirped X-ray pulse is not limited by the total duration of this pulse but can be much shorter. Exactly how much shorter depends on many factors, among them the nature of the chirp itself.

ISSN:1359-6640    

DOI:10.1039/b207968m

出版商:RSC    

年代:2003

数据来源: RSC

摘要:

Prof. Finneyopened the discussion of Dr Smart's paper: How can you ensure that you are not cross-linking between molecules? Could not such cross-linking explain the peptide association you mention on the seventh page of your paper?Dr Smartresponded: The association you refer to occurs before the (i,i + 7)GCN4-p1 is cross-linked. We find that the circular dichroism signal at 222 nm is anomalously small in comparison with previously published values (ref. 22 and 27 of the paper) yet it shows a marked concentration dependence (Table 1 of the paper). The presence of 3 mM DTT would ensure that the sulfhydryl groups of the cysteine residues are reduced, thus precluding the formation of disulfide bonds. As set out in the Discussion and presented in Fig. 7 we believe that the effect is probably due to the mutation of a number of residues that would create a hydrophobic stripe on the surface of the intact coiled-coil.Dr Sagisaid: The cross-linked peptide has an additional spacer which links the two cysteines. Such an artificial spacer may affect the DNA binding properties of the cross linked peptide to the DNA in terms of affinity and/or the “correct” binding.Dr Smartreplied: The peptide we have described here is a variant of GCN4-p1 which lacks the DNA basic binding helix found on the longer GCN4-bZIP. A clear aim of this work is to produce a variant of GCN-bZIP whose DNA binding is under reversible photo-control. The strategy presented here is to control the dimerisation properties of the zipper part of the protein that is distant from the DNA. A clear alternative would be to try to alter the conformation of the DNA binding helix. As explained in the Introduction of the paper based on the results of Vinson and co-workers (ref. 18 of the paper) we expect control of the leucine zipper part to produce a high degree of photo-control. An advantage of controlling an area distant from the DNA is that the binding specificity of the protein is unlikely to be affected.Dr Techertsaid: 23% of the peptides do not isomerise. What are the alternative reaction channels?Dr Smartanswered: We find for the cross-linked (i,i + 7)GCN4-p1X coiled-coil peptide that illumination with 370 nm for long periods of time resulted in only 62% of the azobenzene chromophores switching to thecisstate, the other 38% remaining in atransconformation. This is a feature of using the azobenzene chromophore—the dark-adapted state is generally >99% of thetransisomer whereas the maximum percentage of thecisisomer that can be achieved by irradiation varies but is typically from 70 to 90%.1It is interesting to note that the maximum percentage conversion achievable with the cross-linker used here varies with the application it is used for. In recent work (ref. 9b in the paper) we have shown, in a peptide where the cross-linker is used to link two cysteine residues with a relative spacing of (i,i + 4), that a proportion of 90% can be switched to thecisstate by irradiation. The half-life for thermal relaxation back to thetransstate is also found to be longer than in the application presented here.1 H. Rau, inPhotochemistry and Photophysics, ed. J. F. Rabek, CRC Press, Boca Raton, 1995, p. 119.Prof. J. C. Smithasked: Can one follow the time course for helix folding in these compounds using molecular dynamics and/or fast spectroscopy? Certainly for molecular dynamics (MD) one now has sufficient computer power to sample peptide conformational equilibria and maybe combining MD with your and other experiments one might learn something about helix initiation and propagation.Dr Smartanswered: We have supplied the original cross-linked monomeric peptide (ref. 8 in the paper) to a number of groups for fast time resolved spectroscopic studies (ORD, absorption/photoacoustic spectroscopy and FTIR methods). Initial preliminary results show that it is possible to trigger photoisomerisation of the chromophore using a laser pulse and that the conformational change in the peptide that follows this is fast (<30 ns), although these results need to be confirmed. Your suggestion for molecular dynamics studies is certainly a good one. In this context Watchtveitl and co-authors have just published a study where molecular dynamics simulation is linked to femtosecond time resolved spectroscopy results for a seven residue peptide that is backbone-cyclized with an azobenzene derivative. Upon photoisomerization of the chromophore the peptide switches between two distinct coil conformations (in DMSO) and an encouraging correspondence of results between simulation and experiment is observed.11 S. Spörlen, H. Carsten, H. Satzger, C. Renner, R. Behrendt, L. Moroder, P. Tavan, W. Zinth and J. Wachtveil,Proc. Natl. Acad. Sci. USA, 2002,99, 7998.Mr Terieteasked: Can the observed difference in relaxation of the azobenzene switch be correlated to internal kinetic processes involved in the formation and folding of the helix? Has the system been investigated using NMR spectroscopic methods?Dr Smartreplied: As detailed in the response to the question of Dr Techert both the maximum percentage conversion of the azobenzene chromophore to thecisform and its thermal relaxation lifetime do depend on the ground state structure of the peptide to which it is attached. To what extent the isomerization of the linker and the refolding of the peptide are temporally coupled is presently unknown.We have been studying the original cross-linked monomeric peptide (ref. 8 in the paper) by NMR methods and will report the results soon.Dr Hädenersaid: Is it clear that the wavelengths of the light used to induce isomerisation of the azo groups will be the same for the free crosslinker as compared to the crosslinker attached to the cysteines? In the latter case the isomerisation will be sterically hindered and this may affect the energy of the photon needed to isomerise the azo groups.Dr Smartresponded: We have not observed such an effect. The absorption spectrum of the azobenzene cross-linker before reaction, once attached to a number of peptides or reacted with glutathione, is essentially identical.1As noted in the paper and in previous replies, both the maximum percentage conversion of the azobenzene chromophore to thecisform and its thermal relaxation lifetime do vary with the context. It should be noted that the absorption spectrum of azobenzene derivatives does depend on the nature of the substituent on the ring. Unmodified azobenzene in thetransform has an absorption maximum around 340 nm.21 G. A. Woolley, personal communication.2 H. Rau, inPhotochemistry and Photophysics, ed. J. F. Rabek, CRC Press, Boca Raton, 1995Prof. Wilsonsaid: I am interested in the possibility of gaining more information on the “re-coiling” of the helix in the slow 10–30 min relaxation process. Has this been examined and is there potential there to use this system (admittedly in the presence of the zipper) as a model for protein folding.Dr Smartreplied: The relaxation process that has a half-life of 10 to 30 min is the thermal revision of the azobenzene chromophore from itscisto the dark-adaptedtransform. It is quite possible to use circular dichroism to monitor the secondary structure of the peptide during this revision. However, because conformational changes of peptides occur on time scales that are very many orders of magnitude faster the results would simply reflect the particular proportions ofcisandtransforms at each particular time. It can be noted that for the three different monomeric peptides that we have studied in detail (ref. 8 and 9b in the paper) CD results show isodichroic points. This is consistent with a simple two-state equilibrium between the disordered and helical states of the proteins.Prof. Wilsonsaid: By how much can the relaxation process (fromcis-azobezene/uncoiled helix totrans-azobenzene/coiled helix) be alterede.g.by the use of laser/light flash. Further, by how much is the process speeded up if the azobenzene is removed/excised from the uncoiled protein by chemical means.Dr Smartanswered: In the coiled-coil peptide we have described we have produced a system where thetransform of the cross-linker produces a coiled-coil with a lower helicity than that produced by irradiation with 370 nm light. In monomeric peptides it is possible to initiate photo-isomerisation using a pulse of laser light (as described in a previous reply). It would be difficult to specifically remove the cross-linking compound from the peptide by chemical means without destroying the peptide. In the paper we have described in detail the properties of the GCN4-p1 derivative prior to cross-linking.Dr Hirstasked: In your paper, you report that the helicity of native GCN4 drops by about 25% when the pH changes from pH 7 to pH 5. Why might that be?Dr Smartanswered: We have indeed found this effect, in agreement with the previous results of Sosnick and co-workers (ref. 26 and 27 in the paper), but can offer no explanation. That such a subtle change in pH produces a large effect on the circular dichroism signal of a well-understood coiled-coil is of interest.Dr Grantasked: Have you ever considered using the technique of Raman optical activity (ROA) as it gives more detailed structural information than just CD, although ROA is not a time-resolved technique, if revision back totransis slow enough, it may be possible to employ this technique.Dr Smartreplied: That is a very good suggestion. We are also interested in the possibility of using synchrotron radiation circular dichroism as this offers the potential to acquire more detailed information with good time-resolution.Prof. J. C. Smithasked: A speculative question—various groups are working on using synthetic metal chelates to design sequence-specific DNA cleavage systemsi.e., artificial restriction enzymes. Is there any chance (or interest) in photoactivating such processes?Dr Smartanswered: The chemical nucleases produced by Bailly and co-workers1do not have a peptide component and so our approach is not immediately applicable. However, in this context Komiyama and co-workers have produced impressive results in oligonucleotides incorporating azobenzene in place of a DNA base.2,3Most notably they have produced an oligonucleotide whose binding to DNA can be utilized to photo-regulate the action of DNA polymerase enzyme on the complimentary strand.41 S. Routier, H. Vezin, E. Lamour, J. L. Bernier, J. P. Catteau and C. Bailly,Nucl. Acid Res., 1999,27, 4160.2 X. G. Liang, H. Asanuma and M. Komiyama,J. Am. Chem. Soc., 2002,124, 1877.3 H. Asanuma, X. G. Liang, T. Yoshida and M. Komiyama,ChemBioChem, 2001,2, 39.4 A. Yamazawa, X. G. Liang, H. Asanuma and M. Komiyama,Ang. Chem. Int. Ed., 2000,39, 2356.Prof. Moffatsaid: What would be the ideal set of properties for a light-dependent, artificial transcription factor (or DNA binding protein)?Dr Smartreplied: An ideal light-dependent transcription factor would have:(i) A complete photo-control of the DNA binding process, that is absolutely no DNA binding in the light but completely normal binding upon irradiation.(ii) Use a chromophore that is altered by a long wavelength of light in comparison to azobenzene. This is important to avoid both scattering and radiation damage effects to living cells. Furthermore the chromophore should have a high quantum yield so that only short irradiation would be required.(iii) Have rapid reversible photo switching between the on and off states such that it is possible to turn on the protein for a given time and by using an alternative wavelength. This would allow the equivalent of single turnover experiments on enzymes by watching the effect in a cell of a small “dose” of the active protein.(iv) Be easily produced—most ideallyin vivoby standard molecular biology procedures with no chemical intervention so that it can be assayed in the cell in which it is expressed.Prof. Helliwellasked: What are the applications in biotechnology?Dr Smartanswered: The technology is still in a development phase. We have demonstrated how a photo-isomerisable crosslinker can be used to control the conformation of peptides and coiled-coil molecules. The next stage is to apply the method to recombinant proteins and show how activity can be controlled. The initial application will be to DNA binding proteins and work is in progress.Prof. Moffatsaid: Biological systems have evolved various forms of chemistry for absorbing light and harnessing its energy to drive certain biological processes. Can you learn from nature, to direct your chemical approaches?Dr Smartreplied: Compared to the natural biological systems you refer to our approach must be seen to be crude. The subtlety involved in the mechanisms of the rhodopsins and other photochromic proteins is impressive. Compared to a chemical approach where it is necessary to purify and modify proteinsin vitroa biologically based method of photo-control would have great advantages. For instance if it might be possible to achieve photo-control by using a fusion method where a given target was produced together with a natural photosensitive protein. This would have the advantage of being easily applied toin vivosystems.Prof. Sir John Meurig Thomasopened the discussion of Dr Techert's paper: Your interesting work on the topochemical photodimerization ofp-formyl-trans-cinnamic acid, where the crystalline monomer is converted to a polycrystalline (amorphous?) dimer, prompts me to draw to your attention a very nice example of photodimerization where a single-crystal → single-crystal transformation occurs. This (diffusionless) 2 + 2 photodimerization takes place1in the archetypal molecule, 2-benzyl-5-benzylidenecyclopentanone (BBCP) and in itsp-bromo derivative (BpBrBCP). My colleagues and I were able to record in a time-resolved fashion (on a 4-circle diffractometer) detailed crystallographic measurements on these materials at various stages of solid-state conversion at room temperatures. In particular, for the case of BBCP, the gradual formation of the cyclobutane ring between the two monomers in an incipient dimer could be directly identified2(see Fig. 1 presented here).The gradual formation of the cyclobutane ring between the two monomers of BBCP in an incipient dimer.I am also prompted to raise some more general issues concerning topochemical photo-induced transformations in organic molecular crystals. My own interest in such phenomena began when it emerged that the topochemical postulates of Schmidt and Cahen were, in some cases, vitiated. For example, anthracene may be photo-converted in the solid state to yield the diplanar-dimer, yet the structure of the stable, monoclinic form of anthracene should not permit such a transformation to occur. Even more puzzling was the observation that 9-cyanoanthracene (9CNA) yields the “wrong” dimer,i.e.thetransrather than thecisform, although the packing in the solid state of 9CNA suggests ready formation of thecisdimer, just as your cinnamic acid monomers yield the truxinic acid dimer. Why is this so? It is because crystalline defects hold sway. Excitation energy migrates through the crystal and is trapped at defects, where the local structure is what matters.At stacking faults (bounded by two partial dislocations) in 9CNA juxtaposed incipient dimers are in thetrans, not thecisconfiguration. And in anthracene, certain types of strucutal faults—like those favoured by the application of gentle pressure that facilitates the production of a metastable form of anthracene in which incipient dimers are brought within the distance (ca.4 Å) required for photodimerization—are also implicated in the photoreactivity.There are many other examples (e.g.1,8-dichloro-9-methylanthracene and 1,8-dichloro-10-methylanthracene), where my colleagues and I found that photodimerization yields the “wrong” dimer; and the explanation in each case is to be found in the role of structural faults.5With the refined