摘要:

AbstractIn order to examine the electrostatic forces in globular proteins, pKavalues and their ionic strength dependence of His residues of hen egg white lysozyme (HEWL) and human ly−sozyme (HUML) were measured, and they were compared with those calculated numerically.pKavalues of His 30 residues in HEWL, HUML, and short oligopeptides were determined from chemical shift changes of His side chains by1H−nmr measurements. The associated changes in pKavalues in HEWL and HUML were calculated by solving the Poisson‐Boltzmann equations numerically for macroscopic dielectric models. The calculated pKachanges and their ionic strength dependence agreed fairly well with the observed ones.The contribution from each residue and each α‐helix dipole to the pKavalues and their ionic strength dependence was analyzed using Green's reciprocity theorem. The results indicate that (1) the pKaof His residues are largely affected by surrounding ionized and polar groups; (2) the ionic strength dependence of the pKavalues is determined by the overall charge distributions and their accessibilities to solvent; and (3) α‐helix dipoles make a significant contribution to the pKawhen the His residue is close to the helix terminus and not fully exposed to

ISSN:0006-3525    

DOI:10.1002/bip.360320802

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractElongational flow techniques are used to investigate the birefringent response and flow‐induced molecular scission of monodisperse phage‐DNA samples in aqueous solution. A 4‐roll mill apparatus was used to characterize the solutions ac low stain rates,\documentclass{article}\pagestyle{empty}\begin{document}$ \dot \varepsilon $\end{document}≤ 300 s−1, and the opposed jets apparatus used to study fracture of the DNA molecules at strain rates up to 15 × 103S−1. The molecular weight values were measured before and after fracture in elongational flow using the high‐resolution technique of pulsed field gel electrophoresis (PFGE). The birefringent response incorporates both rigid and flexible components. The birefringence is nonlocalized and rises gradually to a plateau value, similar to rigid‐rod behavior. In addition a certain minimum value in the strain rate is necessary, anonsetvalue\documentclass{article}\pagestyle{empty}\begin{document}$ \dot \varepsilon $\end{document}0, before the signal appears, indicating a flexible component. This behavior is consistent with a hinged‐rod model and is similar to that observed for the protein collagen molecule at elevated temperature. We propose that this type of behavior is likely for multistrand rope‐like macromolecules where localized separation or partial untwisting of the intertwined chains occurs, creating temporary hinges, in accordance with biochemical evidence for sequence‐specific sites of flexibility. Results are presented on the entanglement effects at high concentrations. We have calculated rotational diffusion rates as a function of concentration and molecular weight. Using PFGE to measure the molecular weight profiles, our fracture studies at high strain rates demonstrate chain halving and quartering in accordance with the predictions of the thermally activated barrier to scission theory for

ISSN:0006-3525    

DOI:10.1002/bip.360320803

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe presence of subsaturating levels of a high‐affinity ligand has been demonstrated both by experiment and calculation to have far‐reaching consequences on thermally induced protein denaturation due to the coupling between the protein denaturation and ligand‐binding equilibria. Under such circumstances, a protein may undergo biphasic denaturation even though in the absence of ligand it exhibits a thermogram comprised of a single essentially symmetric endotherm. Up to now, the presence of just 2 maxima in the thermogram has been presented merely as an experimental observation or as the result of equilibrium computations. Here we develop a thermodynamic description of the linkage between these equilibria in which the number of cusps present in the thermogram correlates with the number of resolved steps in the plot of saturation level of remaining native protein vs temperature (i.e., the thermal binding curve). During thermally induced denaturation, the concentration of native protein decreases; thus, the native protein in effect is titrated with ligand at constant total ligand concentration. The free ligand concentration increases substantially through the release of bound ligand by unfolding protein thereby increasing the saturation level of the remaining native protein. The form of this thermal binding curve is a function of the number of ligand‐binding sites on the protein, the magnitudes of the association constants, and the total ligand and total protein concentrations. As a result, the model predicts multiphasic denaturation of a single cooperative unit when the thermal binding curve consists of discrete multiple steps. The presence of only 2 maxima (i.e., a single cusp) in a thermogram for a protein with multiple sites on the native species derives from the form of the thermal binding curve, which in this case is a single‐step sigmoidal plot, andnotfrom the predominant denaturation of unliganded and fully liganded native species. In addition, it is shown that, in general, the contributions from the denaturation of individual native protein species are decidedly non‐two‐state in character; thus, simple deconvolutton should not be carried out. The effects of nonzero values of ΔCPanddΔCP/dTfor denaturation and of changes in enthalpy and in heat capacity for ligand binding, as well as the interaction of ligand with the denatured protein, ar

