Browsing by Subject "Membrane proteins"
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Item Biochemical and functional characterization of zonadhesin: a sperm protein potentially mediating species-specific sperm-egg adhesion during fertilization(Texas Tech University, 2002-05) Bi, MingFertilization is an important and unique process in organisms that generate offspring by sexual reproduction. Several fines of evidence support the idea that glycoproteins in the egg zona pellucida (ZP) mediate species-specific sperm-egg adhesion. In contrast, studies on the complementary molecules in spermatozoa remain controversial. Some potential candidate adhesion molecules have been identified, but none of them is supported by unequivocal evidence. A mosaic protein called zonadhesin is one of them. Zonadhesin was first found in pig sperm membrane extracts based on its ability to bind to the native ZP in a species-specific manner. The zonadhesin cDNA sequence reveals that the protein comprises several extracellular domain types found in other molecules known to mediate cell-cell interactions. Orthologs of pig zonadhesin in three other species possess domain structures similar to pig zonadhesin's, but with variations. Our overall hypothesis is that zonadhesin mediates species-specific adhesion between spermatozoa and the egg ZP. In this dissertation, I report biochemical and functional characterization of pig zonadhesin. I observed that zonadhesin forms disulfidebonded oligomers and multimers, but the pl05/p45 monomer displays a preference for binding to the ZP. Zonadhesin undergoes heterogeneous processing and is targeted to more than one physicochemical compartment in developing sperm cells. Posttranslational processing generates at least three zonadhesin components that are glycosylated with different types of oligosaccharides. Zonadhesin's oligomeric status changes during maturation of spermatozoa in the epididymis. Immunoelectron microscopy demonstrated that zonadhesin localizes to the outer acrosomal membrane and the acrosomal matrix. Moreover, I identified zonadhesin protein in eight additional species by Western blotting, immunofluorescence, or both. Zonadhesin from each of these species localizes to the apical sperm head overlying the acrosome, but the MrS of these proteins' polypeptide chains vary significantly. In addition, a bead adhesion assay was developed for future tests of zonadhesin's ability to bind to homologous or heterologous ZP. Finally, preliminary work on expression of zonadhesin domain(s) initiated future loss- and gain-of-function transgenic studies. Potential practical benefits derived from this work may include development of new contraceptives that function by blocking gamete interactions and diagnostic methods for human fertility and farm animal fecundity.Item Biochemical properties and localization of zonadhesin in equine spermatozoa(Texas Tech University, 2002-12) Breazeale, Kelly RebeccaReproduction in all manunals requires successful completion of recognition events between spermatozoa and the ovum. Failure of gamete recognition may underlie some forms of sub-fertility or sterility in the stallion. This study has focused on comparing biochemical properties and localization of zonadhesin, a sperm-specific adhesion molecule, in Equus cahallus (horse), Equus asinus (donkey) and Equus grevyi (Grevy's zebra). Sperm proteins that function as adhesion molecules are prime candidates for such markers because of their essential functions in fertilization. One well-defined recognition event in mammalian fertilization is the species specific adhesion between spermatozoa and the egg's extracellular matrix (zona pellucida, ZP). Sperm proteins that may function in sperm-ZP adhesion include sp56, spermadhesins, RSA/Sp 17, PH-20, Sp38, and zonadhesin. The majority of these candidate adhesion molecules have been characterized in stallion spermatozoa. Among these proteins, only zonadhesin is capable of binding directly and in a species-specific manner to the zona pellucida. Zonadhesin is a high Mr protein evolutionarily related to von Willebrand factor, mucins, and other known cell adhesion molecules. Zonadhesin is expressed only in developing spermatozoa, and is localized to the apical head of spermatozoa. Zonadhesin has been most extensively characterized in pig spermatozoa. Preliminary studies in our laboratory have detected zonadhesin in spermatozoa of eight mammals, including the horse. This study documents the detection and initial characterization of equine zonadhesm, with an emphasis on properties that may vary across the Equus species and with length of time extended semen is stored. In this study, semen was collected artificially from 23 stallions and one jack (mean age lOyr + .91). Breeds of horses included Quarter Horse, Quarter Horse cross. Thoroughbred and Arabian, in addition to a large standard donkey and a Grevy's zebra. Semen samples were processed, extracted sequentially