Effects of confinement and macromolecular crowding on protein stability and protein folding dynamics

In cells, proteins perform specific tasks in crowded and confined environments; these environments influence their stability and folding dynamics. To investigate these effects of confinement and macromolecular crowding on proteins based on statistical mechanical methods, we have carried out numerical simulations using minimalist models (2-Dimensional HP lattice, Monte Carlo, Brownian dynamics) and 3-Dimensional off-lattice polymer models. For the confinement effects, our results, based on heat capacity calculations, show that the folding temperature increases when box size decreases, indicating that the protein is stabilized. These results are consistent with the experimental observations obtained in Dr. Wei's group. We have also investigated the effects of confinement on the kinetics of refolding and unfolding as a function of temperature and box size. The unfolding time increases as box size shrinks, however, the folding time behaves in a more complicated way.; To investigate the effects of macromolecular crowding, Brownian motions of crowders are included inside a virtual box with periodic boundary conditions. Besides temperature, the concentration of crowders and crowder size are also varied in our simulations. Simulated results indicate that folding temperature increases with the crowder concentration; the protein is thus stabilized by the presence of crowders. However, this increase is not as large as that observed in the case of confinement. Our dynamic studies show that both folding and unfolding times increase with the concentration of crowders in such a way that the equilibrium is shifted towards the folded state. Furthermore, our simulations show that the activation energy of unfolding remains approximately constant as the number of crowders increases.; Based on the concept of depletion force we have also calculated the enhancement of the mechanical force required to unfold ubiquitin molecules in a solution of dextran, the crowding agent. We have employed a 3-D polymer model using the pivot algorithm to calculate the depletion zone and have applied the scaled particle theory for the osmotic pressure of the crowders. Our results are in reasonable agreement with recent measurements carried out in Dr. Yang's laboratory.