The role of backbone hydrogen bonds in the transition state for protein folding of a PDZ domain.

Søren W Pedersen,Greta Hultqvist,Per Jemth,Kristian Strømgaard

doi:10.1371/journal.pone.0095619

Søren W Pedersen, Greta Hultqvist + Show 2 more

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https://doi.org/10.1371/journal.pone.0095619

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Journal: PloS one	Publication Date: Apr 18, 2014
Citations: 45	License type: CC BY 4.0

Affiliation: University of Copenhagen, Uppsala University

Abstract

Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.

Highlights

Protein domains usually fold in a highly co-operative manner with concomitant formation of hundreds of non-covalent bonds
We designed and synthesized five different amide-to-ester mutants of PSD-95 PDZ2, L170l, G171c, F172Q, I174i and G176c as described [21]. Such backbone mutations remove a hydrogen bond formed by the NH of the amide and destabilize the hydrogen bond formed by the amide carbonyl oxygen [3,24]
In the case of PSD-95 PDZ2 [25,26], the amide NH of Phe172, Ile174 and Gly176 are posed for binding a peptide ligand and not directly involved in intra-domain hydrogen bonds

Summary

Introduction

Protein domains usually fold in a highly co-operative manner with concomitant formation of hundreds of non-covalent bonds. Most experimental analyses of folding transition states use the strategy of truncating side-chains of amino acid residues and probing the effect on protein folding kinetics as well as the stability at equilibrium (W value analysis) [2]. This approach provides information on the relative energetics of the interactions of the side-chains in the transition and ground states. Very few proteins have been subjected to this approach, for example WW domain variants, for which the role of hydrogen bonds for overall stability as well as folding mechanism was assessed [5,6]

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Conclusion