摘要:

The general theme of this presentation is nuclear magnetic resonance (NMR) spectroscopy in structural biology. Two aspects of special interest to biological physicists are discussed. These are, firstly, the results of NMR studies on the solvation of proteins and nucleic acids in solution and, secondly, the physical foundations of a novel NMR approach for studies with very large particles in solution, transverse relaxation-optimized spectroscopy (TROSY). ©1999 American Institute of Physics.

ISSN:0094-243X    

DOI:10.1063/1.59892

出版商:AIP    

年代:1999

数据来源: AIP

摘要:

We consider two simple biosystems, both exhibiting biological function at room temperature: (1) hydrated protein powders, and (2) nearly anhydrous enzymes in alcohol, and we propose a common structure of the solvent-protein interface. We extend our previous treatment of percolation to the “blobs, nodes and links” model, to describe the surface as patches of H-bonded OH groups, singly connected in the percolating backbone. A dynamical pathway between surface and core is identified in few strong H-bonds between charged side chains and protein secondary structure. Since recent work in collaboration with F. Bruni and G. Consolini shows dielectric behavior typical of proton glasses, we model proton frustration in blobs by a 2D Ising net, and in collaboration with D. Stauffer we find by Q2R cellular automata that this system fails to reach internal equilibrium. A functional relevance for non-ergodicity in enzymatic activity is proposed. ©1999 American Institute of Physics.

ISSN:0094-243X    

DOI:10.1063/1.59873

出版商:AIP    

年代:1999

数据来源: AIP

摘要:

Systematic, computer-simulated elongation of A- and B-DNA double helices beyond the range of normal room temperature fluctuations provides new insights into recent physical manipulations of single DNA molecules. The calculations include unusual states that are energetically disfavored under equilibrium conditions but that become favored as the DNA is highly stretched or compressed. The variation of potential energy vs. stretching provides a detailed picture of cooperative conformational change that points to the possible role played by the non-canonical states. Dramatic structural changes take place as the energies of different conformers approach one another. In particular, large-scale, concerted changes in base pair inclination, brought about by changes in backbone and glycosyl torsions, offer a model of the observed sharp increase in force required to stretch single DNA molecules more than 1.6 times their canonical extension. Small degrees of over- and under-twisting have limited effects on the over-stretching transition, but influence local “melting” of the double helix. The energy landscapes of the systems evaluated here reveal the energetically economical pathways for molecular deformations operative in DNA processing, such as recombination and transcription. ©1999 American Institute of Physics.

ISSN:0094-243X    

DOI:10.1063/1.59886

出版商:AIP    

年代:1999

数据来源: AIP

摘要:

Cis/trans isomerization of proline residues is known to exhibit high activation energies. These kinetic barriers often dominate the energy landscape of protein folding. There are 6 proline residues (at positions 11, 31, 42, 47, 56 and 117) in staphylococcal nuclease (SNase) [EC 3.1.31.1]. Stopped-flowCD222nmmeasuring the evolution of the secondary structure of protein has detected 5 kinetic barriers in SNase folding (&Dgr;G≠for&tgr;fr<15,&tgr;f116.9,&tgr;f218.5,&tgr;f319.5, and&tgr;fs21.8 kcal/mol) and 3 kinetic barriers in unfolding (&Dgr;G≠for&tgr;ur<15,&tgr;u117.4,&tgr;us21.6 kcal/mol). To investigate systematically how individual proline residues and 6 proline residuesin totocan shape the folding funnel we have expediently constructed 7 proline mutants for study. They are 6 single-proline-substituted mutants (P11A, P31A, P42A, P47A, P56A and P117A) and 1 proline-free mutant (PallA). Study of equilibrium folding/unfolding and stopped-flow kinetics of the wildtype and the 7 mutants of SNase have allowed us to identify sources of 3 main kinetic barriers in the SNase folding. The highest barrier(&Dgr;G≠=21.8&hthinsp;kcal)belongs to the cis/trans isomerization of Pro117. The next barrier(&Dgr;G≠=19.5&hthinsp;kcal)involves synergetic effects of proline residues which limits the rate of folding of the oligonucleotide binding (OB) domain in all 7 proline-containing SNase. For the proline-free mutant (PallA) the OB domain folds rapidly. Furthermore, we have found that the equilibrium folding/unfolding properties of these proline mutants are remarkably similar to that of the wildtype despite their startlingly different folding/unfolding kinetics. These results lead us to conclude that while free energy of folding(&Dgr;GF=−4.5&hthinsp;kcal/mol)provides the driving force, it is the activation energy that forms a conduit or shapes a kinetic funnel for SNase folding. The landscape for SNase folding is extremely rugged. Data support our previously proposed Least Activation Path (LAP) model for protein folding [Su, Z.D. &etal; Proc. Natl. Acad. Sci. USA93, 2539–2544 (1996)]. The LAP concept depicts protein folding as the movement of the unfolded population of protein along deep valleys of the energy landscape to reach the free energy minimum of the native state. An analogy for the LAP model would be flow of water from a highland over the rugged surface of a landscape to reach the lowest point of the ground. The fine features of the landscape will dictate kinetics and pathways of the flow. ©1999 American Institute of Physics.

