Measuring protein stability in the GroEL chaperonin cage reveals massive destabilization.

Ilia Korobko,Gilad Haran,Hisham Mazal,Amnon Horovitz

doi:10.7554/elife.56511

Abstract

The thermodynamics of protein folding in bulk solution have been thoroughly investigated for decades. By contrast, measurements of protein substrate stability inside the GroEL/ES chaperonin cage have not been reported. Such measurements require stable encapsulation, that is no escape of the substrate into bulk solution during experiments, and a way to perturb protein stability without affecting the chaperonin system itself. Here, by establishing such conditions, we show that protein stability in the chaperonin cage is reduced dramatically by more than 5 kcal mol-1 compared to that in bulk solution. Given that steric confinement alone is stabilizing, our results indicate that hydrophobic and/or electrostatic effects in the cavity are strongly destabilizing. Our findings are consistent with the iterative annealing mechanism of action proposed for the chaperonin GroEL.

Highlights

The Escherichia coli GroE chaperonin system, which comprises GroEL and its co-factor GroES, assists protein folding in vivo and in vitro in an ATP-dependent manner (Thirumalai and Lorimer, 2001; Saibil et al, 2013; Hayer-Hartl et al, 2016; Gruber and Horovitz, 2016)
dihydrofolate reductase from Moritella profunda (DHFRMp) was fused to the C-terminus of enhanced green fluorescent protein in order to further destabilize it as observed before for other proteins (Sokolovski et al, 2015; Dave et al, 2016)
The apparent melting temperature, Tm, in bulk solution of DHFRMp alone was found to be about 41.2 (±1.8) ̊C whereas DHFRMp fused to enhanced green fluorescent protein (eGFP) was found to be strongly destabilized with a Tm of 22.8 (±1.1) ̊C (Figure 2). eGFP in the chimera was found to be destabilized in bulk solution relative to eGFP alone, but to a lesser extent, with Tm values of 76.3 (±0.3) and 81.3 (±1.3) ̊C, respectively

Summary

Introduction

The Escherichia coli GroE chaperonin system, which comprises GroEL and its co-factor GroES, assists protein folding in vivo and in vitro in an ATP-dependent manner (Thirumalai and Lorimer, 2001; Saibil et al, 2013; Hayer-Hartl et al, 2016; Gruber and Horovitz, 2016). The GroE system has been studied intensively for more than three decades, but it is still unclear and controversial whether its cavity is a ‘passive cage’ in which protein substrate aggregation is prevented but the folding pathway is unchanged or a chamber in which the folding process is altered in some manner. It is unclear whether encapsulation in the GroE cavity is thermodynamically stabilizing, for example because of confinement, or destabilizing owing, for example, to a diminished hydrophobic effect. GroEL-mediated folding can be affected by the cavity-

Methods

Results

Conclusion