Mechanisms of chaperonin-assisted protein folding

Gardner, Scott David (2021) Mechanisms of chaperonin-assisted protein folding. PhD thesis, Birkbeck, University of London.

Abstract

The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used recent advances in cryoEM to determine cryoEM structures of GroEL, GroEL-ADP·BeFx, and GroEL-GroESADP·AlFx, all bound to the model substrate Rhodospirillum rubrum RuBisCO in a non-native state. The use of the Chameleon vitrification instrument reduced preferred orientation and air-water interface denaturation effects in cryoEM. Previous cryoEM reconstructions of GroEL-substrate complexes have been limited in resolution, and there are no previously reported structures of GroEL-ATP (or ATP analogue) bound to a non-native substrate. Our structures show how the conformation and interactions of a non-native substrate protein change as GroEL proceeds through its reaction cycle. We observe specific charged and hydrophobic GroEL residues forming strong initial contacts with non-native RuBisCO. Binding of an ATP analogue results in an enhanced interaction of substrate with the disordered C-terminal tails of GroEL. The cryoEM structure of GroEL-ADP·BeFx-RuBisCO displays striking asymmetry in the substrate-occupied ring. We observe individual GroEL subunits fulfilling different functions. Four of the GroEL subunits sequester non-native substrate inside the cavity while the remaining three adopt an extended conformation allowing GroEL to efficiently recruit GroES. Our structure explains how GroEL can recruit GroES without releasing bound substrate. The cryoEM structure of GroELGroES-RuBisCO shows a native-like RuBisCO interacting weakly with the walls of GroEL and the inner face of GroES. Additionally, the trans ring of our nucleotide bound structures show a novel conformation that may be related to the negative cooperativity of non-native RuBisCObinding.

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