The Effect of Proline cis-trans Isomerization on the Folding of the C-Terminal SH2 Domain from p85.

Francesca Troilo,Francesca Malagrinò,Angelo Toto,Lorenzo Visconti,Stefano Gianni

doi:10.3390/ijms21010125

Abstract

SH2 domains are protein domains that modulate protein–protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.

Highlights

A detailed description of the mechanism by which a polypeptide chain achieves its functionally active conformation is one of the central problems of protein science [1,2,3,4,5,6]
In the case of the SH2 domains, because a proline in the cis conformation is present in the native state, folding appears to be rather complex
We recently observed that the N-terminal SH2 domain from p85 presents a highly peculiar chevron, with the folding and unfolding arms showing a clear mismatch and the kobs obtained from refolding experiments being at least 1 order of magnitude lower than those expected from unfolding experiments [27]

Summary

Introduction

A detailed description of the mechanism by which a polypeptide chain achieves its functionally active conformation is one of the central problems of protein science [1,2,3,4,5,6] It is well-established that aberrant folding into non-native misfolded conformations may lead to potentially devastating events that lead to severe human pathologies [7,8,9]. Protein misfolding is initiated by the transient accumulation of non-native contacts These interactions may either spontaneously break, with the protein folding towards its native conformation, or drag the polypeptide chain towards long-lived misfolded conformations that may potentially lead to aggregation [7,8,9]. Because of the links between aggregation and disease, the study of misfolding phenomena is of particular importance and much experimental effort has been devoted to understanding them

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Methods

Conclusion