Browsing by Subject "protein engineering"
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Item Directed evolution of phosphotriesterase: towards the efficient detoxification of sarin and soman(Texas A&M University, 2004-09-30) Lum, Karin TienDirected evolution studies were done with PTE for the enhancement of hydrolysis of both sarin and soman analogs. Particular attention was focused on the toxic SpRc and SpSc isomers of the soman analog, and the Sp isomer of the sarin analog. Double substitution libraries yielded several mutants that had enhanced activity for the substrates. Among them was the double mutant, H257Y-L303T, which displayed a 462-fold increase in activity for hydrolysis of the most toxic SpSc isomer of the GD analog in comparison to the wild type. Several other mutants such as the triple mutants H254R-H257A-L303T and H254R-H257S-L303T had enhancements of between 150- and 200-fold, and had also displayed a different order of stereoselectivity relative to the wild type. For these mutants, the order of preferential hydrolysis was such that the SpRc isomer was preferentially hydrolyzed first. In contrast, the order of preferential hydrolysis for the wild type was that the RpRc was hydrolyzed first, followed by the RpSc, SpRc, and then the SpSc isomer. The reversal of stereoselective preference was also seen with the double substitution library members for hydrolysis of the sarin analog isomers. However, there were no significant improvements for sarin analog hydrolysis in these libraries. Among the best mutants obtained were H254G-H257W, H254G-H257R, and H257Y, all of which had catalytic efficiencies on the order of 106 M-1 s-1 for hydrolysis of the Sp isomer. The toxicity for analogs of sarin, soman, and VX was evaluated using Hydra attenuata as a model organism. The toxicity of each compound was assessed quantitatively by measuring the minimal effective concentration within 92 h in H. attenuata. There was a positive correlation between the molecular hydrophobicity of the compound and its ability to cause toxicity. Results from this study indicate the potential for application of this assay in the field of organophosphate nerve agent detection, as well as for the prediction of toxicity of structurally similar organophosphate compounds. The minimal effective concentration for two of the VX analogs was 2 orders of magnitude more toxic than the analog for soman and four orders of magnitude more toxic than the analog for sarin.Item Engineering pH tolerant mutants of a cyanide dihydratase of Bacillus pumilus C1 and identifying constraints on substrate specificity in nitrilases(2009-05-15) Wang, LanThis study generated two cyanide dihydratase (CynD) mutants of Bacillus pumilus C1 with improved activity at higher pH by random mutagenesis. The purpose of this study was to create enzyme variants better suited to degrade cyanide under the harsh conditions of industrial applications. We employed error-prone PCR to construct a library of CynD mutants. A high throughput screening system was developed to screen the library for improved activity. Two mutants were identified that could degrade cyanide at pH10 whereas the wild-type enzyme was inactive at pH9 or higher. The mutants each had three amino acid substitutions compared to the wild-type enzyme. The mutants were also more stable than the wild-type enzyme at 42oC. E327G was identified as one of the key amino acids that are responsible for the improved activity. The goal of the second project was to convert substrate specificity of the Bacillus sp. OxB-1 nitrilase to that of a cyanidase by mutagenesis or construction of hybrid genes. The OxB-1 nitrilase of Bacillus sp. shows a high level of identity with the cyanide dihydratases from B. pumilus C1 and P. stutzeri AK61 but utilizes different substrate. This provides a valuable resource to study the substrate specificity determinants of cyanide degrading enzymes. One deletion mutant and four hybrid proteins were constructed based on the alignment information. The constructed proteins were all unable to degrade cyanide.Item On the structure and assembly of staphylococcal leukocidin: a study of the molecular architecture of beta-barrel pore-forming toxins(Texas A&M University, 2006-08-16) Miles, Jr., George EmmettStaphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other β-PFTs form homooligomeric pores. For example, the staphylococcal α- hemolysin is a homoheptamer. Limited and controversial data exist on the assembly and molecular architecture of the leukocidin pore. In this work, biochemical and biophysical methods were used to characterize the leukocidin pore produced by the LukF (HlgB) and LukS (HlgC) components encoded by Staphylococcus aureus. I demonstrate that LukF and LukS assemble to form an SDS-stable pore on rabbit erythrocyte membranes. In addition, the pore-forming properties of recombinant leukocidin were investigated with planar lipid bilayers. Although leukocidins and staphylococcal α-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS (1 M KCl, 5 mM HEPES, pH 7.4), which is over three times greater than that of α-hemolysin measured under the same conditions. The subunit composition and stoichiometry of a leukocidin pore were determined by two independent methods, gel shift electrophoresis and sitespecific chemical modification during single channel recording. Four LukF and four LukS subunits were shown to co-assemble into an octameric transmembrane structure. The existence of an additional subunit in part explains properties of the leukocidin pore, such as its high conductance. Additionally, this is the first time that either technique has been applied successfully to assess the composition of a heteromeric membrane protein. It is also relevant to understanding the mechanism of assembly of β-PFT pores, and suggests new possibilities for engineering these proteins. In additional studies, the HlyII pore encoded by Bacillus cereus was found to form a homoheptameric transmembrane pore with properties conforming in general with those of other members of the class of β-PFTs. HlyII possesses additional properties which make it an attractive candidate for applications in biotechnology, such as an oligomer with a high thermal stability in the presence of SDS and the ability of the pore to remain open at high transmembrane potentials.Item The role of protein-membrane interactions in modulation of signaling by bacterial chemoreceptors(2009-05-15) Draheim, Roger RussellEnvironmental signals are sensed by membrane-spanning receptors that communicate with the cell interior. Bacterial chemoreceptors modulate the activity of the CheA kinase in response to binding of small ligands or upon interaction with substrate-bound periplasmic-binding proteins. The mechanism of signal transduction across the membrane is a displacement of the second transmembrane domain (TM2) a few angstroms toward the cytoplasm. This movement repositions a dynamic transmembrane helix relative to the plane of the cell membrane. The research presented in this dissertation investigated the contribution of TM2-membrane interactions to signaling by the aspartate chemoreceptor (Tar) of Escherichia coli. Aromatic residues that reside at the cytoplasmic polar-hydrophobic membrane interface (Trp-209 and Tyr-210) were found to play a significant role in regulating signaling by Tar. These interactions were subsequently manipulated to modulate the signaling properties of Tar. The baseline signaling state was shown to be incrementally altered by repositioning the Trp-209/Tyr-210 pair. To our knowledge, this is the first example of harnessing membrane-protein interactions to modulate the signal output of a transmembrane receptor in a controlled and predictable manner. Potential long-term applications include the use of analogous mutations to elucidate two-component signaling pathways, to engineer the signaling parameters of biosensors that incorporate chemoreceptors, and to predict the movement of dynamic transmembrane helices in silico.