GROEL/ES Buffers Entropic Traps in Folding Pathway during Evolution of a Model Substrate

Abstract

The folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen.We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and entropic traps in the folding pathway of the substrate protein.

Conserved and divergent chaperoning effects of Hsp60/10 chaperonins on protein folding landscapes

“…S3 B ). In the presence of ATP and its cochaperone, GroES, GroEL accelerated the refolding rate of sGFP as reported earlier ( 16 ) ( Fig. 3 B ).…”

“…sGFP fluorescence increased with GroEL/ES expression as reported ( Fig. 3 A ) ( 16 ). This increase was more prominent upon overexpression of cfl-EL/ES, the more negatively charged GroEL/ES homolog ( Fig.…”

“…To check if these chaperonins differ in their ability to assist the folding of GroEL/ES substrates in vivo, we used sGFP and MetK. sGFP is a recently described model substrate of GroEL/ES that can quantitatively report the ability of chaperonin assistance in vivo and in vitro ( 16 ), and MetK is a known endogenous substrate of GroEL/ES ( 7 ). Additionally, the folding landscape of sGFP in the presence and absence of GroEL/ES has been well characterized, providing us with a tool to understand the differences between the chaperonins in terms of their ability to change the folding landscape of its substrates ( 16 ).…”

“…sGFP is a slow folding mutant of yeGFP that is rapidly degraded in vivo ( 16 ). With GroEL/ES overexpression, the protein folds efficiently and shows enhanced activity (fluorescence) in vivo.…”

“…Error bars are SDs of three independent experiments. $ Dashed bar for spontaneous folding is from published parameters ( 16 ). ( F ) The calculated ρ values obtained from Arrhenius fitting of sGFP refolding shown in E plotted against centroid charge density for different chaperonins.…”