techniques that Techert, Wulff and coworkers report in their paper, it would be profitable to re-investigate some of these seemingly anomalous instances of topochemical reaction.1 H. Nakanishi, W. Jones, J. M. Thomas, M. B. Hursthouse and M. Motevalli,J. Phys. Chem., 1981,85, 3636.2 J. M. Thomas,Nature, 1981,289, 633.3 M. D. Cohen, J. M. Thomas, J. O. Williams and Z. Ludmer,Proc. R. Soc. London, Ser. A, 1971,324, 459.4 J. M. Thomas, G. M. Parkinson, S. Ramdas, M. J. Gorringe, C. M. Gramaccioli, G. Filippini and M. Simonetta,Nature, 1980,284, 153; see also S. Ramdas, J. M. Thomas, J. O. Williams and G. M. Parkinson,Eighth Molecular Crystals Symp., Santa Barbara, 1977.5 J. M. Thomas,Philos. Trans. R. Soc. London, Ser. A, 1974,277, 251.Dr Techertresponded: Indeed, the suggested 2 + 2 photodimerisation you refer to in ref. 1 came to our attention recently and we decided to work on it in more detail since it is a full single-crystal–single-crystal transformation. In this context it will be particularly interesting to measure and to understand the differences in the kinetics if, as in the presented work, the product state is amorphous (or less ordered) or if the product state is fully periodically lattice ordered.9CNA and other derivatives are also classical and interesting candidates for investigations of topochemical reactions.Prof. Coppensasked: What drives the condensation of the phonon mode into the metastable state you propose. Can you reproduce the intensity changes reproduced in Fig. 12 (of the paper) with the model? What is the percent conversion by each X-ray flash that gives rise to your signal?Dr Techertanswered: In Fig. 1 (of the paper) only theeffectivereaction mechanism with the effective reaction coordinate of the dimerisation process is shown. However, if one looks more carefully into photo-induced dimerisation processes (e.g.taking the Woodward–Hoffmann rules into consideration), then one has to study in more detail the shape of the potential energy hypersurface (PES) of the ground state as well as of the electronically excited state or states in which the sample is excited by 267 nm excitation wavelength. Depending on the interactions of the different PESs of the excited states, level-crossings and adiabatic transitions between the different excited states drive the reaction to particular product states. Here, we think that the proposed metastable state is connected with a local minimum on the excited state PES decaying from there to the product state. This local minimum is populatedviaan optically allowed vibronic transition where the electronic state and one (or more) phonon modes are excited.At the moment we are performing simulations employing density functional theory of this system in order to obtain a more detailed understanding of the found transient signal changes. In this context it is necessary not only to study the geometrical aspects but also to simulate the energy levels of the (different distorted) lattices in the ground state as well as the electronically excited state PESs.Taking the preliminary state of the simulation into consideration, the intensity changes in Fig. 12 are reasonably well reproduced. However, the quality of the measurements as well as the quality of the theoretical simulations have to be improved further in order to obtain a complete and quantitative picture of the dimerisation mechanism.Due to laser damage problems, p-FCA cannot be excited with too high a laser power. From spectroscopic measurements we approximated that a maximum of 5–8% of the chromophores are excited in one laser flash. This corresponds to a relative change in X-ray signal of about 2–4% (maximum) in the reported system.Dr Nibberingasked: Are the phonons that are supposed to play a role in the reaction dynamics of the acoustic or optical type? If they are optical phonons, one could drive these with optical light pulses through the mechanism of impulsive stimulated Raman scattering. As a consequence, the reaction dynamics will be influenced when the phonons are light-driven, and one may hope to perform some form of optical control of the reaction dynamics.Dr Techertanswered: According to the selection rules, the proposed mechanism is based on optically active phonons, which drive the process. Concerning the energies of these phonon modes, frequencies in the THz regime are assumed—as,e.g., in solid beryllium or InSb.1Controlling the reaction dynamics by the impulsive stimulated Raman scattering technique is a very interesting approach, in particular since only a little work has been carried out concerning the vibrational spectroscopy/analysis of this type of reaction.1 D. A. Reis, M. F. DeCamp, P. H. Bucksbaum, R. Clarke, E. Dufresne, M. Hertlein, R. Merlin, R. Falcone, H. Kepteyn, M. M. Murnane, J. Larsson, Th. Misalla and J. S. Wark,Phys. Rev. Lett., 2001,86, 3072.Prof. Bürgiasked: How does the wavelength of the light used for dimerization affect the lack of crystallinity of the product?Dr Techertreplied: This is indeed a very interesting question. At the moment we do not know the answer. Using 267 nm as the excitation wavelength is surely far above the resonant state of the vibronic transition which is necessary to initiate the reaction. Part of the excess energy will be transformed in heating of the sample, which can lead to structural disorder and loosing the crystallinity of the product state. To answer this question, a systematic study of the amorphism of the product state as a function of excess excitation energy is necessary.Prof. Helliwellsaid: Please comment on the X-ray laser potentionalversusradiation damage issues for your particular compounds.Dr Techertcommented: The proposed X-ray FELs will deliver pulses of approximately 1012photons with a duration of about 100 fs that will create an ideal source for the investigation of ultra-fast processes, like structural (re)organisation. The high flux also will enable one to investigate irreversible processes much more easily as well ase.g.nonlinear properties of material. Furthermore the coherence of the XFEL radiation certainly will pave the way to new types of X-ray diffraction experiments.Also Dr Th. Tschentscher, Hamburger Synchrotronstrahlungslabor HASYLAB at the Deutsches Elektronen-Synchrotron DESY, Hamburg.It is clear, however, that sample damage due to the high number of photons must be considered carefully when defining experiments. Here one must take into account the reduced photon flux at the sample due to the throughput of mirrors and monochromator and due to collimation. This flux has to be compared to the photon flux required to measure the effect under investigation. Beam attenuation techniques (as already practised at synchrotrons of the third generation) may be applied to match both values.To estimate the sample damage one has to take into account the particular time structure of the XFEL radiation. The short duration of the pulse, and therefore the short sampling time in diffraction experiments, allows one to extract information before diffusion controlled processes can play a role.Processes occuring at the sub-picosecond timescale therefore are important to consider. If sample damage effects are found to alter the experiments single sample exchange methods can be applied, like liquid jets or sweeping beam techniques. It is also important to consider sample damage due to laser excitation. Here already sample exchange techniques are practised.Dr Techertopened the discussion of Dr Cole's paper: What is the concrete application of the rhenium carbene complex and why is it important to know the triplet structure?Dr Coleresponded: The rhenium carbene complex is one in a series of compounds that has potential as a candidate for a building block as a ‘molecular wire’, and it also has attractive prospects in catalysis (its photolysis might alter the reactivity of the carbene ligand through population of the3MLCT state).Prof. Coppenssaid: You mentioned a life time of 0.5 μs but did not mention the temperature. The life times of excited states of metalloorganic complexes are often quite temperature dependent. Did you obtain the spectra at the same temperature?Dr Colereplied: The emission spectra of the sample were measured at both room temperature and 77 K in dichloromethane solvent and a 4∶1 ethanol–methanol glass matrix, respectively. Their profiles are very similar in both cases thus indicating that the nature of the dominant3MLCT band remains the same at both temperatures,i.e.the lifetime, measured at room temperature, is not significantly altered at 77 K.11 W.-M. Xue, M. C.-W. Chan, Z.-M. Su, K. K. Cheung, S. T. Liu and C.-M. Che,Organometallics, 1998,17, 1622.Prof. Wilsonsaid: We heard that changing temperature dramatically affects the timescales of the process under study. Does it also affect the degree of transformation in the process?A related point—why not “quasi-continuously” pump to achieve higher degrees of transformation and allow higher resolution diffraction experiments (e.g.charge density). Is the problem that molecules are knocked out of the excited state by further irradiation?Dr Coleresponded: It could affect the degree of transformation, but more drastically, it could affect the entire nature of the emissive-state of the sample,e.g.a compound with a dominant3MLCT type emission at room temperature could be overridden by an intraligand (IL) state which dominates at low temperature. (See for example, compound 7 in ref. 1.)By quasi-continuously pump–probing a sample, one is likely to destroy the samplecf.a typical continuous laser provides ∼1018photons per pulse, compared with a pulsed laser of 1014–1016depending on its repetition rate.1 W.-M. Xue, M. C.-W. Chan, Z.-M. Su, K. K. Cheung, S. T. Liu and C.-M. Che,Organometallics, 1998,17, 1622.Dr Chenasked: In your paper, the excited state lifetime was measured in dichloromethane glass at 77 K. However, the diffraction experiments were carried out in the single crystal. The triplet lifetime of the molecule therefore is likely to be shortened. Have you measured the lifetime in the single crystal? With ∼20% excitation states in your paper, how do you deal with triplet–triplet annihilation?Dr Coleanswered: No. Lifetime measurements of samples on a single crystal are practically very difficult although we are presently investigating this as an option. The triplet lifetime may indeed be different in the glass compared with the single-crystal since the solvent viscosity will prevent thermal equilibrium in the rigid 77 K glass in a time comparable with the emission lifetime. Moreover, the nature of the surrounding frozen and disordered solvent environment is likely to affect the lifetime.20% is stated in the paper simply as a likely maximum level of excitation (based on observations from the literature on other samples). The level of excitation for this compound is not reported in this paper, since data refinement is still underway. Therefore, triplet-triplet annihilation is not necessarily a significant effect here.Dr Techertasked: In Fig. 2 of the paper, a laser power of about 50 μJ is used. Does one need these high energies in order to reach a sufficient excitation level in the crystal?Dr Colereplied: Tests were carried out to assess the maximum level of laser power that could be used without destroying the crystal during the experiment. As a result, we arrived at a chosen laser power of 50 μJ. The greater the laser power, the greater the level of excitation and the larger the crystal that one can use for the experiment (c.f.optical penetration depth calculations). Therefore, by using the maximum feasible laser power, we were able to maximise the intensity of X-ray diffraction, as well as the level of excitation, thereby ensuring the best possible contrast between excited and ground state.Prof. Coppensaddressed Dr Techert: You asked why Coleet al. need a power as high as 50 μJ per pulse. We have used 200 μJ per pulse on a 50 μm crystal and found this to give a marginal photon/molecule ratio, so in fact more than 50 μJ per pulse is needed. This leads to the next question. How did you get away with a lower power pulse in the DMABN experiment?Dr Techertresponded: In the experiments reported till now1,2the samples were prepared in such a way that the size of the crystalline powder sample never reached the upper limit of 1 μm guaranteeing an optical density of less than 1. Having such small samples (and calculating the number of chromophores per unit volume), higher laser power than the reported one was not necessary for an efficient excitation.1 S. Techert, F. Schotte and M. Wulff,Phys. Rev. Lett., 2001,86(10), 2030.2 G. Busse, T. Tschentscher, A. Plech, M. Wulff, B. Frederichs and S. Techert, paper presented at this meeting..Prof. Moffataddressed Dr Cole: (i) Are your crystals optically isotropic or anisotropic? (ii) What is the polarization of the incident laser beam? How do (i) and (ii) affect the ability to uniformly stimulate the molecules in the crystal, in all orientations?Dr Coleresponded: (i) They are probably anisotropic. (ii) A vertically polarised beam was used, arriving at the sample from above at an angle of 45 degrees.The level of excitation is therefore likely to vary with changing orientation of the sample, since excitation is based on the transition molecular dipole moment of the molecule. This varying level of excitation may be averaged out, but it is possible to correct for the geometrical effect if one knows the faces of the crystal at two given oscillation angles.Prof. Helliwellcommented: On your data collection strategy, atomic resolution with Laue is possible.11 See D. Gomez de Anderez, M. Helliwell, J. Habash, E. J. Dodson, J. R. Helliwell, P. D. Bailey and R. E. Gammon,Acta Crystallogr., Sect. B, 1989,45, 482–488; M. Helliwell, D. Gomez de Anderez, J. Habash, J. R. Helliwell and J. Vernon,Acta Crystallogr., Sect. B, 1989,45, 591–596.Dr Coleresponded: Yes, atomic resolution is of course possible for small molecule compounds. However, better resolution is generally achieved using the monochromatic oscillatory method, since this is void of any problems of wavelength overlap and the more restricted coverage of the Ewald sphere, common in Laue experiments.Prof. Helliwellsaid: The monochromatic rotating single crystal method will be very sensitive to unit cell changes, unlike Laue geometry, and thus could provoke intensity change errors. (I agree of course that the stroboscopic approach creates a stable transient species for which the conventional monochromatic approach is definitely suited.)Dr Techertsaid: Three years ago we tried to investigate at ID09b of the ESRF time-resolved Laue diffraction on small molecules (in collaboration with Dr S. Arzt). However, at that time with the old set-up, the collected Laue pattern (unit cell size of the system of investigation: about 20 × 30 × 20 Å3) contained 10–20 diffraction spots and we were not able to index the Laue pattern with the available software. Today, the beamline as well as the software have been improved and further attempts would be useful.Prof. Coppenssaid: I would like to clear up a possible misconception about the effect of a cell dimension change on the accuracy of the monochromatic stroboscopic method. In fact two diffraction patterns are measured by alternate light-on light-off measurements; each can be integrated separately with the appropriate cell dimension, if the change is indeed sufficiently large to warrant such a treatment. So there is no reduction in accuracy of the results associated with the change in cell dimensions. It is of course necessary to take a change in cell dimensions into account when transferring ground state molecular geometry from a dark experiment to the light-on structure.Prof. Cernikasked: Are third generation SR sources the best way to achieve short bunch structure as the pulse length is limited by the RF structure? What are the possibilities of using FELs or the lower stimulated femtosecond X-ray1at ALS?1 For example, R. W. Schoenlein, S. Chattopadhyay, H. H. W. Chong, T. E. Glover, P. A. Heimann, W. P. Leemans, C. V. Shank, A. Zholents and M. Zolotorev,Appl. Phys. B, 2000,71, 1–10.Dr Colereplied: The use of FELs are likely to provide X-rays that are too damaging for the crystal, and the ALS developments for achieving femtosecond X-rays are said to give too low flux. I refer to the paper of Moffat presented in this Discussion Meeting where the viability of these options are specifically discussed [see the section ‘Ultra-fast time-resolved X-ray experiments’, part (a)].Prof. Moffatsaid: Femtosecond laser based “slicing” of 100 fs synchrotron X-ray pulses1is indeed feasible; but the number of X-rays emitted by the perturbed “slice” is small; too small for all but experiments that have very strong scatterers or absorbers. The fs free electron laser looks like a more promising route to short X-ray pulses (which are also extremely intense, with full temporal and spatial coherence).1 R. W. Schoenlein, W. P. Leemans, A. H. Chin, P. Volfbeyn, T. E. Glover, P. Balling, M. Zolotorev, K.