Back to GroEL-Assisted Protein Folding: GroES Binding-Induced Displacement of Denatured Proteins from GroEL to Bulk Solution

Natalia Marchenko,Gennady Semisotnov,Victor Marchenkov,Andrey Gorokhovatsky,Tanya Ivashina

doi:10.3390/biom10010162

Abstract

The main events in chaperone-assisted protein folding are the binding and ligand-induced release of substrate proteins. Here, we studied the location of denatured proteins previously bound to the GroEL chaperonin resulting from the action of the GroES co-chaperonin in the presence of Mg-ATP. Fluorescein-labeled denatured proteins (α-lactalbumin, lysozyme, serum albumin, and pepsin in the presence of thiol reagents at neutral pH, as well as an early refolding intermediate of malate dehydrogenase) were used to reveal the effect of GroES on their interaction with GroEL. Native electrophoresis has demonstrated that these proteins tend to be released from the GroEL-GroES complex. With the use of biotin- and fluorescein-labeled denatured proteins and streptavidin fused with luciferase aequorin (the so-called streptavidin trap), the presence of denatured proteins in bulk solution after GroES and Mg-ATP addition has been confirmed. The time of GroES-induced dissociation of a denatured protein from the GroEL surface was estimated using the stopped-flow technique and found to be much shorter than the proposed time of the GroEL ATPase cycle.

Highlights

Studies of protein refolding reactions mainly confirm Anfinsen’s hypothesis that the necessary and sufficient information on the spatial structure of proteins is encoded in their amino acid sequences [1,2,3]
The electron microscopy [9,10] and crystallography [11,12] data show that 14 identical subunits (57 kDa each) of the GroEL chaperonin are arranged into two stacked heptameric toroids forming a cylinder with a central inner cavity of 45 Å in diameter
The studies of GroEL and GroES structural and functional properties have been being in progress for nearly thirty years, but the main question as to how these chaperonins assist the folding of a broad variety of proteins remains unanswered, many aspects of this problem have been solved

Summary

Introduction

Studies of protein refolding reactions mainly confirm Anfinsen’s hypothesis that the necessary and sufficient information on the spatial structure of proteins is encoded in their amino acid sequences [1,2,3]. A number of cellular proteins have been revealed to be required for the formation of the protein native conformation [4,5] Many of these proteins, named molecular chaperones [4], are members of a large group of heat shock proteins (hsps), whose biosynthesis in the cell is enhanced by various cellular stresses [6]. Molecular chaperones facilitate the folding of various proteins both in vivo and in vitro by interacting transiently and non-covalently with non-native early or late folding intermediates and not with the native (rigidly packed) protein. This interaction prevents aggregation of the intermediates or, perhaps, repairs their misfolded conformations. The electron microscopy [9,10] and crystallography [11,12] data show that 14 identical subunits (57 kDa each) of the GroEL chaperonin are arranged into two stacked heptameric toroids forming a cylinder with a central inner cavity of 45 Å in diameter

Methods

Results

Discussion

Conclusion