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42828-AC7
Molecular Simulation and Statistical Mechanical Studies of Pressure Effects on Proteins
Background: The structure, thermodynamics, and dynamics of biomolecules in solution are intimately connected to their function. Much of our understanding of these fundamental aspects of biomolecules has come from quantifying their response to systematic perturbations in the extended thermodynamic space. Over the past decade or so, primarily due to development of high pressure experimental technology, hydrostatic pressure has emerged as an important dimension in which to perturb biological macromolecules and their processes. Experiments show that proteins unfold at high pressures leading to water-swollen molten globule like ensemble of states. In addition, the kinetics of folding-unfolding are significantly slowed down at high pressures. Lastly, protein aggregates or protein-protein complexes (or multimeric units) can be particularly sensitive to pressure, dissociating at moderate to high pressures. That proteins unfold at high pressures appears counterintuitive at first and is also at odds with the prediction of the simple “hydrophobic transfer” model. Thus, fundamental understanding of pressure effects on proteins and their complexes presents an important challenge.
Our studies: We have taken a two-pronged approach to address the above challenging problem.
(1) Firstly, we are focusing on pressure effects on fundamental water-mediated interactions (e.g., hydrophobic interactions) over the extended pressure temperature space. Specifically, we have performed molecular simulation studies to obtain the free energy of hydrophobic interactions (as quantified by methane-methane potentials of mean force in water) over the entire liquid state phase space of water. These calculations show that near ambient temperatures, increasing pressure distinctly weakens the strength of hydrophobic interactions, consistent with lower thermodynamic stability of proteins at higher pressures. Qualitatively, with increasing pressure, the “open” hydrogen bonded structure of liquid water is changed in such a way as to weaken hydrophobic interactions.
More recently, we have studied an exciting aspect of hydrophobicity, namely, its lengthscale dependence. Studies from David Chandler's group at Berkeley have pointed to interesting lengthscale dependencies of hydrophobic hydration with increasing size of the solute. We find that pressure effects on the hydration of small and large solutes are different – the hydration shells of larger solutes (or large hydrophobic patches on proteins) have higher compressibility compared to those of small solutes. This observation is consistent with experimental results that protein-protein complexes with significant hydrophobic interface are more sensitive to pressure. We plan to extend these fundamental studies to protein-protein complexes over the coming year.
(2) Secondly, we have performed atomically detailed simulations of protein Staphyloccocal Nuclease with focus on its volumetric properties and on thermodynamics of pressure unfolding. Given that the timescales of unfolding of proteins at high pressures can range from seconds to hours or longer, brute force molecular dynamics are severely limited to probe such structural transitions. Instead, we developed an algorithm (where water molecules are gradually inserted into protein interior) that generated ensemble of states from fully folded to partially folded to unfolded proteins. By simulating these states in separate simulations, we calculated partial molar volumes of the protein in the folded and unfolded ensemble. These volumetric data, combined with a thermodynamic cycle, allows prediction of the pressure-dependent free energy of unfolding of this protein. Our predictions of free energy for the wild type protein as well as for its mutant are in good agreement with experimental results.
These calculations and similar studies on non-ionic micelles allowed us to accurately measure from simulations the compressibility properties of protein interiors and of micelle cores. We found that whereas micelles are highly compressible (with compressibility about 50% of liquid hexane), protein interiors are comparatively incompressible with compressibilities similar to those of solid polymeric materials. We were able to relate these observations to thermal fluctuations, and find that the width of cavity size distribution in these media correlates well with the observed compressibility (see Molecular Physics, 105:2, 189-199, 2007.)
Currently, we are extending the above studies of pressure effects on water-mediated interactions as well as on protein systems. We are calculating free energies of interactions between solutes of different charge densities over a range of pressures and temperatures. At the macromolecular level, we plan to study other proteins as well as selected protein-protein systems.