-J. Kim, S. Chattopadhyay and C. V. Shank,Science, 1996,274, 236–238.Prof. Moffatopened the discussion of Prof. Helliwell's paper: With the benefit of hindsight, this is a remarkably complicated reaction with numerous chemically-distinct microstates. Nevertheless, the crystals are active. Please comment on the overall suitability and/or desirability of this system for time-resolved studies.Prof. Helliwellresponded: I agree that this is a complicated reaction but hydroxymethylbilane synthase (HMBS, EC 4.3.1.8) is a very important enzyme1,2because of its fundamental position in the metabolism of all organisms, except viruses, in the production of haem, vitamin B12, chlorophylletc. The complexity of the reaction constitutes a challenge, to all available techniques, to examine the enzyme's structure and function if we are to understand it.Time-resolved crystallographic studies are a valuable option for the following reasons: (i) HMBS is a rather slow enzyme (kcat = 0.1 s−1for HMBS fromE. coliunder optimal conditions) and can therefore readily be examined under pre-steady-state conditions; (ii) the substrate can gain access to the crystalline enzyme's active site which is open to the solvent channels of the crystal; (iii) the crystal habit has a favourable shape (thin plates) for quick diffusion of small molecules into the crystal so that the flow-cell method of reaction initiation would work; (iv) the time-dependent changes of electron density are expected to be substantial, as movements of a number of aromatic rings are involved during a single, polymerisation-like, catalytic cycle (see Fig. 1 of our paper); (v) the crystals are relatively resistant to white radiation and the crystal mosaicity remains small also even after substrate diffusion and colour transformation from colourless to red have occurred; (vi) several mutant variants of HMBS have been constructed that exhibit altered reaction kinetics; among these is the Lys59Glu mutant known to accumulate ES2as a predominant intermediate (after 2 min for Lys59Gln HMBS immobilised on an anion-exchange column).3These are technical feasibility reasons of choice. In addition it is worth emphasising that there are no good competitive inhibitors of HMBS known that could allow traditional inhibitor soak experiments followed by crystallographic analysis to determine the location of the inhibitor in the active site in three-dimensional space; a new approach was needed.The fruits of the time-resolved Laue protein crystallography approach thus far are: (i) the three-dimensional binding site for substrate and/or product within the enzyme active site has been experimentally located; (ii) protein engineering results, namely that mutations of Asp 84, Arg 149, Arg 155,i.e.residues proximal to the elongated electron density, which are known to affect the various stages of the enzyme reaction, are supported; (iii) the present results now guide future experiments, involvinge.g.when to freeze-trap the reaction in the crystal, and provide the basis for further efforts to determine the function of the mobile loop of residues 43 to 59. Overall the HMBS enzyme structure–function relationship, including the correlation of electron density changes with the colour transformation from colourless to red in the crystal, has been successfully probed by direct experiment.1 A. R. Battersby, C. J. R. Fookes, G. W. J. Matcham and E. McDonald,Nature, 1980,285, 17–21.2 A. R. Battersby and F. J. Leeper,Chem. Rev., 1990,90, 1261–1274.3 A. C. Niemann, P. K. Matzinger and A. Hädener,Helv. Chim. Acta, 1994,77, 1791–1809.Prof. Wilsonasked: What is the rationale for the difference in timescales for the build-up of enzyme–substrate complexes (2 h in the diffractioncf.2–10 min expected)? Is this a solid state effect (e.g.compared with solution)?Prof. Helliwellanswered: There may be several reasons as to why the elongated electron density builds up slowly in the enzyme active site: (i) the crystal lattice may restrict conformational change(s) for optimal enzyme reaction; (ii) the pH at which the crystals are grown is non-optimal for the enzyme reaction; (iii) the delivery of the substrate to the enzyme active site through the solvent channels of the crystal may cause delay (however, as Helliwellet al.1analyse, the HMBS crystal habit of thin parallelepipeds, 50 μm thick, and the substrate size are estimated to facilitate diffusion of PBG into the crystal in seconds. The flow delivers approximately one substrate molecule per enzyme molecule per second which is far in excess of what Lys59Gln HMBS, even if it were to be saturated with substrate, can turn over at room temperature and pH 5.3). Reason (ii) above is probably the most likely but since the crystals are not stable at the enzyme's optimum pH this is not testable.1 J. R. Helliwell, Y.-P. Nieh, J. Raftery, A. Cassetta, J. Habash, P. D. Carr, T. Ursby, M. Wulff, A. W. Thompson, A. C. Niemann and A. Hädener,J. Chem. Soc., Faraday Trans., 1998,94, 2615–2622.Prof. J. C. Smithasked: Is the reason that the ‘missing loop’ remained undetected that there is static disorder in the crystal at low temperaturesi.e., the loop is trapped in different energy minima, and, if so, might there be a diffuse scattering signature for this disorder?Prof. Helliwellreplied: This is possible but the specific-to-this-loop diffuse scattering signature is likely to be difficult to extricate from other contributors to the X-ray background notably from solvent and the rest of the protein diffuse scattering. Nevertheless it is an interesting suggestion especially if it were possible to make some of the residues on the loop selenomethionine variants (i.e.those that are hydrophobic could be substituted by selenomethionine). Then the diffraction recorded at two wavelengths, at thef′ dip of the seleniumKedge and a 'remote' wavelength, would allow a difference, Δf′, diffuse scattering pattern to be measured. The precise localization of the loop from such measurements would be difficult but could involve matching to computational models to ascertain where the loop might be. This is an interesting suggestion. Thank you.Mr Terietesaid: On the flexible loop, are there any glycines or prolines on dominant start and end positions, where the glycines could work as hinges or the proline cause an alteration between two conformations, throughcis–transisomerisation?Prof. Helliwellreplied: TheE. coliloop amino acid sequence is, starting from residue 43, Thr-Arg-Gly-Asp-Val-Ile-Leu-Asp-Thr-Pro-Leu-Ala-Lys-Val-Gly-Gly-Lys,i.e.finishing with residue 59. So, ‘yes’ there are glycines located at putative hinge positions to facilitate conformational change. Regarding prolines, it is established that these (staying intrans-configuration) tend to restrict conformational change of a polypeptide.1The proline near the centre of the mobile loop may therefore restrict conformational freedom near this part of the loop.Cis–transisomerisation, however, as you suggested, seems unlikely to me, sincecis-prolines are quite rarely found in proteins (within a database of 571 representative proteins, only 5,2% of the Xaa-Pro peptide bonds are incis-, the rest are in thetrans-configuration.2It is notable also that the conserved residues between HMBS fromE. coliandM. tuberculosishere are Thr 43, Gly 45, Asp 46, Pro 52, Ala 54, and Gly 57 (E. colinumbering).1 C. Branden and J. Tooze,Introduction to Protein Structure, Garland Publishing, New York, 1991, p. 259.2 M. S. Weiss, A. Jabs and R. Hilgenfeld,Nature Struct. Biol., 1998,5, 676.Prof. J. C. Smithasked: Is time-resolved solution X-ray scattering sensitive enough to resolve the inter-domain changes you are looking for?Prof. Helliwellanswered: Time-resolved solution small angle X-ray scattering (SAXS), involving synchrotron radiation, could resolve changes in radius of gyration of around 1 Å. This might not be enough but perhaps more helpful is the current practice in SAXS of recording as high angle diffraction as possible. Thus the shape of the protein is estimated.1Inter-domain movements could lead to significant changes of the HMBS protein shape. It is nevertheless a challenging experiment.1 D. I. Svergun,J. Appl. Cryst., 2000,33, 530–534.Prof. Mooresaid: I am interested in the influence of pH in this system because the cofactor has four carboxylic acid groups and the substrate and product also have carboxylic acid groups. (i) Is the enzyme activity pH dependent? (ii) What are the pKavalues of the cofactor, substrate and product substituents? I imagine they will be 5–5.6 in the absence of carboxylate–carboxylate electrostatic interactions (which could be substantial). (iii) Was the pH of the crystallisation solution 5.3? (iv) Do you have a mixture of forms in the crystal, and could this contribute to disorder thus making the ‘loop’ invisible in the electron density map?Dr Hädenerreplied: (i) At 37 °C, the optimal pH for wild-type HMBS fromE. coliis 7.4, with values forkcatandKmbeing 0.1 s−1and 7 μM, respectively, but the enzyme still exhibitsca.20% of its maximal activity at pH 6.2 and at pH 8.7.1,2At pH 7.4, the Lys59Gln mutation does not affectkcatbut it increasesKmapproximately 30-fold.2At room temperature and pH 5.3, the kinetic parameters of crystalline Lys59Gln HMBS can be estimated to beKm ⩾ 1.9 mM andkcat ⩽ 1.9 × 10−3s−1.3(ii) The pKavalues of the substrate (PBG) are known. They are 10.1 (aminomethyl substituent), 4.95 (propionic acid substituent), and 3.70 (acetic acid substituent).4The formation of a favourable ion pair between the negatively charged acetate substituent and the protonated aminomethyl side chain5is probably the reason for the latter low value. The pKavalues of the substituents of the cofactor and of the product (HMB) have not been determined as far as I know. The formation of intramolecular ion pairs is not possible in the HMB case, so you would expect the pKavalues to be around 5 including those of the acetic acid substituents, in the absence of the enzyme and in the absence, as you said, of carboxylate–carboxylate electrostatic interactions. In the case of the cofactor and enzyme-bound PBG and HMB, however, ion-pair formation between the carboxylate groups and positively charged side chains of amino acids may have a substantial influence on individual pKavalues.(iii) This is correct.(iv) This may well be the case. There are a number of charged residue side chains in the mobile loop (aspartates and lysines, two each, and one arginine). They are disordered in the structure of the holoenzyme but during catalysis they may find a suitable charged group for ion pairing among those of the growing oligopyrrole or of the side chains of amino acids.1 G. J. Hart, C. Abell and A. R. Battersby,Biochem. J., 1986,240, 273–276.2 A. Hädener, P. R. Alefounder, G. J. Hart, C. Abell and A. R. Battersby,Biochem. J., 1990,271, 487–491.3 J. R. Helliwell, Y.-P. Nieh, J. Raftery, A. Cassetta, J. Habash, P. D. Carr, T. Ursby, M. Wulff, A. W. Thompson, A. C. Niemann and A. Hädener,J. Chem. Soc., Faraday Trans., 1998,94, 2615–2622.4 S. Granick and L. Bogorad,J. Am. Chem. Soc., 1953,75, 3610.5 P. M. Jordan, inThe Biosynthesis of the Tetrapyrrole Pigments, ed. D. J. Chadwick and K. Ackrill, 1994, Ciba Foundation Symposium, vol. 180, pp. 70–89, Wiley, Chichester.Dr Techertasked: What is the biological reason for the unusually lowkcatvalue of 0.1 s−1?Dr Hädeneranswered: The product of the reaction, hydroxymethylbilane (HMB), is not stable at physiological conditions. With release of H2O, it cyclises to uroporphyrinogen I with a half-life of less than 5 min.1Uroporphyrinogen I, however, is not an intermediate of the biosynthetic pathway leading to the tetrapyrrolic pigments. An isomer of it in which ring D (see Fig. 1 of our paper) is rearranged, uroporphyrinogen III, is the true intermediate.2A companion enzyme, formerly called ‘cosynthetase’, now uroporphyrinogen III synthase, has evolved to take charge of HMB by ring-closing it at high rate to the correct isomer uroporphyrinogen III with release of H2O. Uroporphyrinogen III synthases exhibit relatively highkcatvalues such as 500 s−1for the enzyme fromE. coli.3The evolutionary aspects of the biosynthetic pathway of which this enzyme system is part have been thoroughly discussed by Eschenmoser.41 A. R. Battersby, C. J. R. Fookes, K. E. Gustafson-Potter, E. McDonald and G. W. J. Matcham,J. Chem. Soc., Perkin Trans. I, 1982, 2427–2444.2 A. R. Battersby, C. J. R. Fookes, G. W. J. Matcham and E. McDonald,Nature, 1980,285, 17–21.3 A. F. Alwan, B. I. A. Mgbeje and P. M. Jordan,Biochem. J., 1989,264, 397.4 A. Eschenmoser,Angew. Chem. Int. Ed. Engl., 1988,27, 5–39.Prof. Wattsasked: So the HMBS constitutes the rate limiting and regulating step for the much faster subsequent reactions?Dr Hädenerreplied: This is certainly true regarding the conversion of PBG to uroporphyrinogen III. There are, however, preceding reactions that are precisely regulated to control the entire pathway leading to the tetrapyrroles by preventing even the formation of PBG if it is not needed. This regulation affectse.g.porphobilinogen synthase, 5-aminolevulinate synthase, and, in plants and many anaerobic bacteria, glutamyl-tRNAGlureductase and glutamate-tRNA ligase, which are all inhibited by haem.1A reason for the prevention of PBG being accumulatedin vivomight be its intrinsic lability with respect to oligomerisation.2,31 G. Michal,Biochemical Pathways, Spektrum Akademischer Verlag, Heidelberg, 1999.2 G. H. Cookson and C. Rimington,Biochem. J., 1954,57, 476–484.3 D. Mauzerall,J. Am. Chem. Soc., 1960,82, 2605–2609.Prof. Wattsopened the discussion of Prof. Moore's paper: In Fig. 4 of your paper, you indicate far less α-helix structure from the NMR data, than shown in the modes or crystal structures of related colicins. Why?Prof. Mooreresponded: The only relevant X-ray structure for Fig. 4 is the structure in Fig. 1 of the paper. This shows a high content of helix in the min-R region of the protein. Fig. 4 is the output of the chemical shift index, which is a conservative empirical procedure for obtaining information on secondary structures from NMR chemical shifts. This procedure requires a run of NMR data for four or more sequential residues to all indicate a helix before one is indicated. Because of the incomplete resonance assignment for min-R we cannot provide a complete secondary structure map for it. However, what information the CSI does provide is fully consistent with the X-ray structure of Fig. 1; and with the shape analyses and relaxation time study later in the paper that provides strong confirmation that min-R has a helix–loop–helix structure.Prof. Finneyasked: What relationship do you expect between the two conformers and the structure determined crystallographically? Are the structural differences between the two conformers such that you might expect both of them to be accommodated within the crystal, or force the selection of one of them?Prof. Moorereplied: In connection with the DNase domain of colicin E9 referred to in the Introduction to the paper, one possibility is that there is only a small difference between the two conformers in solution,1with both co-existing in the crystal though the quality of the crystallographic electron-density map is not sufficient to resolve the different conformers. A second possibility is that the crystallisation process selects only one of the two conformers. At present we don't know which happens but we do know from NMR data that the difference between the conformers is very small and so I favour the first possibility. Another possibility is that the X-ray structure was determined at 100 K (ref. 2) while the NMR data were measured at room temperature. Perhaps at 100 K the protein preferentially populates the lowest energy conformer?The story with the conformers on the min-R protein that is the subject of the paper is different. The X-ray structure is only at 3 Å resolution, and it is for a full-length protein while the NMR study was carried out on a fragment of the structure. So the conformational exchange we detect by NMR may be a consequence of min-R being a sub-domain of the coiled-coil or it may be a feature of the coiled-coil itself with the X-ray structure not revealing it because it has a relatively low resolution. We plan to carry out NMR studies of the full-length coiled-coil domain and these should show whether the two conformers are an intrinsic property of it.1 S. B.