ISSN:0006-3525    

DOI:10.1002/bip.360320804

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractA solution of the problem of topology of a hydrogen bond net in a triple helix of collagen is suggested on the basis of an analysis of thermodynamic data on denaturation of phylogenetically different collagen [T. V. Burjanadze (1982), Vol. 21, pp. 1489–1501; T. V. Burjanadze, E. I. Tiktopulo, and P. L. Privalov (1987), Dokl. Akad, Nauk. USSR, Vol. 293, pp. 720–724] as well as on the earlier evaluation of the energy of the OH group of the 4−hydroxyproline bond [A. R. Ward and P. Mason (1973),Journal of Molecular Biology, Vol. 29, pp. 431–435]. It is shown that only the water‐bridged collagen structure [G. N. Ramachandran and R. Chandrasekharan (1968),Biopolymers, Vol. 6, pp. 1649–1661: G. N. Ramachandran, M. Bansal, and R. S. Bhatnagar (1973),Biochimica Biophysica Acta, Vol. 322, pp. 166–171: M. Bansal, C. Ramakrishnan, and G. N. Ramachandran (1975), Proceedings of the Indian Academy of Sciences, Vol. 82, pp. 152–164] can explain both the change of thermostability upon proline hydroxylation [J. Rosenbloom, M. Harsch. and S. Jimenez (1973),Archives of Biochemistry and Biophysics, Vol. 158, pp. 478–484] and its phylogenetic change [T. V. B

ISSN:0006-3525    

DOI:10.1002/bip.360320805

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractA study has been done of the effect of neutral salts (NaCl and CaCl2) on the mechanism of type I collagen triple helix folding and unfolding in concentrated acetic acid solutions (2–8.8M). It is shown that in these conditions, thermoabsorption and secondary structure change in heated solutions proceed in two consecutive stages. Salts exert a different destabilizing effect on different sites of the macromolecule, promoting the detection of a thermostable domain. The presence of a thermostable domain permits one to carry out reversible denaturation of collagen and to study the mechanism of the triple helix folding. Proceeding from the mechanism of the triple helix folding, an assumption has been made on the localization of the thermostable domain and its biological rol

ISSN:0006-3525    

DOI:10.1002/bip.360320806

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractIn order to prevent enzymatic degradation of β‐casomorphin‐5 (1) and morphiceptin, reduced peptide bonds were incorporated at the 2–3 and 3–4 bonds, respectively. The analogues were synthesized by a combination of solid phase methodology and reductive alkylation of resin‐bound peptide amines with Boc‐amino acid aldehydes (Boc:tert‐butyloxycarbonyl) in the presence of NaBH3CN. During reversed phase high pressure liquid chromatography purification, peak shape distortions could be observed. Epimerization was excluded, based on gas chromatography/mass spectroscopy analysis, which indicated acceptable levels of racemization (<3%) in the crude product. Instead, the phenomena could be attributed to slowcis/ transisomerizations originating from the Xxx‐Pro bonds in the sequence. The presence of different conformational isomers was also established by1H‐nmr spectroscopy in DMSO‐d6. All analogues showed high stability in blood plasma, enhanced binding affinity for the μ receptor, and very low binding to the δ receptor. While the Phe3Ψ(CH2‐N) Pro4analogues (3) and (5) displayed agonist activity, the Pro2Ψ(CH2‐NH) Phe3modified analogue (2) showed antagonist activity comparable toD‐Phe‐C

ISSN:0006-3525    

DOI:10.1002/bip.360320807

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractTo optimize antigen–antibody reactions, we have synthesized chemically well‐defined hy‐drophilic microspheres. Proteins or haptens were covalently linked to these carriers. When the microsphere conjugates were agglutinated by the corresponding antiserum, the size of the complex artificially increased during the immunological reaction. After optimizing var‐ious parameters such as the hydrophilic character, repulsion charges, and amount of antigen coupled to the microspheres, we developed a rapid and sensitive immunoassay based on laser light scattering by the co

