The main objective of this project, jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Biological Physics Program in the Division of Physics in the Mathematical and Physical Sciences Directorate, is to establish relationship between the thermodynamics and the structural properties of pressure induced changes in proteins using molecular simulations. Pressure provides a way of shifting equilibrium of protein configurations without increasing thermal fluctuations or changing the system composition. Hydrostatic pressure provides a way of probing the role of hydration in protein stability and dynamics. In spite of the wealth of experimental information available, theoretical and computational studies of pressure denaturation have been limited by system size and the slow relaxation of proteins at high pressures. With the development of enhanced sampling techniques, the use of large distributed computing resources, and the development of coarse-grained models for protein folding, these limitations can be overcome. This project will study the effect of high pressure on the stability of peptides that are known to form alpha and beta hairpin structures to determine the effect that high pressure has on the secondary structure stability. To study the effect of tertiary interactions in protein stability, the PI will study the effect of pressure on the stability of mini proteins (Trp-cage) and a small protein (protein A), two systems with simple folds that involve the packing of secondary structure elements. A major challenge will be to study the effect of pressure on larger proteins, such as ubiquitin and SNase. The studies on large proteins will involve coarse-grained models, combined with umbrella sampling techniques on fully solvated systems. The models will be validated by comparing their results with those obtained in unbiased detailed calculations on smaller systems, and by making predictions that can be tested experimentally. The structure and thermodynamics of the unfolded and transition state ensembles to correlate volume effects measured thermodynamically to the degree of hydration of the various ensembles will be studied in detail. This work will establish a framework for understanding the role of water in protein function and stability, as well as for the interpretation of a number of experimental studies of pressure effects on biological molecules.
This project is inherently interdisciplinary and it will be done in collaboration with theoretical, computational and experimental groups. Software, models, and algorithms developed in this project will be made available to the scientific community. Students at all levels will be trained in the modeling of thermodynamics properties of biomolecular systems. The PI will train graduate and undergraduate students from both Physics and Biology departments. To enhance the participation of under represented minorities in science, the PI will host researchers and students from the University of Puerto Rico Mayaguez and Humacao.
Intellectual merit: Proteins are molecular machines that perform many of the tasks needed in a living cell. Proteins are amino acid polymeric molecules that need to adopt a specific three-dimensional structure in order to perform these tasks. The folding of a protein into its three dimensional structure is a self-assembly process driven by the interactions of polar and non-polar groups in the protein with water, co-solvents, and with other groups in the molecule. The hydration of proteins plays an important role in folding, binding, and catalysis. Pressure and temperature can be used to reversibly change the effect of hydration without changing the composition of the system or the solvent. In particular, high-pressure experiments provide a way of shifting the equilibrium of protein configurations without increasing thermal fluctuations. In spite of the wealth of experimental information available, theoretical and computational studies have been limited by system size and the slow relaxation of proteins at high pressures. With the development of enhanced sampling techniques combined with the use of large distributed computing resources, and the development of coarse-grained models for protein folding, these limitations have been reduced. Theoretical studies of pressure effects on biomolecules will lead to a better understanding of the thermodynamic and structural role of water molecules in folding and binding. The main objective of this project was to gain an understanding of the forces determining the folding of proteins from computer simulations. Of particular interest is to establish the relationship between the thermodynamics and the structural properties of proteins and how these properties change as a function of pressure, temperature and co-solvents. As part of this project we performed the first calculations of a protein pressure-temperature stability diagram. We found that protein stability can be modulated by changes in pressure and temperature in unexpected ways. For example, simulations are capable of describing protein denaturation upon cooling at high pressures (cold denaturation), in addition to the typical denaturation upon heating at low pressure. These results could be relevant for handling proteins in an industrial setting. We also determine the effect of denaturants on protein stability and were able to determine the mechanism by which urea denatures proteins. Urea is simple molecule commonly used to denature proteins and its molecular mechanism has been a subject of considerable debate for over 50 years. Previous simulation studies have sought to elucidate the mechanism of urea denaturation by focusing on the pathway of denaturation, rather than examining the effect on the folding/unfolding equilibrium. We have reported the first reversible folding/unfolding equilibrium of a protein in presence of urea using molecular dynamics simulations. The simulations captured the experimental linear dependence of unfolding free energy on urea concentration. In addition, we found that the denaturation is driven by favorable direct interaction of urea with the protein, through both electrostatic and van der Waals forces. In collaboration with experimental groups in the USA and France, we studied the effect of cavity formation in mutants in a protein (Snase). Our studies consisted of a comprehensive study of this protein by crystallography, optical and nuclear magnetic resonance spectroscopy and simulations. It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. We demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins. Broader Impacts This project is inherently interdisciplinary and the research was done in collaboration with theoretical, computational and experimental groups. The research results were presented at national and international conferences and were published in peer-reviewed journals. Software, models, and algorithms developed in this project have been made available in a web site. This project supported three PhD students and multiple undergraduate students in the PIs laboratory. Graduates PhD students are currently working at the National Institutes of Health, Los Alamos National Laboratory, and Intel Corporation. Many undergraduate students are currently pursuing PhD degrees at leading US universities. The PI recognizes the importance of educating underrepresented minorities in science. He has actively participated in reviews and advisory boards at various minority institutions.
