Rational design of protein stability: effect of (2S,4R)-4-fluoroproline on the stability and folding pathway of ubiquitin.

Maria D Crespo,Marina Rubini,Nediljko Budisa

doi:10.1371/journal.pone.0019425

Maria D Crespo, Marina Rubini + Show 1 more

Open Access

https://doi.org/10.1371/journal.pone.0019425

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Journal: PloS one	Publication Date: May 16, 2011
Citations: 80	License type: CC BY 4.0

Affiliation: ETH Zurich, University of Konstanz

Abstract

BackgroundMany strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a Cγ-exo or a Cγ-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying Cγ-exo puckering.Methodology/Principal FindingsWhile (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the Cγ-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of −4.71 kJ·mol−1 in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub.Conclusions/SignificanceThe results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein.

Highlights

As conformationally restricted amino acid, proline adopts numerous structural and regulatory functions in protein structures
The substitution of all three prolines with its analogs was achieved by the traditional use of the auxotrophic E. coli bacterial strain JM83 that is deficient in proline biosynthesis
ESIMS analyses confirmed that the analog (4R)-FPro was successfully incorporated into human ubiquitin in response to the two proline codons CCG and CCT in the sequence of the ubiquitin gene

Summary

Introduction

As conformationally restricted amino acid, proline adopts numerous structural and regulatory functions in protein structures. The proline ring pucker exerts an effect on the main chain torsion angles; the biasing of a single conformer enables to rationally manipulate the protein backbone conformation by preorganization, with concomitant effects on protein stability. The fluorinated pyrrolidine rings of proline can preferably stabilize either a Cc-exo or a Cc-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying Cc-exo puckering

Methods

Results

Conclusion