using 1% Triton and 2% SDS, and evaluated for protein content via BCA protein assay. Western blots were prepared loading equal amounts of protein from each stallion and membranes were probed with domain specific, DODl and D3, antisera to zonadhesin protein. The D3 bands were consistently located at approximately 95 kD and 60 kD. This band pattern was similar between stallions and across the species. The DODl band pattern showed numerous proteins with molecular weights ranging from approximately 120 kD to 25 kD. In addition, variation in Western blot band patterns was detected in the only known sub-fertile stallion evaluated for this study. Zonadhesin localized by immunofluorescence to the apical head of spermatozoa, in agreement with other mammals that have been studied. The similarity of zonadhesin composition between Equus caballus, Equus sinus and Equus grevyi is consistent with the ability of these species to interbreed. This research represents the first molecular studies on sperm recognition proteins in three Equus species.Item Cell surface interactions between Bdellovibrio and its prey(Texas Tech University, 1984-08) Portnoy Duran, Catherine AnneBdallovibrios are predatory gram-nagativa bacteria which have been suspected of stealing proteins from the outer membranes of their prey. I studied the outer membrane proteins of Bdellovibrio and its prey to show this phenomenon. Both the obligately parasitic strain Bdellovibrio bacteriovorus 109D and tha facultatively parasitic strain Bdellovibrio stolpii were used in these studies. Bedllovibrios ware cultivated on several types or strains of prey for outer membrane protein analysis using SDS-polyacrylamide gel electrophoresis. I found that Bdellovibrio acquires the OmpF major outer membrane protein of its Escherichia coli prey. Growth of bdellovibrios on mutant prey defective in outer membrane protein expression showed that in the absence of tha OmpF protein, the OmpC protein was acquired, but protein 2 was not. These results suggest that there is a specific Bdellovibrio preference for the OmpF protein, and furthermore it shows that our results did not arise from preparatory contamination. It was further determined through kinetic measurement of the disappearance of the OmpF and OmpC proteins from the bdelloplast surface, that Bdellovibrio does have a preference for the prey OmpF protein. Additionally, colicin studies and antibiotic killing kinetic experiments indicated that the prey-derived OmpF protein retained its porin function in the Bdellovibrio outer membrane. In summary, Bdellovibrio acquisition of prey outer membranes is unique, and the concommitant transfer of biological activity from prey outer membrane to Bdellovibrio outer membrane is a novel occurrence.Item Cys-loop receptors: Investigating the nicotinic acetylcholine receptor transmembrane domain and overexpression of a prokaryotic homolog (Gloeobacter violaceus Ion Channel)(2012-05) Pauwels, Jonathan; Jansen, Michaela; Cuello, Luis G.; Knaff, David B.We investigated the thermal motion of the transmembrane domain in murine muscle type nicotinic acetylcholine receptors (nAChR) and developed a bacterial expression system for the overexpression and purification of a prokaryotic ligand-gated ion channel from Gloeobacter violaceus (GLIC). nAChR, along with receptors for γ-aminobutyric acid, serotonin, glycine and glutamate, is a member of the superfamily of pentameric ligand-gated ion channels known as cys-loop receptors and GLIC is a closely related bacterial homolog. In the first set of experiments, we investigated differences measured by disulfide trapping at the 20’ position in the M2 transmembrane helices of nAChR and GABAA receptors. Due to their homology, it is commonly assumed these receptors have a great deal of structural and functional similarity. At the 20’ position in GABAA receptors disulfide trapping has shown inter-subunit crosslinking between both adjacent and non-adjacent (across the channel) residues. Whereas, similar experiments at the 20’ position in nAChR only showed inter-subunit crosslinking between adjacent subunits. In this study, we attempted to elucidate the fundamental difference between nAChR and GABAA receptors that led to these divergent findings. Specifically, we engineered neutralizing residues at the 20’ position in nAChR in an attempt to promote a biochemical environment more favorable to the formation of disulfides. Next, we developed a procedure for the overexpression and purification of GLIC. As a bacterial homolog, GLIC is an invaluable asset in the study of the basic structure and function of cys-loop receptors. After comparison of BL21(DE3), C41(DE3) and C43(DE3) chemically competent cells in the transformation and expression of GLIC, we determined C41(DE3) provided the best expression system for our construct. We used centrifugation and chromatographic techniques to purify GLIC. Our procedure provides large amounts of pure protein