Journal of Molecular Biology
Volume 432, Issue 20, 18 September 2020, Pages 5649-5664
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GROEL/ES Buffers Entropic Traps in Folding Pathway during Evolution of a Model Substrate

https://doi.org/10.1016/j.jmb.2020.08.015Get rights and content

Highlights

  • Made an expression-controlled GFP library to study chaperone-dependence in vivo.

  • Simulated a single step of molecular evolution toward GroEL/ES dependence in vivo.

  • GroEL/ES can prevent non-native contacts and take protein out of entropic traps.

  • The model substrate in vivo use GroEL/ES’ capacity to remove entropic traps.

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.

Introduction

Mutations in a protein sequence may subtly change either thermodynamics of the folding polypeptide or protein-solvent interactions. In vivo, mutations that arise spontaneously may lead to problems in the folding pathway or stability of proteins. This, in turn, may make the proteins either non-functional or dependent on molecular chaperones for folding [1,2].

In Escherichia coli, the most abundant cytosolic chaperone systems consist of the Hsp70 system (DnaK/DnaJ/GrpE), the Hsp60 system (GroEL/GroES), Hsp90 (HtpG), Trigger Factor (Tig) and SecB along with other less abundant chaperones and small heat shock proteins. Properties of the substrates assisted by these chaperone systems have been explored by multiple groups [[3], [4], 5., [6], [7], [8]]. While the DnaK system binds to many proteins and has the potential to stabilize thermosensitive proteome [9], GroEL/ES binds and helps in the folding of a much smaller subset of cellular proteins [6,10]. The mechanism of substrate targeting to the right chaperone is an active field of research underlining the significance of the conformations adopted by proteins in their non-folded states [11,12].

While canonical chaperone-targeting is important for chaperone-dependent wild-type proteins, the accumulation of mutations during evolution may create additional substrates requiring chaperone assistance. Can we predict the type of mutations on a GroEL/ES independent protein that would make it GroEL/ES dependent? Mechanistically, some mutations may increase the propensity of formation of non-native contacts (intramolecular or intermolecular) that need to melt before the protein can fold to its native state (enthalpic trap) while other mutations may increase the flexibility of the non-native states (entropic trap), both of which are folding problems at opposite ends of the spectrum. Do different chaperone systems differ in their capacity to edit the two types of mutations?

GroEL/ES system is capable of removing the entropic trap in its substrates [13,14]. It has also been shown to unfold proteins [15] and remove enthalpic traps in the folding pathway in vitro. Does the chaperonin possess both these activities in vivo? When proteins accumulate mutations, which type of mutations would be preferentially accommodated by GroEL/ES? This is difficult to answer with the previous model substrates as neither the authentic substrates (which precludes understanding the mutational steps that led to its chaperone dependence) nor the slow-folding model substrates (that were not evolved for GroEL/ES dependence in vivo in an unbiased manner) gave us the handle to quantitate folding assistance by GroEL/ES in vivo. Although, Horovitz group made advances in this direction by identifying that mutations in frustrated sequence regions lead to GroEL/ES dependence in vivo, but the biophysical consequence of these mutations on the folding landscape was not investigated [16].

To address this, we sought an unbiased screen using mutagenesis to obtain an in vivo GroEL/ES substrate starting from a spontaneously folding GroEL/ES-independent substrate. We show that the identified substrate is exclusively dependent upon the GroEL/ES system in vivo and in vitro. We find that the unique mutation present in the pool of GroEL/ES dependent protein resulted in an entropic trap in folding that is corrected by GroEL/ES system. We also show that DnaK/DnaJ/GrpE (KJE) system or the GroEL chaperone can take care of enthalpic traps effectively in vitro in an ATP dependent manner. This function of GroEL is essential for folding the substrate protein in vivo. Thus, we posit that the proteins that acquire entropic traps during evolution would be assisted by GroEL/ES system in vivo. While GroEL/ES also possesses the capability to edit folding landscape by preventing the formation of non-native contacts, this chaperoning capacity is not exclusive to GroEL/ES but is also shared by more abundant KJE system.

Section snippets

Isolation of a synthetic GroEL-dependent substrate

A single mutational step may make a spontaneously folding protein chaperone-dependent during evolution. To learn the physico-chemical basis of the mutational step that confers chaperone-dependence, we wanted to mimic this evolutionary step and develop a synthetic substrate dependent on the canonical chaperone GroEL/ES in vivo from a GroEL/ES-independent protein. Comparison of the mutant and Wt protein would help in understanding the type of folding problems that GroEL/ES tends to edit, and the

Strains, Plasmids, and Proteins

E. coli strain DH5α was used for cloning, WT E. coli K-12 (BW25113) strain was used for expression of arabinose inducible pBAD GFP and BL21 (DE3) was used for protein expression and purification. Protein concentrations were determined spectrophotometrically at 562 nm using BCA kit (Pierce-ThermoFisher Scientific). Deletion strains were obtained from CGSC as part of Keio collection [28].

Construction of mutant GFP library

Mutant GFP library was made in arabinose inducible pBAD vector using a random mutagenesis approach by

Acknowledgment

We thank Prof. Hideki Taguchi for the kind gift of pET21 SR1 plasmid. We also thank Kanika Saxena for sharing the starting plasmid with GFP and mCherry. K.M. acknowledges the funding from the Department of Biotechnology (DBT), Government of India (grant number BT/PR28386/BRB/10/1671/2018) and Science and Engineering Research Board (SERB), Government of India, for Core Research Grant (SERB/CRG/2019/006281) and SNU core funding. The work in KC Lab was supported by Swarnajayanthi Fellowship Grant

Data and Software Availability

All data are provided in the manuscript. We did not develop any new software.

Author contribution

Conceptualization: Koyeli Mapa; Supervision: Koyeli Mapa, Kausik Chakraborty; Reagent generation: Satyam Tiwari, Kanika Verma, Anwar Sadat, Aseem Chaphalkar; Experiments: Anwar Sadat, Satyam Tiwari, Kanika Verma, Mudassar Ali, Vaibhav Upadhyay, Anupam Singh, Aseem Chaphalkar, Asmita Ghosh, Rahul Chakraborty, Kausik Chakraborty; Analysis: Arjun Ray (computational), Anwar Sadat (biophysics), Satyam Tiwari (biophysics), Kanika Verma (FACS), Rahul Chakraborty (MS), Kausik Chakraborty, Koyeli Mapa;

Declarations of Interest

The authors declare no competing interests.

Key Resource Table

Reagent or resourceSourceIdentifier
Antibodies
 Rabbit anti-GFPAbcamAB290
Bacterial and virus strains
 E. coli BL21 (DE3)
 E. coli DH5α
 E. coli WT (K-12, BW25113)
F-, DE(araD-araB)567, lacZ4787(del)::rrnB-3, LAM-, rph-1, DE(rhaD-rhaB)568, hsdR514
[28]CGSC#: 7636
  Tig (JW0426–1)
F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), Δtig-722::kan, λ-, rph-1, Δ(rhaD-rhaB)568, hsdR514
[28]CGSC#: 8589
  dnaK (JW0013–4)
F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, rph-1, ΔdnaK734::kan, Δ(rhaD-rhaB)568, hsdR514
[28

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