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Corwin Miller Milya Davlieva Corey Wilson Rafael Couñago Jeffrey C. Myers Yousif Shamoo 《Biophysical journal》2010,99(3):887-896
Success in evolution depends critically upon the ability of organisms to adapt, a property that is also true for the proteins that contribute to the fitness of an organism. Successful protein evolution is enhanced by mutational pathways that generate a wide range of physicochemical mechanisms to adaptation. In an earlier study, we used a weak-link method to favor changes to an essential but maladapted protein, adenylate kinase (AK), within a microbial population. Six AK mutants (a single mutant followed by five double mutants) had success within the population, revealing a diverse range of adaptive strategies that included changes in nonpolar packing, protein folding dynamics, and formation of new hydrogen bonds and electrostatic networks. The first mutation, AKBSUB Q199R, was essential in defining the structural context that facilitated subsequent mutations as revealed by a considerable mutational epistasis and, in one case, a very strong dependence upon the order of mutations. Namely, whereas the single mutation AKBSUB G213E decreases protein stability by >25°C, the same mutation in the background of AKBSUB Q199R increases stability by 3.4°C, demonstrating that the order of mutations can play a critical role in favoring particular molecular pathways to adaptation. In turn, protein folding kinetics shows that four of the five AKBSUB double mutants utilize a strategy in which an increase in the folding rate accompanied by a decrease in the unfolding rate results in additional stability. However, one mutant exhibited a dramatic increase in the folding relative to a modest increase in the unfolding rate, suggesting a different adaptive strategy for thermostability. In all cases, an increase in the folding rates for the double mutants appears to be the preferred mechanism in conferring additional stability and may be an important aspect of protein evolution. The range of overlapping as well as contrasting strategies for success illustrates both the power and subtlety of adaptation at even the smallest unit of change, a single amino acid. 相似文献
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Milya Davlieva James Donarski Jiachen Wang Yousif Shamoo Edward P. Nikonowicz 《Nucleic acids research》2014,42(16):10795-10808
Several protein-targeted RNA aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. We examined the structural accommodation of an RNA aptamer that binds bacterial r-protein S8. The core of the primary binding site for S8 on helix 21 of 16S rRNA contains a pair of conserved base triples that mold the sugar-phosphate backbone to S8. The aptamer, which does not contain the conserved sequence motif, is specific for the rRNA binding site of S8. The protein-free RNA aptamer adopts a helical structure with multiple non-canonical base pairs. Surprisingly, binding of S8 leads to a dramatic change in the RNA conformation that restores the signature S8 recognition fold through a novel combination of nucleobase interactions. Nucleotides within the non-canonical core rearrange to create a G-(G-C) triple and a U-(A-U)-U quartet. Although native-like S8-RNA interactions are present in the aptamer-S8 complex, the topology of the aptamer RNA differs from that of the helix 21-S8 complex. This is the first example of an RNA aptamer that adopts substantially different secondary structures in the free and protein-bound states and highlights the remarkable plasticity of RNA secondary structure. 相似文献
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The structure of the trimeric adenylate kinase from the Archaebacteria Methanococcus mariplaludis (AKMAR) has been solved to 2.5‐Å resolution and the temperature dependent stability and kinetics of the enzyme measured. The KM and Vmax of AKMAR exhibit only modest temperature dependence from 30°–60°C. Although M. mariplaludis is a mesophile with a maximum growth temperature of 43°C, AKMAR has a very broad functional range and stability (Tm = 74.0°C) that are more consistent with a thermophilic enzyme with high thermostability and exceptional activity over a wide range of temperatures, suggesting that this microbe may have only recently invaded a mesophilic niche and has yet to fully adapt. A comparison of the Local Structural Entropy (LSE) for AKMAR to the related adenylate kinases from the mesophile Methanococcus voltae and thermophile Methanococcus thermolithotrophicus show that changes in LSE are able to fully account for the intermediate stability of AKMAR and highlights a general mechanism for protein adaptation in this class of enzymes. Proteins 2010. © 2009 Wiley‐Liss, Inc. 相似文献
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Ulrich Strych Milya Davlieva Joseph P Longtin Eileen L Murphy Hookang Im Michael J Benedik Kurt L Krause 《BMC microbiology》2007,7(1):1-7
Background
Over the past fifteen years, antibiotic resistance in the Gram-positive opportunistic human pathogen Streptococcus pneumoniae has significantly increased. Clinical isolates from patients with community-acquired pneumonia or otitis media often display resistance to two or more antibiotics. Given the need for new therapeutics, we intend to investigate enzymes of cell wall biosynthesis as novel drug targets. Alanine racemase, a ubiquitous enzyme among bacteria and absent in humans, provides the essential cell wall precursor, D-alanine, which forms part of the tetrapeptide crosslinking the peptidoglycan layer.Results
The alanine racemases gene from S. pneumoniae (alr SP ) was amplified by PCR and cloned and expressed in Escherichia coli. The 367 amino acid, 39854 Da dimeric enzyme was purified to electrophoretic homogeneity and preliminary crystals were obtained. Racemic activity was demonstrated through complementation of an alr auxotroph of E. coli growing on L-alanine. In an alanine racemases photometric assay, specific activities of 87.0 and 84.8 U mg-1 were determined for the conversion of D- to L-alanine and L- to D-alanine, respectively.Conclusion
We have isolated and characterized the alanine racemase gene from the opportunistic human pathogen S. pneumoniae. The enzyme shows sufficient homology with other alanine racemases to allow its integration into our ongoing structure-based drug design project. 相似文献6.
Rafael M Cou?ago Milya Davlieva Ulrich Strych Ryan E Hill Kurt L Krause 《BMC structural biology》2009,9(1):53
Background
Bacillus anthracis is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of B. anthracis (Ames) alanine racemase (Alr Bax ), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native Alr Bax structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation. 相似文献7.
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Jannell V. Bazurto Dipti D. Nayak Tomislav Ticak Milya Davlieva Jessica A. Lee Chandler N. Hellenbrand Leah B. Lambert Olivia J. Benski Caleb J. Quates Jill L. Johnson Jagdish Suresh Patel F. Marty Ytreberg Yousif Shamoo Christopher J. Marx 《PLoS biology》2021,19(5)
Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.The known formaldehyde stress response systems involve enzymatic detoxification. Here, the authors show that the formaldehyde sensor efgA plays an important role in the endogenous formaldehyde stress response in Methylorubrum extorquens, halting cell growth in response to elevated levels of formaldehyde, and is found almost exclusively in methylotrophic taxa. 相似文献
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