We report molecular dynamics simulations of the equilibrium folding/unfolding thermodynamics of an all-atom model of the Trp-cage miniprotein in explicit solvent. Simulations are used to sample the folding/unfolding free energy difference and its derivatives along 2 isochores. We model the ⌬G u(P,T) landscape using the simulation data and propose a stablility diagram model for Trp-cage. We find the proposed diagram to exhibit features similar to globular proteins with increasing hydrostatic pressure destabilizing the native fold. The observed energy differences ⌬E u are roughly linearly temperature-dependent and approach ⌬E u ‫؍‬ 0 with decreasing temperature, suggesting that the system approached the region of cold denaturation. In the low-temperature denatured state, the native helical secondary structure elements are largely preserved, whereas the protein conformation changes to an "open-clamp" configuration. A tighter packing of water around nonpolar sites, accompanied by an increasing solventaccessible surface area of the unfolded ensemble, seems to stabilize the unfolded state at elevated pressures.folding ͉ free energy ͉ hydrostatic pressure ͉ simulations T he stability of natively folded proteins in solution is determined by the competition of many effects that reach a balance near physiological conditions. As a consequence, the stability of a protein can be affected in many different ways (1-5). High-temperature denaturation is mostly accompanied by a dramatic loss of protein secondary structure (6). However, elevated pressures (2, 3), changing pH (2), and cosolvents such as salts (7) and osmolytes (4) also affect the stability of the native state, often destabilizing in character but under certain conditions also significantly stabilizing (4). In addition, many globular proteins are also destabilized at low (subzero) temperatures, a process known as ''cold denaturation'' (8). Cold denaturation is experimentally accomplished with the help of elevated pressures (2), leading to a characteristic tongue-shaped P,T-stability diagram, found for many globular proteins (9-15). Hydrophobic forces play a key role in the protein folding process (16-18), but it is the balance of hydrophilic and hydrophobic forces that determines the conformational equilibrium. The notion that proteins under native conditions are only ''marginally stable'' (7) seems to be an important requirement for their ability to explore different conformational substates (19) and hence for protein function. The application of high hydrostatic pressure (20) has been shown to be able shift the equilibrium of conformational states (21, 22), promoting denaturation (23) but also altering the native state (24) and modifying protein-protein interaction (20). Pressure effects on protein structure appear to be determined mostly by changing the balance between hydrophilic and hydrophobic interactions (25)(26)(27).Model peptides and proteins have long been sought as templates for understanding protein structure and function. The Trp-cage miniprotein (28) ...

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