Rational Design of Protein Stability: Effect of (2S,4R)-4-Fluoroproline on the Stability and Folding Pathway of Ubiquitin

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.

4-Fluoroprolines: Conformational Analysis and Effects on the Stability and Folding of Peptides and Proteins

“…Ubiquitin has three native proline residues that were replaced with 4-fluoroproline by using the SPI method to study the influence of proline ring pucker on protein folding [112]. Ubiquitylation serves as a key post-translational modification with a wide variety of cellular effects, notably targeting proteins for proteasomal degradation [113].…”

300-Fold Increase in Production of the Zn 2+ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

“…The technique of laboratory evolution has great potential for improving soluble-protein production and involves subjecting the protein to random mutagenesis and recombination, followed by screening of the many variants that are produced for enhanced expression (9,10). Although there have been a few successful examples of rationally engineering proteins to enhance expression of the soluble form (11), our limited understanding of protein folding and stability currently imposes a limit on this approach. Thus, a simple means by which mutations that increase expression can be identified would be very useful.…”

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

“…27 In contrast, in KlenTaq DNA polymerase and human ubiquitin no expression at all could be detected with the disfavored isomer. 134,135 Thus, not only folding but also biosynthesis of proteins might be affected by the different stereochemistry of the fluorinated proline residue.…”