-M. Whittaker, M. Czisch, R. Wechselberger, R. Kaptein, A. M. Hemmings, R. James, C. Kleanthous and G. R. Moore,Protein Sci., 2000,9, 713.2 C. Kleanthous, U. C. Kühlmann, A. J. Pommer, N. Ferguson, S. E. Radford, G. R. Moore, R. James and A. M. Hemmings,Nature Struct. Biol., 1999,6, 243.Prof. J. C. Smithasked: Could you comment on ways of analysing the contribution of internal motion to NMR spin relaxation in terms of collective dynamical variables, such as that being developed by Brueschweileret al.—this may be a way of linking the relaxation results to the interdomain dynamics you appear to have identified here.Prof. Mooreanswered: I can't give a full answer to your question, partly because the approach advocated by Prompers and Brüschweiler has only recently appeared in the literature.1Nuclear spin relaxation results from the modulation of inter-nuclear interactions by motions of the nuclei, and relevant motional modes are the overall tumbling of the protein and independent nuclear motion, usually described as internal motions. In order to define the internal motions of amino acid residues, which are really the dynamic information we seek, we have to be able to separate the contributions these two factors make to the experimentally determined relaxation parameters. There are a number of approaches for analysing the contribution of internal motions to NMR spin relaxation, with the Lipari–Szabo ‘model-free’ formalism2,3most widely used. The usual starting point for model-free analysis is to decide which residues have relaxation characteristics determined by the overall protein tumbling rate and using these to determine the rotational correlation time.4Those residues with significant internal motion can then be characterised. This benchmarking process is a key stage of the analysis and if it looks as if all the residues have internal motion then alternative procedures, such as reduced spectral density mapping,1,5is employed. This is less powerful than the model-free approach in the details it provides, but for disordered proteins in particular it is the most widely used approach. A key feature of the method described by Prompers and Brüschweiler1is that it does not require separation of the residues into those whose relaxation parameters are affected only by the overall tumbling of the molecule and those with significant additional internal motions. This seems to offer a substantial advance over other methods. However, it does involve molecular dynamics simulations in the analysis so it is not as straight-forward to apply as the model-free or reduced spectral density mapping approaches.1 J. J. Pompers and R. Brüschweiler,J. Am. Chem. Soc., 2002,124, 4522.2 G. Lipari and A. Szabo,J. Am. Chem. Soc., 1982,104, 4546.3 For example, A. G. Palmer III,Curr. Opin. Struct. Biol., 1997,7, 732; V. A. Feher and J. Cavanagh,Nature, 1999,400, 289.4 N. Tjandra, S. E. Feller, R. W. Pastor and A. Bax,J. Am. Chem. Soc., 1995,117,12 562.5 J. F. Lefevre, K. T. Daye, J. W. Peng and G. Wagner,Biochemistry, 1996,35, 2674.Dr Sagisaid: One of the main goals in biocatalysis or bio-molecular recognition processes is to correlate dynamics with the actual biological process (e.g.catalysis, molecular recognition). However, NMR probes only equilibrium states therefore one cannot really probe a real molecular recognition process or catalysis and correlate then with dynamics. How much can we stretch NMR to give direct information about correlating catalysis with dynamics?Prof. Moorereplied: You are right to say that NMR is looking at equilibrium states—to use Prof. Helliwell's analogy, the cliff edge and the beach. NMR is too slow to measure what happens to a body after it has jumped off the cliff until it lands on the beach, but what it can do is to characterise—sometimes in considerable kinetic and structural detail—dynamic processes taking place before the jump. With some proteins such dynamic processes are extremely significant for determining molecular recognition events, and in some enzymes a relationship between such dynamics and catalysis has been suggested. The clearest examples of dynamics affecting molecular recognition involve proteins that only fold into a globular form when they bind to their partner1—so-called intrinsically disordered2or natively unfolded proteins.3Coupling of protein folding to intermolecular complex formation has only been discovered to be a widespread phenomenon with the development of NMR spectroscopy for studying proteins and with estimates that as many as 50% of the proteins in humans contain at least one domain that is intrinsically disordered2this is clearly an important field. The way in which a newly synthesized protein folds from its unfolded state to its folded form is related to intermolecular interactions involving at least one disordered protein—in this case the interacting surface is intramolecular—and again NMR is one of the major experimental methods for determining what is happening to the polypeptide chain at key stages of the folding process. Of course, protein folding is generally very rapid so NMR is limited in the states it can inform on, but it is already clear from NMR studies of unfolded protein chains that some are not random coils, even at low pH4or in high urea of guanidine·HCl concentrations.5Colicin E9 is a member of a related class of natively unfolded protein that interacts with other proteins in that its unfolded domain contains some ordered non-random structure that is highly dynamic and forms the binding epitope for its partner protein.6Our NMR studies have shown that even when a ternary protein complex of 114 kDa is formed that anchors the previously dynamic binding epitope, the residues upstream and downstream of it retain considerable flexibility.6We have speculated that this is because this region is like a fishing line, with many hooks on it to bind to specific proteins in constructing a complex of many proteins; however, the biophysical characterization has moved ahead of the biological studies and additional interaction partners have not yet been identified. Even when the binding of a protein to a partner molecule does not involve folding of the protein, NMR relaxation studies are providing new insights into the molecular recognition events,7which include not only identification of residues that interact with the partner molecule but also information on changes in conformational entropy of the protein on binding obtained in a residue-specific fashion. Investigating catalytic events themselves is as difficult with NMR as it is with any structural technique, and perhaps even more so. However, as indicated above, NMR is giving unique information on the dynamics of enzyme interactions with inhibitors and substrate analogues,7and coupled with other data, including that obtained with site-directed mutagenesis, I believe that NMR will turn out to be valuable for characterising catalytic events. Crucially, as my answer to Prof. Smith indicated, this application of NMR will benefit considerably from being combined with theoretical studies of protein dynamics, which themselves may be tested by spectroscopic examination of mutants.1 H. J. Dyson and P. E. Wright,Curr. Opin. Struct. Biol., 200212, 54.2 A. K. Dunker, C. J. Brown, J. D. Lawson, L. M. Iakoucheva and Z. Obradović,Biochemistry, 2002,41, 6573.3 V. N. Uversky,Eur. J. Biochem., 2002,269, 2.4 J. Yao, J. Chung, D. Eliezer, P. E. Wright and H. J. Dyson,Biochemistry, 2001,40, 3561.5 J. Klein-Seetharaman, M. Oikawa, S. B. Grimshaw, J. Wirmer, E. Duchardt, T. Ueda, T. Imoto, L. J. Smith, C. M. Dobson and H. Schwalbe,Science, 2002,295, 1719.6 E. S. Collins, S. B.-M. Whittaker, K. Tozawa, C. MacDonald, R. Boetzel, C. N. Penfold, A. Reilly, N. J. Clayden, M. J. Osborne, A. M. Hemmings, C. Kleanthous, R. James and G. R. Moore,J. Mol. Biol., 2002,318, 787.7 V. A. Feher and J. Cavanagh,Nature, 1999,400, 289; J. J. A. Huntley, S. D. B. Scrofani, M. J. Osborne, P. E. Wright and H. J. Dyson,Biochemistry, 2000,39, 13 356; R. Ishima and D. A. Torchia,Nature Struct. Biol., 2000,7, 74; A. J. Wand,Nature Struct. Biol., 2001,8, 926; F. A. A. Mulder, A. Mittermaier, B. Hon, F. W. Dahlquist and L. E. Kay,Nature Struct. Biol., 2001,8, 932.Prof. Wilsonsaid: You mentioned that the function in this system involves allowing a 100 Å coiled coil to traverse a 150 Å cell membrane. How are the dynamics you have elucidated related to this function?Prof. Moorereplied: How the dynamics of the helix–loop–helix region of the receptor binding domain we have observed relate to the activity of the colicin is not clear at present. The relatively high degree of flexibility of the inter-helix loop compared to the remainder of the coiled-coil might be connected to the loop being part of the binding site for the BtuB receptor on the outer membrane of the target cell, as indicated by site-directed mutagenesis studies.1,2As we have discussed already (in the discussion with Dr Sagi), flexibility in an intermolecular interaction site may help in the recognition events leading to the complex formation.Perhaps it is more interesting to consider whether the coiled-coil unfurls. This is our current hypothesis3, which assumes that the helices remain largely intact, partly because they are interacting with other proteins in the system (including the outer membrane receptor BtuB and periplasmic Tol proteins). If the helices of the now unfurled coiled-coil remain helices, and one of them extends into the periplasm from the loop of the min-R region attached to BtuB it will not be long enough to cross to the inner membrane unless the periplasm is reduced from its normal size. This occurs at contact points. Alternatively, the helix may partly or completely unwind, which would give it sufficient length to cross the periplasm. This is something we are studying with a combined luminescence spectroscopy and protein engineering approach. In the NMR studies reported in this paper we have detected that the two helices of the min-R helix–loop–helix sub-domain move relative to each other in a way that retains the helix–loop–helix structure; and possibly the two helices slide across the surfaces of each other. Such motion may be related to events that lead to unfurling of the coiled-coil in the colicin, though in our model binding to a receptor is the key event that triggers this unfurling and it is not an intrinsic feature of the unbound colicin.1 S. Soelaiman, K. Jakes, N. Wu, C. Li and M. Shoham,Mol. Cell, 2001,8, 1053.2 C. N. Penfold, C. Garinot-Schneider, A. M. Hemmings, G. R. Moore, C. Kleanthous and R. JamesMol. Microbiol., 2000,38, 639.3 R. James, C. N. Penfold, G. R. Moore and C. Kleanthous,Biochimie, 2002, in press.Mr Terieteasked: Has dynamic data been gathered on the loop, which is believed to be involved in the activity, by using RDCs?Prof. Mooreanswered: Not yet.Prof. Wattsopened the discussion of Prof. Finney's paper: All co-operative transitions have an on-set and completion point. Yours are defined by the dynamic range of the method in some cases, in others, only a single transition is observed. But there must be a tool where the whole dynamic range is concerned and onset and completion are encompassed for this 220 K transition, or perhaps not.Prof. Finneyresponded: We are not aware of any experimental technique that can give such information across the whole dynamic range. Encompassing the whole dynamic range implies, in neutron scattering parlance, elucidation of the elastic incoherent scattering function which is determined by the infinite time limit of the van Hove single particle space–time distribution function. Accurate determination of this is possible in some systems where the relaxation times are well within reach of the instruments concerned. For protein solutions the range of motions is such that it has not yet been possible to reach this limit with neutron techniques alone. A range of experiments will be required.Dr Schmidtsaid: Data shown trace Doster and Settles:1On IN13 and IN6 at the ILL, all data show the same transition temperature despite different time scales. The Mossbauer data from Parak and Knapp2show the same transition point at 180–190 K. What type of motions do you expect, which account for your transitions? Are these transitions motions of the entire molecule, or are these motions of domains of the molecule?1 W. Doster and M. Settles, The dynamical transition in proteins: the role of hydrogen bonds, 1998; Les Houches Lectures: workshop on hydration processes in biology: theoretical and experimental approaches, ed. M. C. Bellisent-Funel and J. Teixera, IOS Press.2 F. Parak and E. W. Knapp,Proc. Natl. Acad. Sci. USA, 1984,81, 7088–7092.Prof. FinneyandProf. J. C. Smithresponded: IN6 and IN13 measure motions on very similar time scales, so you would not expect to get a large change in the apparent temperature of the dynamical transition. The measurements you mention are taken on samples at low hydration, while the ones discussed in our paper are in solution, where the behaviour is likely to be different (as we find it is experimentally). Moreover, the method used to determine the mean-square displacements in the paper cited is unclear. IN13 in principle works only at relatively highQ, where the Gaussian approximation for individual atoms fails—this is required for a model-free determination of mean-square displacements. Concerning the kinds of motions involved, determining these is one of the objects of the work. The experimental technique measures motions of all the non-exchanged hydrogen atoms of the enzymes, and hence averages over the whole molecule.Prof. Sir John Meurig Thomassaid: Your arguments, which seem to me to be convincing, would become irrefutable if you (or others) were to try and do measurements of the onset of anharmonicity and of the change of enzymatic activity underin situconditions. This is obviously not an easy thing to do; but one of the reasons for discussion such as this is to identify crucial (time-resolved) experiments. Could you comment on that?Prof. Finneyreplied: We have tried this. The ‘fastest’ enzyme known is catalase. We have measured its activity down to 173 K, and see no deviation from that expected from Arrhenius behaviour. 