Browsing by Subject "Protein Folding"
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Item A comparative study of HPr proteins from extremophilic organisms(Texas A&M University, 2006-04-12) Syed Ali, Abbas RazviA thermodynamic study of five homologous HPr proteins derived from organisms inhabiting diverse environments has been undertaken. The aim of this study was to further our understanding of protein stabilization in extremes of environment. Two of the proteins were derived from moderate thermophiles (Streptococcus thermophilus and Bacillus staerothermophilus) and two from haloalkaliphilic organisms (Bacillus halodurans and Oceanobacillus iheyensis); these proteins were compared with HPr from the mesophile Bacillus subtilus. Genes for three of these homologous HPr proteins were for the first time cloned from their respective organisms into expression vectors and they were over-expressed and purified in Escherichia coli. Stability measurements were performed on these proteins under a variety of solution conditions (varying pH, salinity and temperature) by thermal and solvent induced denaturation experiments. Stability curves were determined for every homologue and these reveal very similar conformational stability for these homologues at their habitat temperatures. The BstHPr homologue is the most thermostable and also has the highest G25; the stability of other homologues was ranked as Bst>Bh>St>Bs>OiHPr. Other key thermodynamic parameters, like Cp, have been estimated for all the homologues and it was found that these values are identical within errors of estimation. Also, it was found that the values of TS are very similar for these homologues. Together these observations allow us to propose a thermodynamic mechanism toward achieving higher Tm. The crystal structures of the BstHPr and a single tryptophan-containing variant (BstF29W) of this homologue are also reported here. Also reported is a domain-swapped dimeric structure for the BstF29W variant, together with a detailed investigation into the solution oligomeric nature of this protein. The crystal structure of BstHPr is analyzed to enumerate various stabilizating interactions like hydrogen bonds and salt-bridges and these were compared with those for the mesophilic homologue BsHPr. Finally, an analysis of sequence alignments together with structural information for these homologues has allowed design of numerous variants of both Bs and BstHPr. A detailed thermodynamic study of these variants is presented in an attempt to understand the origins of the differences in stability of the HPr homologues.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. [Keywords: protein folding disease, membrane protein folding, cystic fibrosis, bioinformatics, statistical coupling, X-ray structure, correction mechanism, mechanism of drug action]Item Multi-directional Rapidly Exploring Random Graph (mRRG) for Motion Planning(2013-08-27) Nath, Shuvra KantiThe motion planning problem in robotics is to find a valid sequence of motions taking some movable object from a start configuration to a goal configuration in an environment. Sampling-based path planners are very popular for high-dimensional motion planning in complex environments. These planners build a graph (roadmap) by generating robot configurations (vertices), and connecting nearby pairs of configurations according to their transition feasibility. Tree-based sampling-based planners (e.g., Rapidly-Exploring Random Tree, or RRT) start growing a tree outward from an initial configuration of the robot. In this work, we propose a multi-directional Rapidly-Exploring Random Graph (mRRG) for robotic motion planning, a variant of the Rapidly-Exploring Random Graph (RRG). Instead of expanding a vertex in the tree in a single random direction during each iteration, mRRG expands in m random directions. Our results show that growing in multiple directions in this way produces roadmaps with more topologically distinct paths than previous methods. In an environment with dynamic obstacles, moving or new obstacles may invalidate a path from the start to the goal. Hence, roadmaps containing alternative pathways can be beneficial as they may avoid recalculation of new valid paths. One of the important phases in sampling-based methods involves finding candidate nearest neighbors to attempt to connect to a node. Generally, the entire graph is considered to search for the nearest neighbors. In this thesis, we propose a heuristic method for finding nearest neighbors based on the hop limit, i.e., the maximum number of edges allowed in the path from a vertex to its neighbor. The candidate nearest neighbors are found by considering only those vertices within the hop limit. We experimentally show that our hop limit neighbor finder significantly reduces neighbor searching time over the standard brute force approach when constructing roadmaps.Item Protein folding and phylogenetic tree reconstruction using stochastic approximation Monte Carlo(Texas A&M University, 2007-09-17) Cheon, SooyoungRecently, the stochastic approximation Monte Carlo algorithm has been proposed by Liang et al. (2005) as a general-purpose stochastic optimization and simulation algorithm. An annealing version of this algorithm was developed for real small protein folding problems. The numerical results indicate that it outperforms simulated annealing and conventional Monte Carlo algorithms as a stochastic optimization algorithm. We also propose one method for the use of secondary structures in protein folding. The predicted protein structures are rather close to the true structures. Phylogenetic trees have been used in biology for a long time to graphically represent evolutionary relationships among species and genes. An understanding of evolutionary relationships is critical to appropriate interpretation of bioinformatics results. The use of the sequential structure of phylogenetic trees in conjunction with stochastic approximation Monte Carlo was developed for phylogenetic tree reconstruction. The numerical results indicate that it has a capability of escaping from local traps and achieving a much faster convergence to the global likelihood maxima than other phylogenetic tree reconstruction methods, such as BAMBE and MrBayes.