News and Publications

Molecular Dynamics of Proteins and Peptides

C.L. Brooks III, S. Banba,* N. Besley, B. Bursulaya, T. Cleveland, P. Constans-Nierga, M.F. Crowley, K. Damodaran, B. Dominy, M. Feig, Z. Guo, J.D. Hirst, J. Karanicolas, R. Kiyama,** H. Minoux, R.T. Morton, J. Radkiewicz, F.R. Salsbury, J.-E. Shea

* Mitsui Toatsu Chemicals, Inc., Chiba, Japan
** Shinogi & Co., Ltd., Osaka, Japan

Understanding the atomic-level forces that determine the structure of proteins, peptides, and protein-peptide complexes and the processes by which these structures are adopted is essential to complete knowledge of protein and peptide structure and function. To address such questions, we use statistical mechanics, molecular simulation, statistical modeling, and quantum chemistry.

Building atomic-level models to simulate biophysical processes (e.g., protein folding or binding of a ligand to a biological receptor) requires (1) the development of potential functions that accurately represent the atomic interactions and (2) the use of quantum chemistry to aid in characterizing these models. Calculation of thermodynamic properties requires the development and implementation of new theoretical and computational approaches that connect averages over atomistic descriptions to experimentally measurable thermodynamic and kinetic properties.

Interpreting experimental results at a more atomic level (e.g., to obtain a detailed description of structure from circular dichroism spectra or hydrogen exchange experiments on proteins and peptides) leads to the development of theoretical models for these processes. Additionally, the massive computational resources needed to produce atomic-level descriptions of proteins, peptides, and protein-peptide complexes in solution motivate efforts aimed at the efficient use of new computer architectures, including large supercomputers. Each of the objectives and techniques mentioned represents areas of development in our research program in computational biophysics.

COUPLED PROTEIN DYNAMICS AND ENZYME CATALYSIS

The source of the rate enhancement of enzymes continues to be debated, and a number of theories explaining the catalytic power of enzymes have been proposed. Part of this debate includes the importance of protein dynamics. A prevalent theory states that the stabilization of the transition state is the primary determinant of the enhanced rate of catalysis. The arrangement of the residues in the active site is the major contributing factor to the rate enhancement, and protein motions have no effect. Other models invoke vibrational coupling and nonequilibrium solvation and propose that protein dynamics play a vital role in catalysis. The nature of correlated motions between substrates and the protein has a direct influence on reaction rates in this situation. In fact, the forces associated with these motions determine the height of the activation barrier. This finding has been proposed as an explanation for observations for several enzymatic reactions involving electron transfer.

Experimental evidence suggests that protein dynamics may play a role in the dihydrofolate reductase (DHFR) catalytic pathway. Escherichia coli DHFR catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF). X-ray, nuclear magnetic resonance, kinetic, mutagenic, and molecular dynamics data indicate that regions of the 159-residue reductase are highly flexible and undergo conformational changes that occur within the timescale of the catalytic cycle. A key kinetic step in this pathway involves the Michaelis complex, DHFR-DHF-NADPH, shown in Figure 1.

To explore whether dynamics may play a role in the catalytic action of the reductase, we carried out long (>10 ns) molecular dynamics studies of the Michaelis complex and of representative product complexes of DHFR-THF-NADPH and DHFR-THF-NADP⁺. Our results lend new support for the idea that protein dynamics are coupled to catalysis. For Figure 1, we examined the results of correlated motions within the protein (1) when the protein was interacting with the reactant molecules (DHF-NADPH) and (2) after the catalytic hydride transfer had taken place (with THF-NADP⁺). The "correlation maps" indicate that the Michaelis complex is a highly coupled system with anticorrelated motions taking place between the loop regions distant from the site of catalysis; the product complex shows no such coupling.

This surprising result (for the "simple" exchange of 2 hydrogen atoms) is fully consistent with a role for dynamics in catalysis for this system. Furthermore, regions with high correlation with the catalytic region are generally also regions known to be susceptible to mutational effects on the catalytic rate, even though these regions are distant from the catalytic site. This work expands the basis for further investigations of the coupling between dynamics and catalysis in proteins and is an ongoing collaborative effort between our group and groups involved in experimental and theoretical studies at TSRI.