173 K does appear to be well below the onset of anharmonicity for all proteins looked at so far. However, the assay at 173 K requires ∼10 h to obtain measurable product. Nanosecond assays appear to be quite out of reach with current technology.Also Prof. R. M. Daniel, University of Waikato, New Zealand.In these circumstances, where it is not possible to measure both dynamics and activity at the same time, it is crucial that the neutron measurements are made under the same conditions as those under which the activity is measured. This was an important aspect of our experimental protocol: the solvents in which the neutron measurements were made were those in which activity was measured at these temperatures. To ensure that the enzymes were still active after the neutron experiments, the samples were recovered and assayed subsequently, confirming that they retained their activity.Dr Colesaid: The error bar at 200 K on Fig. 1 is notably larger than any other in the graph. Since this temperature pertains to the vicinity of the transition, is the size of the error bar a physical consequence of the transition is some way, or does it show the model breaking down here, or does it just emanate from an experimental effect?Prof. Finneyanswered: The error bars on the points relate to those estimated by the extrapolation procedure described in the paper, and for the point you mention, the errors were larger for experimental reasons. This larger error may relate to the phase behaviour of the solvent, though this is not definite. However, the determination of the temperature of the so-called transition does not really depend on this one point. As is seen from a number of papers in which a transition temperature is assigned, it is obtained rather by drawing straight lines to the data above and below the transition, estimating the transition temperature as that at which these lines cross. The points in the vicinity of the transition have little effect on this geometrical construction.Prof. Wattsaddressed Dr Cole: In any transition phenomenon, there are highly disordered intermediates formed which are neither initial nor final state related. These are often very small in population compared with the majority and can account for significant errors in determination of experimental parameters.Prof. Helliwelladdressed Prof. Finney: (i) Re Fig. 1 of the paper: Isn't the functional assay resting on the last data point (that the enzyme behaviour is Arrhenius over the full temperature range)?(ii) Is the xylanase also from a thermophile (i.e.Zaccai is saying (personal communication) that the stiffness of proteins in extremophiles is higher and therefore you could look at non-extremophiles too)?Prof. FinneyandProf. J. C. Smithresponded: (i) I don't think so. Close examination of the figure shows that there are several data points at temperatures lower than the 220 K transition. These show no deviation from linearity. If there were a deviation from Arrhenius behaviour in this region, these results would have shown it.Also Prof. R. M. Daniel, University of Waikato, New Zealand.(ii) The xylanase was from a thermophile. I would point out however that we started out using thermophiles for these measurements as it was thought that their expected greater ‘stiffness’ might shift the transition temperature upwards, which would ease the enzymology. However, where we have taken measurements on equivalent thermophilic and mesophilic enzymes, we find no measurable difference in their dynamic behaviours as measured by these techniques. Thus we find no evidence for greater ‘stiffness’ as measured by transition temperature of the thermophiles. Furthermore, other comparative activity measurements on non-thermophile enzymes give similar results.1,21 J. M. Bragger, R. V. Dunn and R. M. Daniel,Biochim. Biophys. Acta, 2000,1480, 278–282.2 N. More, R. M. Daniel and H. Petach,Biochem. J.1995,305, 17–20.Prof. Mooresaid: (i) Dehydrating proteins can perturb properties even though they may appear to be active. I recall that Kaminskyet al.1reported freeze-dried cytochromecon dissolving in water undergoes a structural change detected spectroscopically, even though the dehydrated protein retains electron-transfer capability. (ii) In your 70% CH3OH/30% H2O solutions how is the protein solvated? Does it have a water shell surrounded by H2O/CH3OH?1 S. Kaminsky, K. M. Ivanetich and T. E. King,Biochemistry, 1974,13, 4866.Prof. Finneyreplied: (i) There is a significant amount of data looking at changes in protein structure on dehydration. In summary, it seems that these are generally quite small (seee.g.ref. 1 and 2), though they may have functional significance.(ii) This is an interesting observation, and is discussed in an earlier paper on the solvent dependence of the enzyme dynamics.3Interestingly, we found that the dynamics did not vary significantly with methanol concentration except when going to pure D2O. One possible interpretation of this observation is that the solvent in the near neighbourhood of the protein is relatively unperturbed by the change in concentration of the bulk, but there may well be other interpretations of the observation.1 P. L. Poole and J. L. Finney,Biopolymers, 1983,22, 255.2 N.-T. Yu and B. H. Jo,Arch. Biochem. Biophys., 1973,156, 469.3 V. Réat, R. Dunn, M. Ferrand, J. L. Finney, R. M. Daniel and J. C. Smith.Proc. Natl Acad. Sci. USA, 2000,97, 9961.Prof. Moffatsaid: The techniques for determining MSDs are as you state—sensitive to time scale. The activities presented by you are essentially on a single time scale for various enzyme systems. Some proteins exhibit “activity” over a very wide range of time scales, fs,μs → s,e.g.the individual steps in the photocycles of lR or photoactive yellow protein. Could you envisage parallel activity and dynamic measurements on such systems?Prof. Finneyreplied: Enzyme activity measurements are confined to the rate limiting step (as defined by the turnover number), and these certainly vary over at least seven orders of magnitude depending upon the enzyme, and vary by another several orders of magnitude with conditions such as temperature. All other events, including all the other reaction steps, must take place over the same or shorter time scales. Ideally we need to correlate activity with a full dynamic ‘spectrum’ of motions, but all techniques have either time scale windows (often defined at only one end) or are time averaged. But the good news is that activity varies predictably and smoothly (in an Arrhenius sense) with temperature, suggesting that the real need may be to identify the groups/motions involved in activity rather than their time scales.Prof. Wattssaid: Lets try and find out what each approach and method of the papers by Prof. Helliwell, Prof. Moore and Prof. Finney can bring to the systems we have heard about. Loops are ill defined in Prof. Helliwell's crystal structure whereas Prof. Moore defines loops, both of which are functionally important. Prof. Helliwell, what can you do for Prof. Moore's system andvice versa, and Prof. Finney finally, where do you see global dynamics fitting into each of these systems?Prof. Mooreresponded: I have been asked how NMR could help clarify what is happening with the ‘missing’ loop in the X-ray data of HMBS. The first experiment would be to look at15N labelled HMBS. I would expect sharp NH1H–15N HSQC peaks from the loop and broader peaks from the remainder of the protein. The cofactor NH signals may also be sharp. Peaks of the loop could be assigned from triple-resonance spectra of the13C/15N labelled material (2H labelling may also be needed given the 34 kDa size of HMBS—the loop residues should not need this); and backbone relaxation parameters measured for the loop with the15N labelled samples. Monitoring the loop signals during the reaction cycle, and as functions of pH and temperature, should establish whether they are playing a role in the reaction. The pKavalues of the carboxylic acid groups of the cofactor and its adducts might be determined with specifically labelled samples.Prof. Finneycommented: In general terms, the ‘elastic scan’ neutron scattering measurements we used in the work reported here give information on the global dynamics of the protein through the average mean square displacements. As the signal is dominated by the scattering from the hydrogen atoms, it is the motions of the non-exchangeable hydrogens on the protein that will be largely reported. As the contributions of deuterium atoms to the measured signal are much less than those of hydrogens, if the protein were selectively deuterated, then we could focus on the motions of those regions that remained hydrogenated. The time scales of the motions that can be accessed depend on the instrument used, and can broadly range from picoseconds to tens of nanoseconds. Furthermore, by making quasielastic and inelastic scattering measurements, we can also begin to obtain more detailed information on the natures of the motions involved and the density of states.Prof. Wattssaid: New genetically engineered labelling methods permit protein domains to be labelled. The case of PBDG seems an ideal one where NMR visible isotopes placed specifically in the ‘invisible’ loop, could be studied by NMR (solution or solid state in an amorphous or semi-crystalline state) to resolve structures, or an ensemble of structures. Ligand labelling too, could help here.Prof. Moorereplied: Probably yes, a set of atomic coordinates can be determined. However, I wonder if this is what is really needed. Given the probable dynamic nature of the loop there are likely to be large uncertainties in an ensemble of structures for this. I would put the effort into the relaxation characteristics of the loop under various conditions. Perhaps the NMR and X-ray data combined might be data used in MD calculations rather than as an attempt to define the “structure” of the complete enzyme as a static entity.Prof. Helliwellcommented: A simulation approach perhaps could be the best benefit of NMR information on the loop,i.e.to resolve where the HMBS loop is, and its excursions. Also, the hypothesis of the role of inter-domain movements of the 3-subunit protein in the enzyme reaction could be assessed. At present, it seems, simulation cannot readily approach longer time scales than ∼10 ns simulations and such a loop might be expected to have slowly moving aspects.Prof. Helliwelladdressed Prof. Moore: Re: Fig. 11 and the 3 Å X-ray structure: crystal annealing could improve the resolution of the protein crystallographic study andBfactor estimates would be much better (to compare with yourT1/T2ratios).Prof. Moorereplied: We clearly need a better X-ray structure and more complete NMR data to fully characterise the structural dynamics of these colicins, but the comparison of X-rayBfactors andT1/T2ratios is suggestive. Remember that the X-ray data is for a full-length protein whilst the NMR data is for a fragment. The reduction in theT1/T2ratio for residues 4 and 5, and beyond residue 70, of min-R (Fig. 11(a)) are because they are its N and C termini while the corresponding residues of colicin E3 (Fig. 11(b)) are in the middle of the coiled-coil helices!Prof. Finneysaid: Neutron scattering measurements are essentially limited to probing global dynamical processes on time scales of ∼1 ns or faster. Yet processes relating to the enzyme activity are likely to be occurring on slower time scales. Mössbauer offers timescales of ∼10−7s, but requires the presence of Fe and cannot be effectively used under solution conditions. NMR would seem to offer a way forward to both (a) probing longer time scales under realistic solution conditions and (b) (through selectively focussing on particular nuclei) probing motions locally through the enzyme. How realistic are such measurements, and how long a programme of work would be needed to characterise fully the dynamics of an enzyme of, say 50 kDa?Prof. Mooreresponded: Let me rephrase your question into 3 parts and then answer them in turn. (i) Can NMR fully characterise the motions in small proteins (say up to 20 kDa)? (ii) How long would it take to do so? (iii) How feasible is it for an enzyme of 50 kDa mass?I assume you will want to do this over the temperature range used in your neutron scattering studies to look at the dynamic transition. The first point to note is that the NMR approach I described is for solutions; solid-state NMR of proteins is a rapidly developing field but cannot yet provide a global description of protein structure and dynamics in the way that you want. The reason size of the protein is important is that the overall tumbling time of the molecule in solution is a key determinant of the nuclear relaxation properties, with slow tumbling producing efficient relaxation and broad lines. Along with the size of the protein, the viscosity of the solvent is important. If the water–alcohol mixtures you use have a viscosity at 200 K and above that allows relatively sharp NMR lines to be observed, you will be set to get the data you need with a small enzyme. This will have to be produced by an expression system that allows isotopic labelling with13C and15N; most NMR groups employ bacterial systems growing on minimal media for this. Starting from scratch with13C/15N labelled samples, it will take 6–8 weeks to collect and analyse spectra that allow the NH resonances observed in the1H–15N HSQC spectrum to be assigned. A further 2 weeks data collection with15N labelled samples allows the backbone15NT1andT2and1H–15N nuclear Overhauser enhancements1to be collected at one field strength. It is likely that the level of detail you want will require the relaxation parameters to be determined with at least one other spectrometer operating at a different field strength to the first. This should also take 2 weeks. The dynamics of the side chains can be determined by13C relaxation times,1,2and also with selective2H labelling,1and both of these require samples with different isotope labelling patterns to those needed for the assignments and for15N relaxation measurements. However, provided the samples are sufficiently stable once you have them you can readily carry out a temperature dependence study. Provided the protein does not undergo substantial temperature-induced conformation changes it is unlikely that you will have to repeat much of the assignment work with13C/15N labelled samples at each temperature. So, assuming a stable protein of less than 20 kDa at a concentration of about 1 mM it may take a year to measure the temperature dependence of the15N relaxation properties.2H and13C relaxation analyses will take longer. Probably you should concentrate on the backbone characteristics first to determine if the dynamic transition is reflected in these. If you pick a protein that is already well-studied by NMR then you will save time; for example, the 14 kDa hen egg white lysozyme may be suitable.An enzyme of 50 kDa presents major problems because of slow tumbling in solution3. Proteins of this size have been studied by NMR but in general, in order to obtain full assignment of the NH1H–15N HSQC spectrum the non-exchangeable protons of the protein have to be replaced by2H. NMR experiments to measure the relaxation properties of the backbone will be somewhat different to those for similar proteins in which deuteration has not been done but unlike the latter proteins, which are routinely studied in many laboratories by these approaches, deuterated proteins are still in the hands of relatively few groups. Moving onto side chain dynamics, deuterated proteins will require specific labelling protocols; for example, introducing protonated methyl groups of particular amino acids into an otherwise deuterated protein. So, the methodology may all be in place for you to characterise the global dynamics of a 50 kDa enzyme over a wide range of temperatures but note that although various groups have described methods that may lead to the fold of a large protein being determined by NMR,4a high-resolution NMR structure of a monomeric protein greater than 30 kDa has not been reported.1 A. G. Palmer III,Curr. Opin. Struct. Biol., 1997,7, 732.2 J. Engelke and H. Rüterjans, inBiological Magnetic Resonance: Structure Computation and Dynamicsin Protein NMR, Kluwer Academic/Kluwer Publishers, New York, 1999, vol. 17, p. 357–417.3 K. Pervushin,Quart. Rev. Biophys., 2000,33, 161.4 B. T. Farmer III and R. A. Venters,J. Biol. NMR, 1996,7, 59; B. O. Smith, Y. Ito, A. Raine, S. Teichmann, L. Ben-Tovim, D. Nietlispach, R. W. Broadhurst, T. Terada, M. Kelly, H. Oschkinat, T. Shibata, S. Yokoyama and E. D. Laue,J. Biol. NMR, 1996,8, 360; W.-Y. Choy, M. Tollinger, G. A. Mueller and L. E. Kay,J. Biol. NMR, 2001,21, 31.