and can easily be adapted to chimeric GLIC proteins for downstream functional and structural studies.Item Genetic analysis of an Escherichia coli K-12 LPS mutant defective in expression of the major outer membrane porin proteins: a dissertation in medical microbiology(Texas Tech University, 1991-08) Burns-Keliher, Lisa LNot availableItem Identification of proteins from Erwinia chrysanthemi involved in animal pathogenesis(Texas Tech University, 2000-05) Anderson, C. ToddErwinia chrysanthemi (EC 16) is a Gram-negative bacterial phytopathogen that causes soft rot disease on a variety of fruit and vegetable-producing plants. The pathogenicity of EC 16 is related to its ability to secrete pectolytic enzymes directly onto the cuticle of the host plant via the type HI secretion system. The type III secretion system is among the virulence factors that have made us increasingly aware of the common mechanistic processes utilized by bacterial pathogens to attack and gain nutrients from either plant or animal host organisms. Crucial to EC 16's ability to deliver its degradative enzymes to the target plant is its ability to associate with the plant surface. Several different genera of bacteria utilize attaching and effacing (AE) proteins to achieve an intimate relationship with their host tissue or organism. An oligonucleotide probe related to Escherichia coli (K12) genomic sequence, which bears homology to numerous bacterial attaching and effacing proteins, was used to probe an EC 16 genomic DNA library in order to detect possible genes that may contribute to the virulence of the bacterium. Cosmid pLAFR 2020 was identified as harboring DNA complementary to the probe. A series of oligonucleotide primers was designed to amplify the desired 7.5 kbp fragment from purified pLAFR 2020 DNA via polymerase chain reaction (PCR). The fragment was cloned and sequenced. Nucleotide sequence analysis indicated that the cloned fragment had approximatly 85% homology with a genomic segment from E. coli K12, which bears strong similarity to numerous bacterial AE proteins and invasins. The translation product from this sequence would have a greater than 60% identity to an AE homologue fragment from enteroaggregative enterohemorrhagic (EAEH)E. coli. Expression of bacterial virulence factors is known to be induced by exposure of the pathogen to host cells or host-cell-nutrient environment. In order to determine if the AE-like DNA is expressed in EC 16, several growth conditions were used as mducers. The proteins from the bacteria grown under the different conditions were separated by SDS-PAGE, and the gels were stained with silver stain. Novel protems were present in each different induced sample (70, 94, and greater than 205 kDa), but most notable of these is the approximately 94- kDa protein present in samples grown in media containing pectin, fetal bovme serum (FBS), or in the presence of human colon adenocarcinoma derived cells. Immuno-blot analysis utilizing anti-Intimm polyclonal antisera revealed that this protein bears immunologic identity to the EPEC Intimin protem.Item Localization and functional interactions of fibronectin and associated basement membrane proteins during embryonic heart development(Texas Tech University, 1984-08) Kitten, Gregory TThe early embryonic heart is composed of two, cylindrical epithelial layers, an inner endothelium and an outer myocardium. The cardiac jelly (CJ), an a cellular accumulation of extra cellular matrix (ECM), fills the space between the two layers. All cardiac endothelial cells (EC) do not follow an identical course of differentiation. Some of the EC of the atrio ventricular (AV) and outflow tract (OT) regions undergo an epithelial-mesenchymal transition to form mobile cardiac mesenchymal (CM) cells while in other regions (e.g., ventricle), EC differentiate along the lines of a typical vascular endothelium. The mechanisms controlling the biphasic differentiation of EC and the subsequent migration of CM cells are poorly understood. Although the CJ lies between two epithelia and is spatially equivalent to a basement membrane (BM), it has not traditionally been considered to be organized into a BM-like structure. The potential significance of this observation lies in the possibility that BM, or their individual components (i.e., fibronectin (FN), laminin (LH), type IV collagen, and heparan sulfate proteoglycan (HSPG)), may function as the regulatory site o f " epithelial differentiation and morphogenesis. The temporal changes in the localization and the functional involvement of the BM components FN, LM, type IV collagen, and HSPG were investigated with respect to (1) EC attachment and differentiation and (2) CM cell attachment and migration. A cryofixation technique was developed in order to determine the in situ immunohistochemical distribution of the BM components in the CJ. Results indicated that the CJ exists IJS the fusion between a larger, myocardially derived BM and