Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli.

Rageia Elfageih,Renuka Kudva,Grant Kemp,Jan-Willem De Gier,Gunnar Von Heijne,Alexandros Karyolaimos

doi:10.1002/pro.3927

Rageia Elfageih, Renuka Kudva + Show 4 more

Open Access

https://doi.org/10.1002/pro.3927

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Abstract

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm.

Highlights

Our results suggest the existence of at least two disulfide-stabilized, cotranslational folding intermediates that form when ~330 and ~360 residues of mature PhoA have emerged into the periplasm
Force-profile analysis (FPA) as a tool to study protein folding in the periplasm
FPA is based on the principle that cotranslational folding of a protein domain fused an arrest peptides (APs) can generate a pulling force on the AP that reduces its stalling efficiency [44,45,46,47,48,49,50,51,52,53]

Summary

Introduction

Protein secretion across the inner membrane in the Gram-negative bacterium Escherichia coli has been classically thought to proceed post-translationally, orchestrated by an interplay between the signal peptide of the protein, cytoplasmic chaperones such as Trigger Factor and SecB, the ATPase SecA, and the SecYEG translocon [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17].Post-translational export commences when SecA recognizes a secretory protein via its signal sequence, and targets it to the inner membrane [18,19,20,21,22]. Our results suggest the existence of at least two disulfide-stabilized, cotranslational folding intermediates that form when ~330 and ~360 residues of mature PhoA have emerged into the periplasm.

Results

Conclusion