ISSN:1359-6640    

DOI:10.1039/b207969k

出版商:RSC    

年代:2003

数据来源: RSC

ISSN:1359-6640    

DOI:10.1039/b207970b

出版商:RSC    

年代:2003

数据来源: RSC

摘要:

Prof. Moffatopened the discussion of Dr Martlew's paper: The Roman and Syrians made superb glass two millennia ago—but not window glass. Who is the “Faraday” of window glass making? When was the modern process invented? Where? What were the key novel features?Dr Martlewreplied: It may have been the expansion of the Roman Empire to these chilly islands which provoked the Roman glassmakers to develop methods of creating glass sheets to block up apertures in buildings. Casting molten glass onto a flat rock slab then rolling it out into a sheet was a technique used by the Romans, to create a translucent rather than transparent pane, appropriate to the glare of the Mediterranean lands!Glass blowing was invented at some time about the beginning of the Christian Era, and naturally provided an easy way to make objects of spherical or cylindrical symmetry or derivatives therefrom. Blowing a glass sphere then flattening it to make a window was quite challenging, and three methods developed. One involved manipulating the sphere to make it into a cylinder, which could then be split axially and flattened to make a flat sheet. A second method (which never became really popular) involved blowing the glass into a suitable mould to create a bottle with four flat sides and a flat base; one could then cut these up to create small flat sheets of glass. The third method, known in England as the Crown Glass process, used the centrifugal forces generated by spinning a sphere on its supporting rod to open the hot glass shape into a flat disc from which small window panes could be cut. The individual inventors of these techniques are not known.By the middle of the twentieth century continuous glassmaking on an industrial scale had replaced these ancient hand methods of production. Two main kinds of process were used. If cheap window glass was needed it could be drawn vertically upwards from a tank of molten glass in a continuous ribbon, being cooled and annealed in a tower before being cut into handlable sheets at the top. Glass thickness was limited to about 4 mm, but the product answered domestic needs very well, aside from the optical distortion generated in the forming process.Thicker glass with better optical characteristics was in demand for shop windows and the large windows and mirrors desired by the affluent classes. Polished plate glass to satisfy this demand was made by continuously rolling the stream of molten glass to create a ribbon as thick as needed. To remove the surface scarring introduced by the rolling process it was then necessary to grind and polish both sides of the glass to create optically perfect plates, a tremendously expensive process, and very wasteful of glass.When the float process was announced in 1959 it was revolutionary in that it amalgamated the strengths of each of the earlier processes. The perfect flatness of polished plate glass was achieved by floating the molten glass on a layer of molten tin. Because the new ribbon didn't touch anything solid whilst it was still deformable, the as-formed surfaces were as brilliant as those of sheet glass drawn up from the melt.We may not know the individual inventors of antiquity, but we do know that the “Michael Faraday” of industrial window glass production was undoubtedly Sir Alastair Pilkington.Prof. Hounslowasked: You show particle tracks, yet your velocity maps indicate a great deal of mixing. Would there not be a distribution of residence times?Dr Martlewanswered: There is indeed a very wide spread of residence times over all possible particle tracks. Mixing does occur, but with the generally very high viscosities of the molten glass and the very low Reynolds numbers involved, mixing tends to be slow and relies heavily on chemical diffusion. Homogenising the glass and avoiding the visible effects of non-uniformity of refractive index in the product has exercised glassmakers for several thousand years.For the present work it has been necessary to prepare several thousand particle tracks for each case, to establish which tracks within the furnace result in the smallest residence times. Dissolution of sand grain residues is favoured by long times and higher temperatures. Generally the particle tracks which have the smallest times yield the worst dissolution efficiency; temperatures are not sufficiently high along these tracks to compensate. For the exploratory research presented here the method of calculation is sufficiently cumbersome to make it necessary to limit the number of tracks studied to a very few. Having established the method and demonstrated some usefulness, the next step is to code the calculation within the 3DGLASS post processor module, so that more complete explorations can be carried out economically.Dr Colesaid: When you add impurities to the glass in order to generate a particular colour of glass, how will this affect the reference data that you have regarding the stability/quality of the glass relating to composition? Do you thus obtain glasses of certain colours being more stable/fragile,etc.than clear glass?Dr Martlewreplied: We have talked already about the corrosive nature of molten glass. Though prejudicial to the longevity of the capital equipment needed to make glass products industrially, this attribute enables us to explore many different compositional variants in the quest for desirable properties and performances.Colouring oxides (notably those of the transition elements) dissolve fairly readily in the molten glass, unconstrained by any rigid stoichiometry. Ligand field effects then do the rest, creating the colour. More often than not, commercial colours require only very small percentages of colouring oxides, so the key macroscopic physical properties are not affected.Prof. Helliwellasked: I would like to make a connection with Greaveset al.'s paper at thisFaraday Discussionand ask how the new characterisation details are informing new or better products. Also are the SR central facilities currently available, adequate, or needing further development to meet your needs as an industrialist?Prof. Wilsonasked: You mentioned the importance of the surface layer. Could you comment on the use of surface scattering methods to examine the nature of glass surfaces and interfacese.g.neutron or X-ray reflection methods, both specular and off-specular.To follow up—is this important enough to justify obtaining access to these centralised, often expensive, facilities?Dr Martlewanswered: I may not comment in any detail about novel products which may be emerging from better understanding of glass structures, particularly the structure of glasses within the vicinity of the glass surface. However it is well known that the vagaries of the strength of glass products may be rationalised in terms of Griffith flaws (these are envisaged as sub-microscopic flaws in the glass surface which concentrate any tensile stress being experienced, starting a crack and causing failure). Any techniques which can be used to investigate glass structure at the molecular level are very desirable. With any such technique, however, it seems that the larger the scale of examination the more expensive is the experimental facility. My personal view is that we need to be looking at mesoscale structures in order to gain the working understanding that is needed to be the fundamental foundation of new commercial products. In the current economic climate, manufacturing industry cannot justify spending large amounts of private money on equipping for such techniques. If the United Kingdom is to take the lead in these matters, I believe that some way of financing such expensive facilities needs to be undertaken at Government level to support British industry in the increasingly competitive global marketplace. Having created such capital resources, the Government must ensure that the accountancy conventions used to finance their application to industrial exploitation must be friendly to the industrial budgetary processes. Sadly, the pressures now faced by industry in the current climate mean they can no longer be thought of as any kind of “cash cow”!Dr Maycommented: At the ILL (Institut Laue-Langevin), there are no “tickets” to get neutron beam time. You just need to propose an experiment, and you can perform it for free if it is accepted by the relevant subcommittee. The condition is that the results get published. However, one has to pay for secret research.Dr Martlewreplied: Thank you for this helpful comment. The crunch is often the tension between the publication requirement to obtain access to centrally funded capabilities, and the secrecy necessary to protect intellectual property. Defensible IP rights often crucially depend on being able to demonstrate a stance of confidentiality during the early period of the inventive step. Publication of results is directly opposed to this stance.Prof. Finneyopened the discussion of Prof. Greaves' paper: (1) The INEL detector resolution is quite poor, and you are getting a very restricted range inqfrom it atλ = 1.5 Å. Is this adequate to be sure of phase identification from one peak for each of two phases (stuffed quartz and spinel)?(2) Similarly, the detector limits you to only a small part of the Debye–Scherrer rings and hence quantifying amounts of each of the two phases on the basis of integrating under one peak leaves you open to problems of preferred orientation. How can you be sure your quantative phase estimates are not significantly affected by preferred orientation?(3) In Section 4 you say that the proportions of amorphous content are estimated from integrating the amorphous background. (a) How good do you think this estimate is? (b) Do you have the data available? It is not plotted with the other quantities in Fig. 4 of the paper. (c) Can the variation of the “amorphous content” give a useful constraint on the accuracy of the other quantities (e.g.by some kind of sum rule of total material quantity)?Prof. Greavesanswered: (1) The identification of phases were first made throughex situmeasurements using standard high resolution powder diffraction with a fullqrange. The INEL system on the station 8.2 at the Synchrotron Radiation Source was then used to providein situfingerprints. It is worth reporting that in the last few weeks the RAPID II curved multi-wire proportion counter has been commissioned at the SRS on station 6.2. This has a much-improved resolution compared to the INEL, a count rate three decades higher and an overlap geometry with respect to the SAXS detector offering a fullqrange to the smallest angles. In future it will be possible to identify phasesin situas well as to track their development as a function of time and temperature.(2) Of course we cannot be sure. However, in recent measurements at the ESRF on BM26B we found that the intensity of the quartz pattern was extremely erratic compared to that of spinel, strongly supporting the view that nucleation of stuffed quartz occurs at the surface and for monolithic specimens is prone to preferred orientation. Regarding the quantification of phases at each stage, we have not used the intensity of diffraction lines but have relied on the good agreement between Cr EXAFS and SAXS data (Porod regime plus Invariant) to determine the crystalline fraction,v.(3) We have chosen here not to useIamorph(t) to square withInlm(t) from the two phases present because the composition of the glass is changing. In the past, though, we have explored the sum rule approach in crystallisation from gels, for example, where the crystalline phase totally replaces the amorphous one or in amorphisation where the reverse occurs (e.g.refs. 1 and 2).1 J. C. Fernandes, D. A. Hall and G. N. Greaves,Mater. Sci. Forum, 1996,228–231, 411–416.2 G. N. Greaves, inFrontiers of High Pressure Research II: Application of High Pressure to Low Dimensional Novel Electronic Materials, NATO Advanced Research Workshop, Kluwer, Dordrecht, 2001, p. 53.Dr Sankarcommented: The main limitation in the angular range is primarily due to thein situhigh temperature facility which has a small window region that permits only up toca.60° 2&thetas;. In addition only a few strong reflections appear in this 2&thetas;range.It appears from Fig. 4 (top) of your paper that the stuffed quartz phase is stable. It has been observed in other cordierite-forming systems, for example Mg2+-exchanged zeolite B, that the stuffed quartz phase appears above 900 °C and disappears as soon as the cordierite phase starts to appear. It appears from these studies that the stuffed quartz phase is unstable, whereas this phase appears to be stable in this investigation. Is that due to the presence of chromium?Prof. Greavesresponded: Our view is that the stuffed quartz phase here is not nucleated from Cr at these temperatures but from defects at the glass surface, which will therefore offer some physical stability. However, with accurate unit cell parameters, the composition of stuffed quartz can be determined from the tabulations of Schreyer and Schairer (ref. 12 of the paper) and may not always coincide with stoichiometric cordierite Mg2Al4Si5O18. From additional unpublished data the stuffed quartz composition in Cr-doped cordierite glass heat treated at higher temperatures than reported falls on the quartz-rich side of cordierite. This may well be because of the twist in the residual glass composition resulting from the earlier formation of spinel (see eqn. (8) of the paper). Also, energy dispersive XRD results on powders of Cr-doped cordierite glass have shown that for isothermal crystallisation at 1250 °C, 200 °C above the temperatures employed here, the stuffed quartz phase is eventually replaced by cordierite, but that this conversion is slow, suggesting that the stability of stuffed quartz relates to the fact that it does not have the composition of cordierite. In the example of Mg-exchanged zeolite B, as far as I understand, the composition of the stuffed quartz phase is closer to Mg2Al4Si5O18which may explain why the final conversion to cordierite is more rapid and your observation that the stuffed quartz in this case is unstable.Prof. Wilsonsaid: I am interested in the fact that the time profiles for evolution of the various types of scattering are similar, indicating that equilibration occurs on all length scales simultaneously. That is, are the formation of 210 Å particles and the formation of crystalline phases governed by the kinetics of a single process?Prof. Greavesreplied: Yes, insofar as the spinel phase is concerned, as this almost completely dominates the SAXS. If the composition determined from the final Cr EXAFS spectrum, MgCr0.18Al1.82O4, is the same at nucleation, then the single process is the one described by eqn. (8) in the paper. This of course raises the question as to whether the changing glass composition remains uniform on all length scales as growth advances.Prof. Ryanasked: The data presented in the paper are truly beautiful and show scattering patterns that look like the form factor of a sphere. The paper indicates that the phase that is growing has a different composition to the glass and that the scattering is dominated by the local Cr concentration. Detailed analysis of the scattering is required, however, as the nucleation and growth process has a dense particle surrounded by a depletion layer, the shape of the depletion layer depending on the thermodynamic driving force for crystallisation and the diffusion coefficients of the components. This effect is not so important at the beginning of the crystallisation and when the reservoir of Cr is exhausted the equilibrium structure could well be dense spherical structures in a uniform background. It is in the intermediate stages (Fig. 1) that a more complex electron density profile needs to be considered.Plot of electron density throughout the growth process.Prof. Greavesanswered: I agree, we have just looked at the start and finish. With improved data coming from the ESRF BM26B and in due course from the SRS 6.2, we can look forward to the possibility of exploring the complete progression from nucleation to full devitrification of Cr.Prof. Hounslowsaid: (1) You indicate in the paper that the scaling of radius of gyration with time indicates a diffusion limited process, but that at long times depletion of free Cr slows the process. Does the overall balance on Cr indicate that the amount of free Cr decreases to zero?