Single-Molecule Spectroscopy Study of Crowding-Induced Protein Spontaneous Denature and Crowding-Perturbed Unfolding-Folding Conformational Fluctuation Dynamics

The effects of molecular crowding on protein folding-unfolding processes are of importance for understanding protein function and structure dynamics in living cells. The enhancement of protein stability as a result of reduced entropic effect in the presence of molecular crowding is well understood both experimentally and theoretically. However, because of the complexity and interplay between various interactions existing in an equally favored environment of protein folding and unfolding conformational dynamics, such a simple reduced entropic enhancement model does not suffice to describe protein folding conformational dynamics under a protein crowding condition. In this paper, we report our observation on that single protein molecules spontaneously denature into unfolded proteins and folding-unfolding fluctuations in solution of crowding reagent Ficoll 70. We have identified that such protein dynamics involves a combined mechanism of polymer-polymer interaction, entropic effects, and protein solvation dynamics. We characterize the protein folding-unfolding dynamics by using single-molecule spectroscopy to obtain detailed molecular dynamic scale information on the protein folding-unfolding conformational fluctuation dynamics. Our findings suggest that the complex unfolding dynamic processes are spontaneous denature of single protein molecules induced by molecular crowding effect which has been elusive for analysis in ensemble-averaged measurements. Furthermore, the energy needed for the spontaneous unfolding is at the biological accessible force fluctuation level, which suggests a strong implication of significant human health relevance and importance. The new knowledge of the inhomogeneous protein unfolding processes can serve as a step forward to a mechanistic understanding of human diseases associated with molecular crowding, protein aggregates, fibril formation, as well as gene translational regulation processes typically under a molecular crowded local environment.