an attenuated, endothelial associated BM. Except for FN, the individual BM components were not all present during early stages. Instead, they appeared in a sequential manner, suggesting that all components of an adult-type B'A are not required to initiate the assembly of a structural and functional BM during development. In the AV and OT, FN lippeared as a progressively expanding gradient of material with the greatest density nearer the myocardium. An in vitro collagen gel bioassay was employed to directly test whether FN or other BM components play a role in EC and CM cell attachment, differentiation, and migration. Biochemical analysis and results from experiments using probes directed against the BH components indicated that mora than one mechanism of attachment, involving FN and/or HSPG, exist during EC development and CM cell migration.Item Membrane binding characteristics of choline-o-acetyltransferase in rat hippocampal nerve terminals(Texas Tech University, 1992-12) Smith, L. KeithCholine-0-acetyltransferase (ChAT; EC 2.3.1.6.) is the enzyme which catalyzes the formation of the excitory neurotransmitter acetylcholine. Although most of the ChAT in central cholinergic nerve terminals is soluble, some is also non-ionically associated with membranes. The major objective of this dissertation research was to determine how this membrane-bound detergent soluble form of ChAT (D-ChAT) was anchored to membranes in rat hippocampal nerve terminals. I tested the hypothesis that D-ChAT was anchored to membranes by a glycosylphosphatidylinositol (GPI) anchor. ChAT appeared to possess three characteristics common to many GPI-anchored proteins: (1) phosphatidylinositol specific-phospholipase C (PI-PLC) selectively released it from membranes, (2) PI-PLC treatment converted it from a detergent soluble into an aqueous form, and (3) an antibody to an epitope on the GPI anchor (anti-CRD) immunoreacted with the cytosolic form of ChAT (S3-ChAT) on Western blots. I also tested the possibility that D-ChAT might be one of the first intracellular GPI-anchored proteins described by internalizing PI-PLC into synaptosomes and determining if it would release D-ChAT from membranes into the cytosol. Internalized PI-PLC increased the amount of ChAT found in the cytosol, while reducing the amount of D-ChAT associated with membranes. In some tissues, endogenous glycosyl-phosphatidylinositol-specific phospholipase Cs (GPI-PLCs) function to release GPI-anchored proteins from membranes. To determine if an endogenous GPI-PLC might function to release D-ChAT from membranes, I utilized the GPI-PLC inhibitor zinc. Zinc inhibited an endogenous temperature-dependent release of ChAT from rat hippocampal minces, an endogenous release of ChAT from intracellular membranes into the cytosol of synaptosomes, and an endogenous conversion of ChAT from a detergent into an aqueous form in a plasma membrane enriched subcellular fraction (P4). These results suggest that some of the D-ChAT in rat hippocampal nerve terminals is GPI-anchored intracellularly, and that an endogenous GPI-PLC-like enzyme releases it from membranes into the cytosol.Item Regulation of biofilm formation and outer membrane protein expression in Vibrio cholerae by iron(2008-08) Craig, Stephanie Anne; Payne, Shelley M.Vibrio cholerae, a natural inhabitant of aquatic environments and the causative agent of the diarrheal disease cholerae, requires iron for survival. Since one of the key factors in the survival of V. cholerae in the environment is the formation of biofilms, we determined the effect of iron on this aspect of the pathogens lifestyle. Since wild type V. cholerae forms a much more robust biofilm in the presence of exogenous iron we tested mutants in iron transport and regulation and found that a mutation in the gene encoding an iron-regulated small RNA, RyhB, was clearly attenuated in the biofilm assay. We determined through microarray analysis that the ryhB mutant has altered regulation of genes involved in many systems that may be involved in biofilm formation including amino acid biosynthesis, the TCA cycle, motility and chemotaxis, and the expression of outer membrane proteins. Due to the pleiotropic regulatory effects of RyhB, it is unlikely that any one individual gene or system regulated by RyhB is the cause of the biofilm defect, but rather the sum effect of the regulatory changes is decreased biofilm formation. Additionally, we discovered that the outer membrane protein, OmpT, is positively regulated by iron and Fur. Generally, when Fur has acted as a positive regulator in previous studies, it has been ultimately shown to do so by negatively regulating the negative regulator, RyhB. However, the positive regulation of ompT by Fur is independent of RyhB. While CRP, a positive regulator of ompT expression, did not affect iron-dependent regulation of ompT, over-expression of the negative regulator ToxR abolishes the iron and Fur dependent regulation. Sequence