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

BACKGROUND: Many 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 C(γ)-exo or a C(γ)-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 C(γ)-exo puckering. METHODOLOGY/PRINCIPAL FINDINGS: While (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 C(γ)-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/SIGNIFICANCE: The 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

<i>n</i>→π* interactions in proteins

Hydrogen bonds between backbone amides are common in folded proteins. Here, we show that an intimate interaction between backbone amides likewise arises from the delocalization of a lone pair of electrons (n) from an oxygen atom to the antibonding orbital (π*) of the subsequent carbonyl group. Natural bond orbital analysis predicted significant n→π* interactions in certain regions of the Ramachandran plot. These predictions were validated by a statistical analysis of a large, non-redundant subset of protein structures determined to high resolution. The correlation between these two independent studies is striking. Moreover, the n→π* interactions are abundant, and especially prevalent in common secondary structures such as α-, 3(10)-, and polyproline II helices, and twisted β-sheets. In addition to their evident effects on protein structure and stability, n→π* interactions could play important roles in protein folding and function, and merit inclusion in computational force fields