ISSN:0006-3525    

DOI:10.1002/bip.360320808

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractMelittin (MLT), a 26‐residue cationic (net charge +5 at pH 7.2) peptide from bee venom, is well known to be a monomeric, approximately random coil; but when its charges are reduced by titration, by acetylation (net charge +2) or succinylation (net charge −2), or by screening by salt, it goes over to tetrameric α‐helix. The conversion is promoted by raising the peptide concentration. The tetramer is held together by hydrophobia forces. We have changed the net charge to −6 by acylation with acetylcitric anhydride (a new acylating agent); this anionic derivative forms tetrameric helix at neutral pH, without salt, and at relatively low concentration, conditions under which the cationic MLT does not become helical. Thus, a high net charge is not sufficient to prevent association and helix formation. We have synthesized an anionic melittin analogue of MLT (E‐MLT; net charge −4) in which all five lysine and arginine residues are replaced with glutamate, and acetyl and succinyl derivatives of E‐MLT (net charges −5 and −6). All three of these are resistant to helix formation. They require much higher NaCl or NaClO4concentration for helix formation than does MLT. Even CaCl2, MgCl2, and spermine. 4HC1 are less effective in helicizing E‐MLT than MLT. MLT, at pH 7.2, shows increasing helix as the peptide concentration increases (8‐120 μM), but E‐MLT and its acyl derivatives do not. MLT and acylated MLTs in the helical tetramer show both cold‐ and heat‐induced unfolding, with maximum stability near room temperature. At high temperature, a significant amount of residual structure remains. Heating (to 100°C) monomeric MLT (i.e., MLT at low concentration) or E‐MLT results in a monotonic increase in negative ellipticity. In 1.0MNaCl, E‐MLT (at sufficiently high concentration) also shows cold and hot unfolding. The results are discussed in respect to charge–charge and charge‐dipole interactions, and hydrophobic effects. E‐MLT is also discussed in relation to proteins of halophilic bacteria, which have higher proportions of anionic residues than do

ISSN:0006-3525    

DOI:10.1002/bip.360320809

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe conformation and ion‐binding characteristics of a cyclic octapeptide, cyclo(Ala‐Leu‐Pro‐Gly)2, in a liphophilic solvent, acetonitrile, have been studied using CD and nmr spectroscopy. The peptide binds preferentially to divalent cations such as calcium, magnesium, and barium. The conformations of the free cyclic peptide and its calcium complex are very similar with well‐defined β‐ and γ‐turns. The cyclic peptide readily forms equimolar and possibly 2 : 1 (peptide:cation) complexes with d

ISSN:0006-3525    

DOI:10.1002/bip.360320810

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractThe central ingredient of any structure modeling tool is a molecular model or force field that accounts for proper geometry and energy calculation. For protein and peptide modeling based on entire amino acids as building blocks, we describe a peptide force field in which each amino acid is represented by a single point in space, taken at the position of the α‐carbon atom. Apart from the positional coordinates, these units carry the two torsional angles φ and Ψ as additional degrees of freedom to account for the orientations of the peptide links. While some of the energy terms are analogous to expressions in atomic force fields, the presence of the angular variables leads to fundamental differences with new features and additional terms with no atomic counterparts.The force field reproduces secondary structure elements with very good accuracy. Globular parts of tertiary packing stay near the experimental structures with a rms deviation in C‐α positions of 0.1–0.3 nm and about 25° in φ and Ψ depending on the size of the structure. A tendency for larger discrepancies is observed in exposed loops or terminal segments the conformations of which may be strongly influenced by neighboring domains.Finally, a scope of possible applications is presented. They range from modeling activities, such as model building by homology, to coarse scanning of conformation space in conformation analysis and structure determination. An extension to a dynamics model would offer the possibility to eliminate the less interesting high‐frequency modes that in all‐atom force‐field dynamics absorb most of the co

ISSN:0006-3525    

DOI:10.1002/bip.360320811

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY