Computational Studies of Stability, Dynamics, and Water Sorption Behavior of Proteins at Extreme Conditions

Abstract

The main challenge in formulating biological therapeutics is to have proteins fully regain their native structures and functionalities after exposure to conditions such as low temperatures and extreme states of dehydration during lyophilization. This dissertation describes computational investigations of proteins at such extreme conditions, with specific focuses on understanding water sorption behaviors, low-temperature stability and dynamics, and folding/unfolding mechanism of proteins. We use advanced molecular-simulation and data-analysis techniques to obtain microscopic understanding of protein-water interactions at low temperature and dehydrated conditions.

In the first part of this dissertation, we use our novel computational technique to investigate water sorption behaviors of amorphous protein matrices. We show that the water sorption isotherms are qualitatively and quantitatively very similar for the Trp-cage miniprotein 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 investigate the microscopic origin of hysteresis between the adsorption and desorption branches by explicitly comparing protein-water interactions in adsorption and desorption processes. We find that significant differences in hydration behavior between adsorption and desorption manifest themselves at the individual amino acid level, in particular around polar or charged residues. We then present the effects of structural constraints on proteins on the water sorption isotherms, by modulating the disulfide linkages and backbone connectivity of the proteins. Disulfide linkages greatly reduces the extent of hysteresis by significantly restricting structural changes of the proteins, and removal of backbone connectivity causes shifting of hysteresis towards the lower humidity regime and enhancement in water uptake capacity.

In the second part of this dissertation, we perform extensive replica-exchange molecular dynamics simulations to investigate cold denaturation of Trp-cage miniprotein. The ubiquity of cold denaturation in globular proteins has broad implications for not only developing preservation strategies for biological materials but also understanding the evolution of freeze-tolerant organisms. We find that Trp-cage cold denatures into a compact, partially folded state with alpha-helix intact, which is accompanied by hydration of buried hydrophobic residues at the core of Trp-cage.

In the third part of this dissertation, we investigate low-temperature dynamical transitions in protein matrices with varying degrees of hydration. We identify two distinct temperatures where transitions in protein dynamics occur. Thermal motions are harmonic and independent of hydration level below the first transition temperature at 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 that decreases as the hydration level increases. Upon heating above this dynamical transition temperature, 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.

Finally, we use diffusion maps, a nonlinear dimensionality reduction technique, to systematically characterize the folding/unfolding pathways of Trp-cage miniprotein. Conventional order parameters provide structural information but fail to capture the underlying dynamics of protein folding process. By embedding simulation trajectories in diffusion maps space, we identify two folding pathways and intermediate structures that are consistent with previous experimental and computational studies, demonstrating that this technique can be employed as an effective way of analyzing and constructing protein folding pathways from molecular simulations.