Browsing by Subject "Protein Folding"
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Item Biophysical and Biochemical Characterization of a REC Domain: Unfolded to Folded Transition of EL_LovR(2014-08-18) Ocasio, Victor J.; Hendrixson, David R.; Gardner, Kevin H.; Sperandio, Vanessa; Rizo-Rey, JoséProkaryotes frequently use two component systems to couple environmental stimuli to adaptive responses. These pathways use histidine kinases to detect environmental cues, harnessing these to control phosphorylation of the receiver domain of the response regulator, which convert this signal into a physiological response. Knowledge of how phosphorylation shifts receiver domains between their inactive and active states is limited, chiefly assembled from several prototypical receiver domains that switch between two similar and well-folded structures. However, it remains unclear how general these observations apply to other receiver domains, particularly for full-length proteins. Here we present a blue light-regulated two-component system from the marine α-proteobacterium Erythrobacter litoralis HTCC2594. The sensor domain of the 3 histidine kinases found in E. litoralis contain a LOV (Light-Oxygen-Voltage) domain, part of the widely used PAS (Per-ARNT-Sim) family of environmental sensors. Interestingly, one of the histidine kinases (EL362) contains a naturally occurring glycine to arginine mutation in the LOV domain that prevents chromophore binding, resulting in a "blind" histidine kinase. Reverting the arginine to a glycine residue allows blue light to trigger the autophosphorylation of EL362 and subsequent phosphotransfer towards the cognate response regulator EL_LovR. This arrangement of RRs is reminiscent of similar systems used in other bacterial general stress responses, most of which have been characterized entirely with genetic methods. Notably, EL_LovR is a single domain response regulator proposed to play a critical role in shutting off such systems via a potent phosphatase activity. Size exclusion chromatography, light scattering and NMR experiments show that phosphorylation and Mg(II) transitions EL_LovR between unfolded and folded monomeric states. Parallel functional assays show that EL_LovR has a fast dephosphorylation rate, consistent with its proposed function as a phosphate sink. Taken together, our findings provide evidence that EL_LovR undergoes drastic conformational changes that have not been seen in other response regulators, likely with effects on its autophosphatase activity. In conclusion, our work expands the kinds of conformational changes and regulation used by receiver domains, critical components of bacterial signaling systems.Item The CFTR Folding Pathway: Implications for the Identification and Development of CF Therapeutics(2012-07-20) Mendoza, Juan Luis; Thomas, Philip J.The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is a member of the ABC transporter superfamily, important for Cl- conductance at the apical cell membrane. Loss-of-function of CFTR leads to Cystic Fibrosis (CF), a fatal genetic disease affecting 70,000 people worldwide. There are hundreds of CF causing mutations with the most common being ΔF508, present in at least one allele in 90% of CF patients. CFTR, comprising of 1480 amino acids, folds into five domains important for forming the channel through the membrane, and the regulation of channel function. F508 is located in Nucleotide Binding Domain 1 (NBD1) and is predicted to be at the interface with Intracellular Loop 4 (ICL4) of Transmembrane Domain 2 (TMD2). Studies of the isolated NBD1 demonstrate that the ΔF508 mutation impacts the folding pathway and stability of the domain. Misfolding of NBD1 contributes to the trafficking defect of the intact protein and subsequent loss-of-function. Conversely, second-site suppressor mutations, which more than compensate for defects of the mutant NBD1 domain, only partially rescue CFTR trafficking, suggesting that the deletion also affects other steps along the folding pathway. The aim of this work was to identify positions in CFTR critical for defining the folding pathway. We used a computational approach and two in vitro folding assays to monitor folding of the isolated NBD1 domain and trafficking of full-length CFTR. These data establish a correlation between the folding of the isolated NBD1 domain and maturation of full-length CFTR. Further, NBD1 second-site suppressor mutations in the ΔF508, F508K (NBD1/ICL4 interface disrupting mutation), and R1070W (ΔF508 NBD1/ICL4 interface stabilizing mutation) backgrounds suggest that ΔF508 CFTR is defective in two steps of CFTR biogenesis: 1) stability and efficiency of folding of the NBD1 domain, and 2) NBD1/ICL4 docking. We demonstrate that efficient rescue of ΔF508 CFTR requires correction the two distinct defects. This work has implications for the discovery and development of CF therapeutics by providing a framework for understanding the observed ceiling in the efficacy of either suppressor mutations or corrector compounds, which likely correct a single defect.Item