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Design of proteins with desired thermal properties is important for scientific and biotechnological applications. Here we developed a theoretical approach to predict the effect of mutations on protein stability from non-equilibrium unfolding simulations. We establish a relative measure based on apparent simulated melting temperatures that is independent of simulation length and, under certain assumptions, proportional to equilibrium stability, and we justify this theoretical development with extensive simulations and experimental data. Using our new method based on all-atom Monte-Carlo unfolding simulations, we carried out a saturating mutagenesis of Dihydrofolate Reductase (DHFR), a key target of antibiotics and chemotherapeutic drugs. The method predicted more than 500 stabilizing mutations, several of which were selected for detailed computational and experimental analysis. We find a highly significant correlation of r = 0.65–0.68 between predicted and experimentally determined melting temperatures and unfolding denaturant concentrations for WT DHFR and 42 mutants. The correlation between energy of the native state and experimental denaturation temperature was much weaker, indicating the important role of entropy in protein stability. The most stabilizing point mutation was D27F, which is located in the active site of the protein, rendering it inactive. However for the rest of mutations outside of the active site we observed a weak yet statistically significant positive correlation between thermal stability and catalytic activity indicating the lack of a stability-activity tradeoff for DHFR. By combining stabilizing mutations predicted by our method, we created a highly stable catalytically active E. coli DHFR mutant with measured denaturation temperature 7.2°C higher than WT. Prediction results for DHFR and several other proteins indicate that computational approaches based on unfolding simulations are useful as a general technique to discover stabilizing mutations. 相似文献
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Julianne H. Grose Kelsey Langston Xiaohui Wang Shayne Squires Soumyajit Banerjee Mustafi Whitney Hayes Jonathan Neubert Susan K. Fischer Matthew Fasano Gina Moore Saunders Qiang Dai Elisabeth Christians E. Douglas Lewandowski Peipei Ping Ivor J. Benjamin 《PloS one》2015,10(10)
Small Heat Shock Proteins (sHSPs) are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2), which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait) and a human cardiac library (prey) coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 “cardiac interactome” to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID). A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is tempting to speculate that small molecule activators of HspB2 might be deployed to mitigate mitochondrial related diseases such as cardiomyopathy and neurodegenerative disease. 相似文献
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Whitney E. Boland Emily DeCrescenzo Henriksen Joy Doran-Peterson 《Applied and environmental microbiology》2010,76(17):6006-6009
Two pectate lyases were identified from Paenibacillus amylolyticus 27C64; both enzymes demonstrated activity on methylated pectin in addition to polygalacturonic acid. PelA is in a subclass of the pectate lyase family III. PelB shows some features of pectate lyase family I but is highly divergent.Pectinases have many industrial applications, including uses in food and textile production (9, 12). Additionally, pectinases are important for the degradation of biomass, where pectin can comprise a significant portion of plant structure (5, 6). The degradation of pectin requires methylesterases and depolymerases. Pectin methylesterases are responsible for the hydrolysis of methylester linkages from the polygalacturonic acid (PGA) backbone (24), while pectin depolymerases act upon the polygalacturonate backbone and belong to one of two families, polygalacturonases or lyases. Polygalacturonases hydrolytically cleave the polygalacturonate chain, while lyases cleave by β-elimination, giving a Δ4,5-unsaturated product (10, 19). There are two types of lyases: pectate lyases (PLs), which cleave unesterified polygalacturonate, and pectin lyases, which cleave methylesterified pectin.Paenibacillus amylolyticus strain 27C64, isolated from the larval hindgut of the aquatic crane fly, Tipula abdominalis, possesses a wide range of lignocellulose-degrading enzymes. This study describes two pectate lyases from P. amylolyticus that display unusual activity by combining traits of pectate and pectin lyases (2, 7, 21, 22). 相似文献
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Rapid extraction of mean emotion and gender from sets of faces 总被引:1,自引:0,他引:1