(2) For a process with monodisperse particles whose growth is limited by diffusion, is it not surprising that no Ostwald ripening is seen?Prof. Greavesreplied: (1) TheR2gvs.tplot is very sensitive to errors inRg. Nevertheless the initial linear rise is clear, as is the levelling off around the stage at which the intensity ofIspinellines are beginning to saturate. The evidence for the eventual removal of Cr from the glass matrix comes from the composition of the spinel and from the agreement of the crystalline fraction,v, from eqn. (8) in the paper with that obtained from SAXSviaeqn. (7) in the paper.(2) Absolutely, but it needs stressing that the greatest degree of monodispersion occurs part way through the heterogeneous growth process,i.e.before all the Cr is crystallised and probably at the point at whichR2gvs.tceases to be linear.Prof. Ryancommented: The formation of monodisperse particles during thermally induced phase separation of a mixture of two polymers has been observed.1Mixtures of polyisoprene and ethylene–propylene copolymer were studied using time-resolved elastic light scattering. For off-critical quenches highly monodisperse spheres were observed whose radii grow with a power law. The monodispersity and growth law are rationalised as a heterogeneous nucleation process in a similar manner to the paper under discussion.1 Cumming, Andrew, Wiltzius, Pierre, Bates and S. Frank,Phys. Rev. Lett., 1990,65(7), 863–866.Prof. Helliwellasked: How has this detailed structural characterisation improved the knowledge of the function of glass?Prof. Greavesanswered: The traditional functions of glasses and ceramics, many with ancient pedigree, have generally developed from the craft sciences. What combinedin situstructural methods, like those presented here, can now provide is direct observation of the development of high temperature structural chemistry as the optimum conditions that result in a particular function are reached. With serendipity new functions may emerge. For example, fine glass ceramics have been developed for their optical transparency, mechanical and thermal properties, for which monodispersion is not critical. The discovery of monodispersed nanocrystals in Cr-doped cordierite glass, however, promises a bulk quantum dot system with the function of non-linear optical response mouldable into any shape.Dr Dentopened the discussion of Dr Chen's paper: What are the differences between the electrochemically generated species and the laser induced species?If the edge data are the same, then how can the EXAFS be different, especially given the signal to noise limitation?Dr Chenreplied: (1) Electrochemically generated Cu(ii) species from the starting Cu(i)(dmp)2+species had one electron removed from the Cu(ii), so the total number of electrons in this species is one less compared to the starting material. The laser excited Cu(i)(dmp)2+, however, underwent an intramolecular charge separation process where one electron was transferred from Cu(i) to one of the ligands, forming the Cu(ii) species. Therefore, the total number of electrons was unchanged. In fact, the metal–ligand charge transfer (MLCT) transition is a reversible process. As the excited state decayed, the intramolecular charge separation recombined.(2) The edge data for the MLCT state and the electrochemically generated Cu(ii) species are almost identical, indicating the generation of Cu(ii) species by the laser. However, the edge data for the ground state and the laser excited state are not the same, as indicated by Fig. 5 in the paper. The EXAFS data in Fig. 6 of the paper which we were comparing were the ground state Cu(i)(dmp)2+and laser excited mixture with 80% ground state and 20% MLCT state. They are different and from these differences, we extracted the MLCT structure.Prof. Coppensasked: (1) With acetonitrile you get a shortening on the Cu–N bonds on excitation, while in toluene you get a lengthening. Is it possible that you are looking at the complex before exciplex formation, as we calculate the 4-coordinate Cu(ii) excited state to have a C–N bond length shortening of 0.04 Å?(2) Do you identify the very short-time species visible in the fast laser experiment with the excited state before exciplex formation?Dr Chenanswered: (1) The process of thermal equilibration of the MLCT state takes place on a subpicosecond to a few picosecond timescale. Therefore, it would be too fast for current 100 ps X-ray pulses to probe.(2) In a recent fs pump–probe laser transient spectroscopic study, we observed two very short components in the kinetic decay trace in addition to the ns longer lifetimes commonly referred to in the literature. We measured transient spectra of Cu(dmp)2+in acetonitrile and in ethylene glycol, where both measurements gave a subpicosecond rise time that was longer than our instrumental response time, and a few picoseconds fast decay time. We are in the process of further investigating the origins of these components in the kinetics of the MLCT state. It is likely that they may be related to generation of the thermally equilibrated MLCT state and exciplex formation.Prof. Evanssaid: (1) This is a very impressive experiment, but the intrinsic problem is the 2 shell fit for the 2 Cu–N sites. Have you examined the correlation between these 2 shells to make a good error estimate?(2) It seems surprising that an expansion is observed in the Cu–N distance. The MLCT process would remove an electron from a t2M–L σ* orbital in Cu(i) and transfer it into a phen π* ligand. So the Cu(ii) transient centre would be expected to have a smaller covalent radius.(3) The BArF anion is an extremely weak binder, with the aryl fluorines the only plausible donors. Acetonitrile, or a linear triatomic ligand, will provide a distinct multiple scattering fingerprint to make it identifiable if it is the fifth ligand.Dr Chenreplied: (1) Yes. In our data analysis, we assumed two Cu–N distances that were associated with the ground state and the MLCT state respectively. From the transient optical absorption experiment and the model calculation with experimental parameters, such as laser pulse energy, sample concentration, sample dimension,etc., we obtained the fraction of the excited state at around 20%. Therefore, in data fitting, we fixed the ratio of the ground state and excited state to 80%vs.20%. TheE0edge shifts in the fittings for the two Cu–N bond distances were kept the same. The difference in Cu–N bond lengths resulted from such fittings with the above precautions.(2) That's a very good point. However, the coordination of Cu also changed from tetrahedral to penta-coordinated. I would expect the steric hindrance could force the Cu–N bond to be longer despite the effects that you mentioned.(3) We are not sure what the fifth ligand was at this point. In the acetonitrile case, you raised a good point.Dr Techertasked: Despite the fact that triplet annihilation processes quench the population of the triplet state (and therefore the EXAFS difference signal), how would this annihilation process change the structure of the energy-transferring moieties and therefore the EXAFS signal?Dr Chenanswered: The triplet–triplet annihilation due to the adjacent molecules being simultaneously excited will certainly quench the excited state population and shorten the lifetime. Thus, the laser excitation will become less efficient and laser photons will be wasted. More importantly, the excited state annihilation will change fundamental aspects of the photoexcitation of the molecules as the interactions between the same kind of molecules can no longer be neglected. The triplet–triplet annihilation could also result in singlet states at a much later time than the initial photoexcitation. Therefore, it is the fundamental aspects of photochemistry that have been changed due to the strongly interacting molecules. Of course, the XAFS signals expected will no longer be limited to those from isolated molecules due to their photochemical reactions, but come from a mixture of isolated and strongly interacting molecules. Therefore, we can no longer claim the signals are from excited molecules of a certain kind, but from a collection of molecules with a certain configuration.Dr Sagicommented: Doing a single energy experiment may reveal some correlation between the structural kinetics and the overall kinetics of the system, thus proving that one can identify distinct features in the single energy experiments on EXAFS or edge regions that are directly associated with the structure, for example the transition from one coordination number to another during the excitation process.Dr Chenreplied: In small molecules rather than proteins, the actual atomic movements after the photoexcitation take place in fs or a few ps. After that, the excited state is thermally equilibrated. Therefore, strictly speaking, our experiment is not time resolved, but provides snapshots of the excited state. However, there are circumstances where different excited states could be generated sequentially, such as a singlet state initially generated by photoexcitation, and a triplet generated by intersystem crossing from the initially generated singlet. In this case, we will have to adjust the time delays corresponding to the optimal population of the singlet and the triplet states respectively in order to capture their structures. If this is successful, we will be able to associate certain distinctive spectral features that appear at a certain time after the photoexcitation to particular excited states.Prof. Helliwellasked: How often does the Advanced Photon Source (APS) offer the “special timing mode”? Is it a limiting factor?Also the APS operates a “top up mode”. How do you allow for that in your experimental design?Dr Chenresponded: APS provides the hybrid fill timing mode about 4 weeks per year. It was split into about one week at a time. So we have the special timing mode every three months. The “top-up” mode from the APS did not affect our experiments, because we average over a long time, and fluctuation caused by the top-up was not obvious to us. We like this mode because it provides 30% more X-ray photon flux.Prof. Coppensopened the discussion of Prof. O'Hare's paper: We have some experience with hydrothermal synthesis in crystal engineering, where one works with multicomponent (>2) systems. Since we cannot predict which phases will be formed, the stoichiometry, and therefore the products change with time. Have you looked at cases where completely different phases are formed as the cooling process proceeds?Prof. O'Harereplied: We have not observed any changes in crystalline phase composition of these reactions on cooling.Prof. Wilsonasked: What is the limitation in time resolution for the neutron and X-ray experiments? How well is this matched to “typical” reaction rates in your processes,i.e.do you need second-scale time resolution?Prof. O'Hareanswered: The current setup using the GEM diffractometer at the ISIS facility gives us a time resolution of minutes while the synchrotron at the SRS gives us a time resolution of seconds. For most hydrothermal reactions and conventional solid–solid processes this is adequate. For other reactions we would ideally need better time resolution.Prof Evanssaid: Have you been able to investigate the solution phase pre-nucleation processes by other techniques, say by high temperature NMR?Prof. O'Harereplied: We have not. Prof. Taudelle and co-workers at the University of Strasbourg have recently usedin situNMR to complement our studies on gallium and aluminium phosphate crystallisation.Prof. Ryanasked: What evidence is there for precursor structures in hydrothermal synthesis?Prof. O'Hareresponded: We cannot detect precursor structures in our experiment. We need crystalline domains which can diffract X-rays.In situNMR experiments by Taudelleet al.suggest the existence of four-membered Al2P2rings in solution prior to crystallisation of aluminium phosphates.Mr Robbsaid: You seem to follow the structure by Bragg peak evolution. Is the data you collect sufficiently good for complete structure refinement and, for example, can you obtain atomic displacement parameters for your material and what does this tell you?Prof. O'Harereplied: The energy dispersive X-day diffraction data we measure is not suitable for complete structure refinement. Different X-ray photon energies are absorbed from the incident and diffracted white X-ray beam by the cell materials and the sample. This means that we cannot define the incident beam profile.Prof. Finneyasked: How much do we know about the variation of conditions within the cell? If there is significant non-uniformity (e.g.in temperature or concentration), is there scope for probing spatially as well as temporally?Prof. O'Harereplied: We try to stir the contents of the hydrothermal cell using a magnetic stirrer which attempts to average out any non-uniformity in temperature or local reagent concentrations.Prof. Cerniksaid: Have you used the information from the three-element SSD to show crystallisation differences within the hydrochemical cell? The intensity overlaps should sample different regions of the crystallite distribution and therefore pinpoint non-homogeneous growth.Following on, do you translate the sample during the experiment to probe preferred regions of synthesis?Prof. O'Hareanswered: The three element detector does allow us to look at differentd-spacing ranges simultaneously. In the systems we have studied using the three element detector where these regions overlap the variation in the intensity of the common Bragg reflections is identical which suggests isotropic crystal growth.Prof. Moffatasked: Might the geophysicist's style of large-volume press that affords simultaneous control of pressure and temperature be of use to your style of experiments?Prof. O'Harereplied: The reagents used in hydrothermal synthesis are quite corrosive viscous gels (acid or alkali) and I wonder whether this would be compatible with their equipment.Prof. Hounslowasked: (1) Fig. 2 of the paper contains a curve fit—of what form?(2) Can you quantify the rates in terms of rate constants and orders of reactions?(3) Are Avrami kinetics appropriate in this case?Prof. O'Hareresponded: (1) The fit is to the Avrami–Erofe'ev equation:α = 1 − exp{−(k(t − t0))n}.(2) Yes, the Avrami–Erofe'ev fit gives us an exponent,n, and a rate constant,k.The value ofn, the Avrami exponent, is believed to contain information about the mechanism of reaction. The model was originally developed to describe the growth of crystallites in solid–solid reactions, and assumes the formation of nucleation sites in a uniform mixture of reagents from which crystal growth occurs.