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

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances

Characterization of a cold-active esterase from Lactobacillus plantarum suitable for food fermentations

Lactobacillus plantarum is a lactic acid bacteria that can be found in numerous fermented foods. Esterases from L. plantarum exert a fundamental role in food aroma. In the present study, the gene lp-2631 encoding a putative esterase was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp-2631 protein has been biochemically characterized. Lp-2631 exhibited optimal esterase activity at 20 °C and more than 90% of maximal activity at 5 °C, being the first cold-active esterase described in a lactic acid bacteria. Lp-2631 exhibited 40% of its maximal activity after 2 h of incubation at 65 °C. Lp-2631 also showed marked activity in the presence of compounds commonly found in food fermentations, such as NaCl, ethanol, or lactic acid. The results suggest that Lp-2631 might be a useful esterase to be used in food fermentations. © 2014 American Chemical Society.This work was financially supported by grants AGL2011-22745 and BFU2010-17929 (MINECO), S2009/AGR-1469 (ALIBIRD) (Comunidad de Madrid), and RM2012-00004 (INIA). M. Esteban-Torres is a recipient of a JAE predoctoral fellowship from the CSIC.Peer Reviewe

Peptic fragments derived from α₁AT that display enhanced deuterium incorporation in M α₁AT.

<p>(A) The kinetics of deuterium incorporation into M α₁AT (black) and Z α₁AT (red) of peptic fragments which show significant increased deuterium uptake in M compared to Z α₁AT. Insert- Crystal structure of M α₁AT (PDB: 1QLP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102617#pone.0102617-Elliott1" target="_blank">[24]</a> indicating the location of peptic fragments with decreased exchange highlighted in blue. The individual data points are the average of three independent experiments for clarity the error bars are not shown.</p

The structure and sequence of α₁AT.

<p>(A) Ribbon diagram of M α₁AT (PDB: 1QLP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102617#pone.0102617-Elliott1" target="_blank">[24]</a> is shown and the peptic fragments used in this study are highlighted in red. The insert shows a close up view of the region around Glu342, the site of the Z mutation. Figures are prepared using PyMol (2002). The PyMOL Molecular Graphic System, San Caros, CA, U.S.A.).</p