16 research outputs found
Folding kinetics of the lipoic acid-bearing domain of human mitochondrial branched chain α-ketoacid dehydrogenase complex
AbstractA reversible two-step (native state↔denatured state) folding mechanism based on equilibrium and stopped flow experiments is proposed for urea denaturation of the lipoyl-bearing domain (hbLBD) of human mitochondrial branched chain α-ketoacid dehydrogenase (BCKD) complex. The results from this circular dichroism (CD) and fluorescence study have ruled out populated kinetic or equilibrium intermediates on folding pathway of this β-barrel domain under experimental conditions. Both studies suggested mono-exponential kinetics without any burst phases. Moreover the thermodynamic parameters ΔGH2O and m obtained from the kinetic analysis are consistent with the equilibrium measurements
Predicting the viability of beta-lactamase: How folding and binding free energies correlate with beta-lactamase fitness.
One of the long-standing holy grails of molecular evolution has been the ability to predict an organism's fitness directly from its genotype. With such predictive abilities in hand, researchers would be able to more accurately forecast how organisms will evolve and how proteins with novel functions could be engineered, leading to revolutionary advances in medicine and biotechnology. In this work, we assemble the largest reported set of experimental TEM-1 β-lactamase folding free energies and use this data in conjunction with previously acquired fitness data and computational free energy predictions to determine how much of the fitness of β-lactamase can be directly predicted by thermodynamic folding and binding free energies. We focus upon β-lactamase because of its long history as a model enzyme and its central role in antibiotic resistance. Based upon a set of 21 β-lactamase single and double mutants expressly designed to influence protein folding, we first demonstrate that modeling software designed to compute folding free energies such as FoldX and PyRosetta can meaningfully, although not perfectly, predict the experimental folding free energies of single mutants. Interestingly, while these techniques also yield sensible double mutant free energies, we show that they do so for the wrong physical reasons. We then go on to assess how well both experimental and computational folding free energies explain single mutant fitness. We find that folding free energies account for, at most, 24% of the variance in β-lactamase fitness values according to linear models and, somewhat surprisingly, complementing folding free energies with computationally-predicted binding free energies of residues near the active site only increases the folding-only figure by a few percent. This strongly suggests that the majority of β-lactamase's fitness is controlled by factors other than free energies. Overall, our results shed a bright light on to what extent the community is justified in using thermodynamic measures to infer protein fitness as well as how applicable modern computational techniques for predicting free energies will be to the large data sets of multiply-mutated proteins forthcoming
Author Correction: Molecular mechanism of K65 acetylation-induced attenuation of Ubc9 and the NDSM interaction
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper
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COA6 is structurally tuned to function as a thiol-disulfide oxidoreductase in copper delivery to mitochondrial cytochrome c oxidase
In eukaryotes, cellular respiration is driven by mitochondrial cytochrome c oxidase (CcO), an enzyme
complex that requires copper cofactors for its catalytic activity. Insertion of copper into its catalytically
active subunits, including COX2, is a complex process that requires metallochaperones and redox proteins including SCO1, SCO2, and COA6, a recently
discovered protein whose molecular function is unknown. To uncover the molecular mechanism by
which COA6 and SCO proteins mediate copper delivery to COX2, we have solved the solution structure of
COA6, which reveals a coiled-coil-helix-coiled-coilhelix domain typical of redox-active proteins found
in the mitochondrial inter-membrane space. Accordingly, we demonstrate that COA6 can reduce the
copper-coordinating disulfides of its client proteins,
SCO1 and COX2, allowing for copper binding.
Finally, our determination of the interaction surfaces
and reduction potentials of COA6 and its client proteins provides a mechanism of how metallochaperone and disulfide reductase activities are coordinated to deliver copper to CcO.Fil: Soma, Shivatheja. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Morgada, Marcos N. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Morgada, Marcos N. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina.Fil: Naik, Mandar T. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Naik, Mandar T. Brown University. Department of Molecular Pharmacology, Physiology, and Biotechnology; United States.Fil: Boulet, Aren. University of Saskatchewan. Department of Biochemistry, Microbiology and Immunology; Canada.Fil: Roesler, Anna A. University of Saskatchewan. Department of Biochemistry, Microbiology and Immunology; Canada.Fil: Dziuba, Nathaniel. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Ghosh, Alok. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Ghosh, Alok. University of Calcutta. Department of Biochemistry; India.Fil: Yu, Qinhong. University of California. Department of Chemistry; United States.Fil: Lindahl, Paul A. Texas A&M University. Department of Biochemistry and Biophysics; United States.Fil: Lindahl, Paul A. Texas A&M University. Department of Chemistry; United States.Fil: Ames, James B. University of California. Department of Chemistry; United States.Fil: Leary, Scot C. University of Saskatchewan. Department of Biochemistry, Microbiology and Immunology; Canada.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Vila, Alejandro J. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Área Biofísica; Argentina.Fil: Gohil, Vishal M. Texas A&M University. Department of Biochemistry and Biophysics; United States