Cotranslational Folding of CFTR(2013-05-31) Patrick, Anna Elizabeth; Thomas, Philip J.; Albanesi, Joseph P.; Gardner, Kevin H.; Jiang, YouxingThe life of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the cell is dictated by its biogenesis, cellular trafficking, regulated function, and destruction. Cystic fibrosis (CF) is the direct result of perturbations in these processes. Treatment of CF mandates the understanding of the molecular events leading to CFTR loss-of-function. The mechanisms by which different mutations throughout the CFTR protein result in misfolding are unclear. The correction of these processes and ultimately the treatment of CF require elucidation of these mechanisms. In this report, CF-causing mutations in the first transmembrane (TM) spanning domain (TMD1) that result in CFTR misfolding are examined. First, the G85E and G91R mutations in TM1 are shown to have different molecular pathologies. For G85E, TM1 is destabilized in the membrane by the ionizable side chain, which correlates with temperature insensitive ER accumulation. By contrast, G91R does not destabilize TM1, which correlates with temperature sensitive ER accumulation. Both mutants were then identified to perturb TMD1 in a manner recognizable by the cell. Finally, consistent with propagation of these defects, all multidomain CFTR constructs were recognized and degraded in the cell. Other mutations in the interdomain interface between TMD1 and the cytosolic nucleotide binding domains (NBDs) did not perturb TMD1, but affected multidomain constructs containing four domains, which can traffic from the ER. Notably, the interface mutants that change a hydrophobic residue to a basic residue increased levels of early multidomain constructs, suggesting a tradeoff between transient stability and later formation of interdomain interactions. The major cellular monitoring of most mutants occurs after TMD2 is present. In most current models, CF-causing mutations like ?F508 are shown to perturb interdomain interactions before TMD2 is produced. However, evidence presented here suggests these interactions are not important until after TMD2 production. The comparison of TM1 mutants and other mutants supports specific domain interactions in the hierarchical folding model. Taken together, the data herein generate a model of CFTR folding that begins with TMD1. Interdomain interactions then become important in a four domain construct, and the final domain confers additional stability and increases cellular trafficking. Multidomain misfolding clearly plays a role in the molecular pathology of CF, thus, a more detailed understanding of this process as globally outlined above is required to generate novel ways to rescue mutant CFTR.Item A New Approach to Optimize a Protein Energy Function on a Folding Pathway Using Gō-Like Potential and All-Atom, Ab Initio Monte Carlo Simulations(2016-01-19) Safronova, Aleksandra; Goldsmith, Elizabeth J.; Grishin, Nick V.; Otwinowski, Zbyszek; Rice, Luke M.Prediction of a protein structure is important for understanding the function of a protein. The process of protein structure prediction employs the approximation of a protein free energy that guides protein folding to the protein's native state. A function with a good approximation of the protein free energy should allow estimation of the structural distance of the evaluated candidate structure to the protein native state. Currently the energy optimization process relies on the correlation between the energy and the similarity to the native structure. The energy function is presented as a weighted sum of components which are designed by human experts with the use of statistical analysis of solved protein strictures. Values of the weights are derived through the procedure that maximizes the correlation between the energy and the similarity to the native structure measured by a root mean square deviation between coordinates of the protein backbone. Two major components are required for a successful ab initio modelling: (1) an effective energy function that discriminates the native protein structure out of all possible decoy structures; (2) an efficient sampling algorithm that quickly searches for the low-energy states. In this dissertation a new method for energy optimization is proposed. The method relies on a fast sampling algorithm and targets successful protein folding. The weights for energy components are optimized on a found with the Gō potential energy fast folding pathway. The Lennard-Jones potential, the Lazaridis-Karplus solvation potential, hydrogen bonding potential are used in the optimization algorithm. The optimized weights successfully predict all α and α/β proteins. The proposed strategy is conceptually different from the existing methods that optimize the energy on solved protein structures. The developed algorithm is a novel concept that allows the optimization of a more complex functional combination of the energy components that would improve the prediction quality.Item [News](1989-01-18) West, MikeItem [Southwestern News](2001-05-10) Echeverria, Ione