(3) I am not sure; it is a standard analysis but I am not confident that it actually tells us much about the chemistry.Prof. Ryanasked: What is the path length through the cell?Prof. O'Harereplied: 20 mm.Prof. Sir John Meurig Thomasopened the discussion of Prof. Ryan's paper: One wonders whether by changing the conditions (in a well-defined fashion) of precipitation so as to produce aragonite (instead of calcite) you would get the same kind of precursor “phases” being formed.Prof. Ryanreplied: We suspect that a poorly ordered precursor phase could well be found prior to the precipitation or aragonite. We have been able to produce mixed aragonite calcite mixtures under conditions where a precursor phase is observable but cannot distinguish whether there are two precursor phases.Prof. Finneycommented: I would like to add a comment concerning metastable forms occurring in the early stages of crystallisation which then transform to the stable form.The system is the formation of normal hexagonal ice Ih from ice II (a phase stable at higher pressures) recovered to ambient pressure at 77 K.1Neutron powder diffraction on the resulting structure looks like the so-called cubic form Ic, though the resolution of the measurements showed (a) the presence of the 100 hexagonal peak and considerable broadening of the higher angle shoulder of the 111 (cubic) peak. Moreover, various peaks showedhkl-dependent broadening suggesting not only a small particle size of about 160 Å, but also significant stacking faulting. On heating to around 160 K, the structure transforms to normal hexagonal ice, without evidence of any significant disorder.The simple interpretation of these results would be that the ambient pressure ice phase nucleates first as very small crystallites of the cubic phase, which then transforms as the crystallites grow on heating to the normal stable hexagonal structure ice Ih. However, considering that the difference between the cubic and hexagonal structures is that of different stackings of the hexagonal layers (ABCABC stacking for cubic, ABABAB for hexagonal), an explanation that takes account of the varioushkl-dependent broadenings observed suggests that the initial small crystallites may not be identifiable as cubic ice Ih but a structure of random stacking of hexagonal layers. In fact, work on colloidal systems shows similar diffraction features to those observed in the ice case, in particular the simultaneous presence of 111 cubic and 100 hexagonal features, and a high angle shoulder on the 111 peak. Only when the temperature is increased do the molecules have sufficient mobility to begin to anneal out the irregular stackings to approach the stable hexagonal structure.Thus there is evidence in even this apparently simple system for a metastable phase forming in the early stages of crystallisation. In this case, the metastable phase is clearly not amorphous (though such might have occurred earlier but not been detected), but it is, in comparison to the stable phase, clearly disordered. Noting electron microscope observations three decades ago of icosahedral structures formed in the early stages of growth of an even simple crystal (fcc gold), we can perhaps suspect that an initial metastable (with respect to the equilibrium phase) structure may be a common occurrence. In fact, there is perhaps little good reason to expect very small microcrystallites to have the structure of the stable phase, considering the large surface/volume ratio compared to the equilibrium (extended) crystal.1 P. N. Pusey, W. van Megen, P. Bartlett, B. J. Ackerson, J. G. Rarity and S. M. Underwood,Phys. Rev. Lett., 1989,63(25), 2753–2756.Prof. Ryananswered: Thank you for pointing out the relationship to the field of supercooled water and colloidal crystals. The formation of dense, random hexagonally-close-packed structures prior to cubic crystallisation is indeed observed in a wide variety of systems and has been predicted theoretically (see for example refs. 1 and 2).1 I. Kusaka, D. W. Oxtoby and Z.-G. Wang,J. Chem. Phys., 2001,115(15), 6898–6906.2 V. Talanquer and D. W. Oxtoby,J. Chem. Phys., 1998,109(1), 223–227.Prof. Finneyasked: Distinguishing a genuinely amorphous structure from a very small crystal by the presence of clear Bragg peaks can often be controversial: once the “crystallite” is <3–4 unit cells across, the peak broadening may make it difficult to distinguish the scattering from that of an amorphous structure. How can you be sure that your amorphous precursor phase really is genuinely amorphous and not a very small crystalline nucleus?Prof. Ryanreplied: This is indeed a difficult problem and was the matter of considerable debate atFaraday Discussion112.1My colleague Nick Terrill did some modelling, using the scattering geometry of beamline 8.2, and estimated that Bragg peaks could be resolved from a collection of 3 × 3 × 3 unit cells at 1% by volume. Obviously there is considerable line broadening with such small crystals and the wide angle scattering from a metastable, lower-order precursor phase would be even more difficult to resolve. There are some semantic problems in the polymer crystallisation field and the distinction between a collection of chains and a nucleus is often blurred by beliefs.1 A. J. Ryan, J. P. A. Fairclough, N. J. Terrill, P. D. Olmsted and W. C. K. Poon,Faraday Discuss., 1999,112, 13–29.Prof. Evanscommented: The local site symmetry of the carbonate anion differs considerably between calcite and aragonite such that these forms can be differentiated by IR spectroscopy. Perhaps this technique could be used to probe the structure of the pre-crystallisation phase.Prof. Ryananswered: We are currently working on the use of FTIR and FTIR microscopy to differentiate between aragonite and calcite in our crystalliser geometry. We would anticipate probing the structure of the precursor phase by IR or Raman spectroscopy in the not-too-distant future.Prof. O'Hareasked: Have you performed an Avrami-type analysis of the crystallization data described in Fig. 13b of the paper?Prof. Ryanreplied: We have indeed performed an Avrami-type analysis on our inorganic crystallisation data but, as was pointed out by Prof. Hounslow, this is not an appropriate model to use for these crystallisation conditions. I bow to his superior knowledge in this regard and leave it to him to explain the reason why the Avrami model is inappropriate and describe the correct model to use.Dr Chensaid: I am impressed by your results of simultaneous collection of SAXS/WAXS data. As a general question, I would like to ask your perspective on using such techniques on other systems, such as protein folding, crystallisation and molecular self-assembly to form nanostructure materials?Prof. Ryanreplied: Thank you, we have worked hard at developing the SAXS/WAXS instrumentation to such a level that it is widely applicable to a wide range of systems. A good account of the work on protein in solution can be obtained from the Daresbury Annual Reports and in refs. 1–6.1 M. Hirai, H. Iwase, T. Hayakawa, K. Miura and K. Inoue,J. Synchrotron Radiat., 2002,9(4), 202–205.2 W.-Y. Choy, F. A. A. Mulder, K. A. Crowhurst, D. R. Muhandiram, I. S. Millett, S. Doniach, J. D. Forman-Kay and L. E. Kay,J. Mol. Biol., 2002,316(1), 101–112.3 W. Zheng and S. Doniach,J. Mol. Biol., 2002,316(1), 173–187.4 R. Russell, I. S. Millett, S. Doniach and D. Herschlag,Nat. Struct. Biol., 2000,7(5), 367–370.5 D. I. Svergun, G. Zaccai, M. Malfois, R. H. Wade, M. H. J. Koch and F. Kozielski,J. Biol. Chem.2001,276(27), 24 826–24 832.6 D. I. Svergun, M. V. Petoukhov and M. H. J. Koch,Biophys. J., 2001,80(6), 2946–2953.Prof. Helliwellcommented: Regarding the literature on protein assembly, protein crystal nucleationetc.I refer you to the following authors:(a) M. H. J. Koch: assembly of multi-macromolecular structures(b) A. Tardieu: protein crystal nucleation(c) D. I. Svergun: combined SAXS/WAXS and SANS for protein fold discoveryProf. Coppensopened the discussion of Prof. Harrison's paper: The discrepancy between the thermal parameters and the cell dimension temperature seems paradoxical, as expansion is driven by anharmonicity, which increases when the thermal parameters increase. So the discrepancy must be in the experiment. My question is: were the crystals in the microwave experiment and the independent measurement the same, or could there be a difference in crystal quality and thus extraction? Or could such a difference be a result of the microwave treatment?Prof. HarrisonAlso Dr Whittaker, University of Edinburgh.replied: The neutron diffraction experiments were performed on the same sample of BaTiO3, first with conventional heating to 1173 K and then, after slow cooling back to 295 K, with microwave heating. We would not expect that this treatment would lead to any significant change in crystallinity between the conventional and microwave heating measurements, and therefore one would expect little change in factors such as extinction and strain broadening as a consequence of the heat treatment. Indeed, when the sample was remeasured at 295 K just before the microwave heating measurement, the peak widths were essentially unchanged. It should be stressed that the limited time available for neutron data acquisition led to relatively fast scans, and the precision of many of the refined parameters is less than ideal. The parameter that appears to show the most distinct change when the microwave and conventional heating are compared is the peak width parameter,σ1, as described in our paper.Prof. O'Hareasked: (1) Have you looked at the effects of using variable microwave frequencies?(2) Do different solids absorb the microwave energy in different ways?(3) Do the thermal parameters give you any indication of the types of motion that may be excited by the microwave field?Prof. Harrisonreplied: (1) No we haven't: so far our work has been restricted to frequencies of 2.45 GHz, which is one of the small number of frequencies allocated for non-telecommunications work. Microwave sources for those other prescribed frequencies are far more expensive, and the range of devices available at those frequencies is much more limited.(2) Absolutely: there are several distinct mechanisms of microwave heating, treated in detail in standard references (for example refs. 1 and 2). The most important modes of heating involve coupling of the microwave radiation with phonons (for example the present case of BaTiO3) or mobile species (electrons, for example in metals, or ions, for example in fast-ion conductors), but defects may also play an important role,3as may the coupling between the magnetic component of the microwave field and materials with high magnetic susceptibility (such as some ferrites).(3) In the first instance one would expect rapid equilibration of microwave energy among the various modes, which one would expect to be manifested through increased thermal parameters as an increase in anisotropic displacement parameters, just as one expects with conventional heating. However, one could imagine cases where the thermal parameters respond to the microwave field in a manner that is different from what one would expect from a simple increase in temperature. Where the rate at which particular modes are excited by microwaves is significantly higher than the rate of repartition, and where these modes are either polarised (as might happen when a single crystal is exposed to a linearly polarised microwave field) or in the case of a molecular solid localised to a particular region of the constituent molecules, it is conceivable that there is an anomalous response of either certain components of the thermal parameters, or of thermal parameters for a particular group of atoms.1 D. M. P Mingos and A. G. Whittaker, inChemistry Under Extreme or Non-Classical Conditions, ed. R. van Eldik and C. D. Hubbard, Wiley, Chichester, 1997, ch. 11, pp. 479–514.2 Y. V. Bykov, K. I. Rybakov and V. E. Semenov,J. Phys. D.: Appl. Phys., 2001,34, R55–57.3 J. H. Booske, K. I. Rybakov, V. E. Semenov, S. A. Freeman, J. H. Booske and R. F. Cooper,Phys. Rev. B, 1997,55, 3559–3567.Prof. Wilsonsaid: (1) Is it possible to decouple microwave heating effects from the electric-field induced issues?(2) Can you envisage a use for some sort of “spatial scanning” resonance radiography as a means ofin situtemperature measurement, helping to pin down the temperature fluctuations locally within the samples?Prof. Harrisonresponded: (1) Yes. This has been in a particularly elegant piece of work to probe this effect,1the essence of which was to study ion migration between similar compounds of different elemental composition in a linearly polarised microwave field. The result was an enhanced diffusion coefficient at a given temperature in the direction of polarisation.(2) I could imagine that such a technique could provide a spatially resolved probe of temperature, but the length scales involved could well be too large for some of the large and highly localised thermal gradients that can arise in microwave-heated systems, so it is likely to provide only a partial solution to some of the problems we presented.1 A. G. Whittaker and L. Cronin, inProceedings of the Second International Conference on Microwave Chemistry, ed. A. Gourdenne, 2000, Institut National Polytechnique de Toulouse, on behalf of AMPERE, Cambridge, UK.Prof. Wilsonsaid: I am interested in the coupling of the microwave frequencies with directional modes within the sample: are these compatible, and is such coupling possible?Presumably the energy redistribution within the sample/system results from partitioning of “thermal” motion into the various modes.Prof. Harrisonreplied: The microwave radiation couples with the solid through a multiphonon process, such that radiation whose frequency is of the order of GHz is able to excite modes whose frequency is the order of THz (see for example ref. 1). If the microwave field is linearly polarised, one would expect the phonons that are excited to be anisotropic. However, the energy put into these modes is rapidly repartioned through anharmonic processes with a time-scale typical of the phonon frequencies, that is of the order of THz (ps). For a recent consideration of this sort of process, see ref. 2.1 M. Sparks, D. F. King and D. L. Mills,Phys. Rev. B, 1982,26, 6987–7003.2 Y. V. Bykov, K. I. Rybakov and V. E. Semenov,J. Phys. D.: Appl. Phys., 2001,34, R55–57.Dr Techertasked: What is the expected order of magnitude of the phonon–phonon coupling ine.g.BaTiO3?Prof. Harrisonanswered: I don't know, and I imagine that it would be very difficult to determine this quantity accurately. When a material such as BaTiO3is heated with microwaves, phonons are excited through a multiphonon process, and this energy is then repartitioned over the phonon spectrum through anharmonic effects. One therefore needs to know which phonons are involved, and then calculate the appropriate anharmonicity, which is not trivial for a material containing heavy atoms. However, it has been estimated that for electric field strength and temperatures typical of those used in microwave processing, deviations of the population of high-energy phonons from Boltzmann values are very small indeed, implying that this process is fairly efficient in redistributing energy.11 Y. V. Bykov, K. I. Rybakov and V. E. Semenov,J. Phys. D.: Appl. Phys., 2001,34, R55–57.Dr Techertasked: Since the water in crystalline protein samples cannot rotate freely, as in solution, the energies of the corresponding libration modes should be shifted to higher frequencies. Do any calculations exist concerning these values?Prof. Harrisonreplied: The motion of water molecules bound to protein molecules in a variety of ways has been the subject of extensive studies by NMR with complementary modelling, and reveal a range of amplitudes and correlation times for libration (see for example refs. 1 and 2) extending from relatively fast, small-amplitude motion with a correlation time of the order of 0.07 ps, to slower, larger-amplitude motion correlated with the motion of portions of the molecule, with correlation times in range 1–10 ps. Note that pure water has modes in the region 0.05 ps that have been attributed to libration of H-bonded molecules.1 V. P. Denisov, K. Venu, J. Peters, H. D. Hörlein and B. Halle,J. Phys. Chem. B, 1997,101, 9380–9389.2 V. P. Denisov and B. Halle,J. Am. Chem. Soc., 1995,117, 8456–8465.Dr Grantcommented: Michael Levitt published a paper in 19851covering calculations (NMA) he made on bovine pancreatic tripsin inhibitor (BPTI) and he found that the collective motions of groups of up to ten residues have frequencies of 2 cm−1to 10 cm−1. 2 cm−1 ≃ 60 GHz, only about one order of magnitude larger than microwave frequency (2.45 GHz for a domestic microwave oven). Therefore the discrepancy between the THz frequency of many of the modes in a protein, and microwave oven and mobile phone frequency (1.8 GHz) is not such a large gap to bridge. The other point is that water in a protein is known to play an integral role in the unfolding and folding of a protein and water obviously couples with 2.45 GHz microwave radiation, so maybe we should be looking at the role of H2O in conformational changes induced by microwaves as providing a bridge to causing structural changes in the host biomolecule.1 M. Levitt, C. Sander and C. Stern,J. Mol. Biol., 1985,181, 423.Prof. Helliwellsaid: (1) You stimulate me to wonder whether the scattering pattern of sectioned brain, with and without microwaves, has been measured.(2) Assessing the risk of mobile phones, another possible “sample” would be the impact of microwaves on the inner ear. The physiology of the inner ear is studied using guinea pig inner ear, as it is easy to section.11 F. Mammano, IUPAB 'Prof. Harrisonanswered: (1) I don't believe that experiment has been tried yet, and I imagine it would be difficult to get conclusive information about microwave-induced changes in brain tissue from such measurements because of the complexity of the sample. At the moment the relatively small amount of work that is being done on the effect of microwaves on materials or biological systems, and which involves spectroscopic or structural measurements, has involved relatively simple model systems.(2) This would indeed be an interesting system for study in that it has been shown to be sensitive to irradiation; however, it is also a relatively complex system and I think it will be some time before conclusive measurements could be performed on it.

ISSN:1359-6640    

DOI:10.1039/b207971m

出版商:RSC    

年代:2003

数据来源: RSC