Crowd Control: Effects of Macromolecular Crowding on Early Events in Protein Folding

Abstract

Earlier time-resolved far-UV TRCD/TRORD studies have shown that electron-transfer triggered folding of reduced cytochrome c (redcyt c) from the GuHCl-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a sub-microsecond time scale, while the remaining population folds more slowly [Chen et al. (2003) J. Phys. Chem. A, 107,8149-8155]. With the addition of 220 mg/mL dextran 70 (spherical-like structure), the population of the fast folding step is greatly reduced [Chen et al. (2012) Biochemistry 51, 9836-9845]. However, this fast phase is not significantly affected when sucrose is used to introduce the same microviscosity as that of the dextran solution. These results, as well those from corresponding coarse-grained simulations, suggest that excluded volume effects, but not viscosity, due to macromolecular crowding compacts the unfolded state ensemble such that cyt c's access to fast-folding conformations is decreased in crowded conditions. Presented here will be studies that examine the effects of other crowding agents of different size and shape (rod-shaped Ficoll 70) and concentration (100 mg/mL dextran 70) on the kinetics of redcyt c folding. How the His33-Fe-His18 heme configuration, which was previously correlated with the fast folding population under buffer conditions, is affected by the presence of macromolecular crowders that facilitate compaction of the unfolded state is also examined.