analysis has revealed a possible Fur box approximately 70 base pairs upstream of the transcriptional start site in a region that overlaps both a ToxR binding site and a CRP binding site in the ompT promoter. We propose the model that in iron-replete environments under ToxR repressing conditions, such as when amino acids are limiting, Fur can further increase the expression of ompT.Item Scoring functions for protein docking and drug design(2014-05) Viswanath, Shruthi; Elber, RonPredicting the structure of complexes formed by two interacting proteins is an important problem in computation structural biology. Proteins perform many of their functions by binding to other proteins. The structure of protein-protein complexes provides atomic details about protein function and biochemical pathways, and can help in designing drugs that inhibit binding. Docking computationally models the structure of protein-protein complexes, given three-dimensional structures of the individual chains. Protein docking methods have two phases. In the first phase, a comprehensive, coarse search is performed for optimally docked models. In the second refinement and reranking phase, the models from the first phase are refined and reranked, with the expectation of extracting a small set of accurate models from the pool of thousands of models obtained from the first phase. In this thesis, new algorithms are developed for the refinement and reranking phase of docking. New scoring functions, or potentials, that rank models are developed. These potentials are learnt using large-scale machine learning methods based on mathematical programming. The procedure for learning these potentials involves examining hundreds of thousands of correct and incorrect models. In this thesis, hierarchical constraints were introduced into the learning algorithm. First, an atomic potential was developed using this learning procedure. A refinement procedure involving side-chain remodeling and conjugate gradient-based minimization was introduced. The refinement procedure combined with the atomic potential was shown to improve docking accuracy significantly. Second, a hydrogen bond potential, was developed. Molecular dynamics-based sampling combined with the hydrogen bond potential improved docking predictions. Third, mathematical programming compared favorably to SVMs and neural networks in terms of accuracy, training and test time for the task of designing potentials to rank docking models. The methods described in this thesis are implemented in the docking package DOCK/PIERR. DOCK/PIERR was shown to be among the best automated docking methods in community wide assessments. Finally, DOCK/PIERR was extended to predict membrane protein complexes. A membrane-based score was added to the reranking phase, and shown to improve the accuracy of docking. This docking algorithm for membrane proteins was used to study the dimers of amyloid precursor protein, implicated in Alzheimer's disease.R. DOCK/PIERR was shown to be among the best automated docking methods in community wide assessments. Finally, DOCK/PIERR was extended to predict membrane protein complexes. A membrane-based score was added to the reranking phase, and shown to improve the accuracy of docking. This docking algorithm for membrane proteins was used to study the dimers of amyloid precursor protein, implicated in Alzheimer’s disease.Item Temperature dependent release of BB' subunits of DNA-dependent RNA polymerase from folded nucleoids of Escherichia coli dnaA [superscript] ts mutants and location of the 1c gene of bacteriophage PA-2(Texas Tech University, 1980-05) Eaton, Leslie C.Not availableItem The tolC homologue of Erwinia chrysanthemi(Texas Tech University, 1998-08) Johnson, Oswald LeonardThe tolC gene of £. coli plays a role in bacterial resistance to large hydrophobic reagents. A cosmid (pLAFR1051) fi-om an Erwinia chrysanthemi genomic library capable of rescuing an Escherichia coli mutant sensitive to sodium deoxycholate was identified. Southern analysis identified DNA sequences from this cosmid that hybridized to the E. coli tolC gene. E. coli TolC antiserum was observed to interact in immunoblots with a 46-kDa polypeptide from E. chysanthemi. Cellular fractionation studies localized the polypeptide to the outer membrane of the bacterial cell. Cosmid pLAFR1051 was also observed to encode this polypeptide by immunoblot analysis. Nucleotide sequence analysis of subclones from pLAFR1051 hybridizing to the E. coli tolC gene identified an open reading frame of 1417 base pairs. The deduced amino acid sequence suggests that a polypeptide of 46-kDa could be encoded by this ORE. Analysis of the deduced amino acid sequence indicated the presence of a signal peptide and strong hydrophilic character. The amino acid sequence also indicated 76 % identity to the E. coli TolC protein. The cloned tolC homologue from E. chrysanthemi allowed an E. coli tolC strain to grow on sodium deoxycholate.