ISSN:0094-243X    

DOI:10.1063/1.59874

出版商:AIP    

年代:1999

数据来源: AIP

摘要:

The long time dynamics and folding of residues of oligopeptides is simulated with a Monte Carlo method in torsion angle space using two different types of moves generating local and global conformational changes, respectively. Due to the rigidity of the molecular model with only torsional degrees of freedom, the energy function from conventional molecular dynamics (MD) has to be adjusted. We find that polyalanine prefers an alpha-helical structure, the sequence AA&ellip;.A GG AA&ellip;.A adopts a helix-turn-helix motif, and VV&ellip;.V GG VV&ellip;.V, preferentially forms an anti-parallel beta-sheet. With conventional MD these results require much more CPU-time or may not be obtainable. By analyzing the geometry and energetics of the obtainable structures, it will be explained how the residues talk to each other to reach their preferred folding pattern. ©1999 American Institute of Physics.

ISSN:0094-243X    

DOI:10.1063/1.59893

出版商:AIP    

年代:1999

数据来源: AIP

摘要:

Functional selection of sequences according to the local configuration at the active sites is simulated with a spin-glass like model. Starting from a random peptide, the unique native-like conformation emerges with high efficiency with a relatively small number of steps of selection. The functional selection on the active sites is sufficient for sequences to evolve to have the folding ability and relieves the need of exhaustive search in the sequence space. ©1999 American Institute of Physics.

ISSN:0094-243X    

DOI:10.1063/1.59897

出版商:AIP    

年代:1999

数据来源: AIP

摘要:

We present a computational method forde novoligand design based on simulated annealing optimization of an empirical free energy function which has been derived from regression analysis of protein-ligand complexes. Structures are built by joining together molecular fragments from the Available Chemicals Directory. The algorithm navigates through the space of molecular structures, conformations and rigid-body transformations. The diversity of the generated structures can range from completelyde novoto products of a combinatorial synthetic reaction scheme and docking of single molecules. The synthetic feasibility of the proposed molecules is considered by the incorporation of penalty terms to reduce undesirable connectivity and chemical features. Test results for tripsin are presented. ©1999 American Institute of Physics.

ISSN:0094-243X    

DOI:10.1063/1.59894

出版商:AIP    

年代:1999

数据来源: AIP

摘要:

Induced transitions play a key role in many cellular processes, both as mechanisms of specific ligand binding as well as mediators of energy transduction. Transitions induced by ligands are understood to be processes in which intrinsic binding energy is used topay foran energetically-unfavorable conformational change. The transition pathway is commonly represented as a two-step reaction in which ligand attachment to the protein “causes” it to change conformation. Here we seek a better understanding of induced transitions by looking at how binding energy is usedduringan induced transition. We focus on the allosteric protein globule itself and look at changes in globule structure and energy from both classical biochemical as well as physical points of view. We consider both the sequenceandtime dependence of changes in globule structure and energy. We find that ligand-induced transitions may be regarded as two-step processes of energy transformation: In the first step, the presence of steric misfit leads to the input of potential energy to the system and the conversion of binding energy into a driving force for globule rearrangement. Presence of an activation energy barrier hindering rearrangement leads to storage of this energy in the globule. In the second step, fulfillment of latent bonding interactions and relaxation to the high-affinity conformation leads to output of the stored energy via dissipation and, in the case of transducing proteins, external work. ©1999 American Institute of Physics.

ISSN:0094-243X    

DOI:10.1063/1.59895

出版商:AIP    

年代:1999

数据来源: AIP

摘要:

Under physiological conditions, proteins continuously fluctuate among a large number of conformational substates which can be represented by local minima in a multidimensional potential energy landscape. The complex topography of the landscape gives rise to an extremely broad distribution of characteristic times for conformational motions, spanning more than fifteen orders of magnitude, from fast bond librations to slow global unfolding. Here we focus on motions occurring on time scales larger than microseconds, which are particularly relevant to biomolecular function. For carbon-monoxy myoglobin, the photosynthetic reaction center of purple bacteria, and bovine pancreatic trypsin inhibitor, large-scale conformational changes have been measured over a wide temperature range. In all three cases, the rate coefficients governing the slow conformational changes depend strongly on temperature, and the dynamics can be understood with a model that assumes diffusional motions on a rugged energy land-scape with a random amplitude distribution around an average of about 10 kJ/mol. ©1999 American Institute of Physics.

ISSN:0094-243X    

DOI:10.1063/1.59896

出版商:AIP    

年代:1999

数据来源: AIP

摘要:

The normal-mode refinement of X-ray crystallographic data opened a new possibility to analyze the mean-square displacements in a protein molecule. A comparison of the X-ray structure of myoglobin at several temperatures with Mo¨ssbauer data is performed. In the low-temperature regime below 180 K the iron mean-square displacements obtained by Mo¨ssbauer spectroscopy are in good agreement with a normal-mode analysis. The X-ray mean-square displacements at the position of the iron, after the motion originated from the external degrees of freedom are subtracted, have practically the same temperature dependence as those from Mo¨ssbauer spectroscopy. The difference between the X-ray mean-square displacements and those predicted by normal-mode analysis measures the distribution of molecules into conformational substates. Above 180 K the Mo¨ssbauer effect indicates fluctuations between conformational substates. The relaxation from a Fe(III) conformation to a Fe(II) conformation is shown for superoxide dismutase ofPropionibacterium shermanii. ©1999 American Institute of Physics.

ISSN:0094-243X    

DOI:10.1063/1.59883

出版商:AIP    

年代:1999

数据来源: AIP