title: Effects of macromolecular crowding on burst phase kinetics of cytochrome c folding.
abstract: Excluded volume and viscosity effects of crowding agents that mimic crowded conditions in vivo on "classical" burst phase folding kinetics of cytochrome c are assessed in vitro. Upon electron transfer-triggered folding of reduced cytochrome c, far-UV time-resolved circular dichroism (TRCD) is used to monitor folding under different conditions. Earlier work has shown that folding of reduced cytochrome c from the guanidinium hydrochloride-induced unfolded ensemble in dilute phosphate buffer involves kinetic partitioning: one fraction of molecules folds rapidly, on a time scale identical to that of reduction, while the remaining population folds more slowly. In the presence of 220 mg/mL dextran 70, a synthetic macromolecular crowding agent that occupies space but does not interact with proteins, the population of the fast folding step for cytochrome c is greatly reduced. Increasing the viscosity with sucrose to the same microviscosity exhibited by the dextran solution showed no significant decrease in the amplitude of the fast-folding phase of cytochrome c. Experiments show that the unfolded-state heme ligation remains bis-His in the presence of dextran 70, but coarse-grained simulations suggest that the unfolded-state ensemble becomes more compact in the presence of crowders. We conclude that excluded volume effects alter unfolded cytochrome c such that access to fast-folding conformations is reduced.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
authors.forename: Eefei
authors.initials: E
authors.lastname: Chen
authors.forename: Alexander
authors.initials: A
authors.lastname: Christiansen
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Margaret S
authors.initials: MS
authors.lastname: Cheung
authors.forename: David S
authors.initials: DS
authors.lastname: Kliger
authors.forename: Pernilla
authors.initials: P
authors.lastname: Wittung-Stafshede
mesh_terms: D002942:Circular Dichroism; D045304:Cytochromes c / Q000737:chemistry*; D007700:Kinetics; D046911:Macromolecular Substances; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet
chemical_list: D046911:Macromolecular Substances; D045304:Cytochromes c