The functional native states of globular proteins become unstable at low temperatures, resulting in cold unfolding and impairment of normal biological function. Fundamental understanding of this phenomenon is essential to rationalizing the evolution of freeze-tolerant organisms and developing improved strategies for long-term preservation of biological materials. We present fully atomistic simulations of cold denaturation of an α-helical protein, the widely studied Trp-cage miniprotein. In contrast to the significant destabilization of the folded structure at high temperatures, Trp-cage cold denatures at 210 K into a compact, partially folded state; major elements of the secondary structure, including the α-helix, are conserved, but the salt bridge between aspartic acid and arginine is lost. The stability of Trp-cage's α-helix at low temperatures suggests a possible evolutionary explanation for the prevalence of such structures in antifreeze peptides produced by coldweather species, such as Arctic char. Although the 3 10 -helix is observed at cold conditions, its position is shifted toward Trp-cage's C-terminus. This shift is accompanied by intrusion of water into Trp-cage's interior and the hydration of buried hydrophobic residues. However, our calculations also show that the dominant contribution to the favorable energetics of low-temperature unfolding of Trp-cage comes from the hydration of hydrophilic residues.cold denaturation | Trp-cage miniprotein | protein folding T he functional native states of globular proteins that are stable near physiological conditions become labile when changes in temperature, pressure, and solvent composition alter their environment. The partial or complete unfolding of secondary and tertiary structure associated with this loss of stability can strongly affect protein behavior, leading to significantly impaired biological function (1, 2). Denaturation upon heating is a ubiquitous and well-studied phenomenon in which proteins gain configurational entropy and unfold as a result of increased kinetic energy. By contrast, the mechanisms responsible for pressure-induced unfolding, and for denaturation of globular proteins at low temperatures, remain incompletely understood (3-5).Fundamental understanding of cold denaturation is important due to its ecological implications and relevance to industrial processing of proteins and biological materials. Freeze-tolerant organisms such as the Arctic char, for example, thrive in subfreezing habitats where cold denaturation can occur (6). Biopharmaceuticals are also exposed to cold conditions that can result in denaturation when they are lyophilized into freeze-dried solids to prolong their shelf life (7,8). Natural cryoprotectants such as sugars and polyols stabilize proteins against denaturation in cold-weather species (6), and similar compounds have been used to mitigate the damaging effects of freeze-drying in pharmaceutical formulations (7, 9).Cold denaturation was first reported by Hopkins in 1930 (10). Brandts (11) subsequently observed ...

Understanding the mechanisms by which proteins fold from disordered amino-acid chains to spatially ordered structures remains an area of active inquiry. Molecular simulations can provide atomistic details of the folding dynamics which complement experimental findings. Conventional order parameters, such as root-mean-square deviation and radius of gyration, provide structural information but fail to capture the underlying dynamics of the protein folding process. It is therefore advantageous to adopt a method that can systematically analyze simulation data to extract relevant structural as well as dynamical information. The nonlinear dimensionality reduction technique known as diffusion maps automatically embeds the high-dimensional folding trajectories in a lower-dimensional space from which one can more easily visualize folding pathways, assuming the data lie approximately on a lower-dimensional manifold. The eigenvectors that parametrize the low-dimensional space, furthermore, are determined systematically, rather than chosen heuristically, as is done with phenomenological order parameters. We demonstrate that diffusion maps can effectively characterize the folding process of a Trp-cage miniprotein. By embedding molecular dynamics simulation trajectories of Trp-cage folding in diffusion maps space, we identify two folding pathways and intermediate structures that are consistent with the previous studies, demonstrating that this technique can be employed as an effective way of analyzing and constructing protein folding pathways from molecular simulations.

We present the first simulation study of the impact of protein matrix structure on water sorption along with a new computational method to hydrate and dehydrate protein systems reversibly. To understand the impact of the underlying structure of the protein matrix on the hydration process, we compare three types of protein substrates comprised of Trp-cage miniproteins with varying degrees of monomer translational and orientational order and monomer denaturation. We show that the water sorption isotherms are qualitatively and quantitatively very similar for the Trp-cage matrices independently of the underlying degree of disorder, which is consistent with the experimental observation that the qualitative features of water sorption isotherms are nearly universal for globular proteins. We also show that the Trp-cage matrices with varying disorder share similar trends in volumetric swelling, solvent accessibility, and protein-water hydrogen bonding during the sorption processes, while hydrogen bonding between protein molecules depends sensitively on the matrix characteristics (crystal, powder, and thermally denatured powder). Volumetric swelling, solvent accessibility, and protein-water hydrogen bonds exhibit no hysteresis when plotted as a function of hydration level and are thus controlled exclusively by the protein's water content.

We investigate computationally the dynamical transitions in Trp-cage miniprotein powders, at three levels of hydration: 0.04, 0.26 and 0.4 g water/g protein. We identify two distinct temperatures where transitions in protein dynamics occur. Thermal motions are harmonic and independent of hydration level below Tlow ≈ 160 K, above which all powders exhibit harmonic behavior but with a different and enhanced temperature dependence. The second onset, which is often referred to as the protein dynamical transition, occurs at a higher temperature TD that decreases as the hydration level increases, and at the lowest hydration level investigated here (0.04 g/g) is absent in the temperature range we studied in this work (T ≤ 300 K). Protein motions become anharmonic at TD, and their amplitude increases with hydration level. Upon heating above TD, hydrophilic residues experience a pronounced enhancement in the amplitude of their characteristic motions in hydrated powders, whereas it is the hydrophobic residues that experience the more pronounced enhancement in the least hydrated system. The dynamical transition in Trp-cage is a collective phenomenon, with every residue experiencing a transition to anharmonic behavior at the same temperature.

Fundamental understanding of protein stability away from physiological conditions is important due to its evolutionary implications and relevance to industrial processing and storage of biological materials. The molecular mechanisms of stabilization/destabilization by environmental perturbations are incompletely understood. We use replica-exchange molecular dynamics simulations and thermodynamic analysis to investigate the effects of ionic liquid-induced perturbations on the folding/unfolding thermodynamics of the Trp-cage miniprotein. We find that ionic liquid-induced denaturation resembles cold unfolding, where the unfolded states are populated by compact, partially folded structures in which elements of the secondary structure are conserved, while the tertiary structure is disrupted. Our simulations show that the intrusion of ions and water into Trp-cage's hydrophobic core is facilitated by the disruption of its salt bridge and 3-helix by specific ion-residue interactions. Despite the swelling and widening of the hydrophobic core, however, Trp-cage's α-helix remains stable. We further show that ionic liquid disrupts protein-protein and protein-water hydrogen bonds while favoring the formation of ion-protein bonds, shifting the equilibrium of conformational states and promoting denaturation near room temperature.