MECHANISMS OF PROTEIN FOLDING

Understanding the means by which a linear amino acid sequence adopts its functional 3-dimensional structure is a key challenge to scientists in many disciplines, from biology to physics. We are using statistical mechanics and computer simulation to elucidate the principles that govern this process. Our explorations of the links between protein topology and the overall mechanism of protein folding are providing new insights that fuel further investigation of theoretical models and experiments. Our specific focus during the past year was the folding mechanism of simple single-domain proteins with different overall topology.

We computed first-principles free-energy landscapes for the folding of a number of distinct protein topologies: an all helical protein, a mixed a/ß motif, an all ß-sheet protein, and a small de novo designed ß-sheet protein. These free-energy surfaces present a landscape for folding that connects topology with mechanism. For the helical protein, folding, which is dominated by local interactions, is downhill with concomitant collapse and formation of native tertiary structure as indicated by a "diagonal" band in the free-energy surface projected onto coordinates such as the radius of gyration that describe the overall size of the protein and coordinates such as the number of native contacts that describe the closeness of the structure to the native state. The folding of the protein with the mixed a/ß motif and of the all ß-sheet protein involves initial collapse, with only small amounts of native tertiary structure being formed, and then a "search" through compact conformational states for the native structure.

According to these findings, and to other ongoing work in our laboratory, this general picture is correct. That is, for proteins with more delocalized topologies, such as ß-sheet structure, folding includes a collapse phase before the acquisition of native structure; more localized structures can fold with concomitant collapse and formation of native structure. Our research in this area is coupled to the theoretical and experimental developments ongoing in other laboratories at TSRI and in the La Jolla area.

PUBLICATIONS

Brooks, C.L. III, Gruebele, M., Onuchic, J.N., Wolynes, P.G. Chemical physics of protein folding. Proc. Natl. Acad. Sci. U. S. A. 95:11037, 1998.

Bursulaya, B.D., Brooks, C.L. III. The folding free energy surface of a three-stranded ß-sheet protein. J. Am. Chem. Soc., in press.

Dominy, B.N., Brooks, C.L. III. Development of a generalized Born model parameterization for proteins and nucleic acids. J. Phys. Chem. B 103:3765, 1999.

Dominy, B.N., Brooks, C.L. III. Methodology for protein-ligand binding studies: Application to a model for drug resistance, the HIV/FIV protease system. Proteins 36:318, 1999.

Hirst, J.D., Dominy, B., Guo, Z., Vieth, M., Brooks, C.L. III. Conformational and energetic aspects of receptor-ligand recognition. In: Rational Drug Design. Parrill, A.L., Reddy, M.R. (Eds.). American Chemical Society Press, Washington, DC, 1999, p. 12. American Chemical Society Symposium Series.

MacKerell, A.D., Jr., Brooks, B., Brooks, C.L. III, Nilsson, L., Roux, B., Won, Y., Karplus, M. CHARMM: The energy function and its parameterization. In: Encyclopedia of Computational Chemistry. Schleyer, P.v.R., et al. (Eds.). Wiley, New York, 1998, p. 271.

Mohanty, D., Dominy, B.N., Kolinski, A., Brooks, C.L. III, Skolnick, J. Correlation between knowledge-based and detailed atomic potentials: Application to the unfolding of the GCN4 leucine zipper. Proteins 35:447, 1999.

Radkiewicz, J.L., Brooks, C.L. III. The role of protein dynamics in enzymatic catalysis. J. Am. Chem. Soc., in press.

Shea, J.-E., Nochomovitz, Y.D., Guo, Z., Brooks, C.L. III. Exploring the space of protein folding Hamiltonians: The balance of forces in a minimalist ß-barrel model. J. Chem. Phys. 109:2895, 1998.

Shea, J.-E., Onuchic, J.N., Brooks, C.L. III. Exploring the origins of topological frustration: Design of a minimally frustrated model of fragment B of protein A. Proc. Natl. Acad. Sci. U. S. A., in press.

Vieth, M., Hirst, J.D., Brooks, C.L. III. Do active site conformations of small ligands correspond to low free-energy solution structures? J. Comput. Aided Mol. Des. 12:563, 1998.

Vieth, M., Hirst, J.D., Dominy, B.N., Daigler, H., Brooks, C.L. III. Assessing search strategies for flexible docking. J. Comp. Chem. 19:1623, 1998.

Vieth, M., Hirst, J.D., Kolinski, A., Brooks, C.L. III. Assessing energy functions for flexible docking. J. Comp. Chem. 19:1612, 1998.