title: Rational design of protein stability: effect of (2S,4R)-4-fluoroproline on the stability and folding pathway of ubiquitin.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Institute of Molecular Biology and Biophysics, ETH-Hönggerberg, Zürich, Switzerland.
authors.forename: Maria D
authors.initials: MD
authors.lastname: Crespo
authors.forename: Marina
authors.initials: M
authors.lastname: Rubini
mesh_terms: D002942:Circular Dichroism; D015195:Drug Design; D005462:Fluorine Radioisotopes / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology; D011487:Protein Conformation; D017510:Protein Folding / Q000187:drug effects*; D055550:Protein Stability / Q000187:drug effects*; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005462:Fluorine Radioisotopes; D025801:Ubiquitin; C431803:fluoro-proline; D011392:Proline