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Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein existing in both soluble monomeric conformation as well as integral-membrane form. The role of the domain interface in the conversion between these species was investigated. Bioinformatics-based analysis was undertaken to compare and contrast the domain interfaces of dimeric GSTs with their monomeric homologues CLIC1 and CLIC4. The mutants CLIC1-M32A and CLIC1-E81M were used as experimental case studies on the role of domain-domain interactions in the stability and folding of CLIC family proteins. A consensus interface was revealed with the prominent interaction being a conserved inter-domain lock-and-key type motif previously studied in class Alpha GSTs (Wallace et al., 2000). A number of domain-interface interactions were found to be unique to the CLIC family and as such thought to play a role in the conversion of these proteins from their soluble form to an integral membrane form. Overall the domain interfaces of monomeric CLIC1 and CLIC4 did not differ significantly from the domain interfaces of dimeric GSTs. The removal of the unique CLIC family salt-bridges between Arg29 and Glu81 and the cavity forming domain interface mutation Met32Ala did not induce significant changes in the conformational flexibility of the native state. The true role of the Arg29-Glu81 salt-bridges was masked by the introduction of stabilizing hydrophobic contacts. Removal of the inter-domain lock-and-key interaction destabilized CLIC1 significantly with concomitant loss in cooperative folding that resulted in the stabilization of a molten globule-like species. This intermediate state was less stable and less structured than the equilibrium intermediate of wtCLIC1 at pH 5.5. However the bulk of the structures found to unfold during intermediate-species formation was the same in mutant and wild-type proteins. It was concluded that formation of the membrane-competent form of CLIC1 involves re-structuring of the N-terminal thioredoxin domain that takes place after destabilization of the salt bridges connecting h1 and h3 and uncoupling of the inter-domain lock-and-key motif.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. October 2014. / The chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. Dysfunction in membrane insertion has been implicated in several pathologies including apoptosis, cancer and homeostatic imbalance. The transmembrane domain (TMD) is implicated in membrane penetration and pore formation and is therefore a key target for understanding amphitropism in CLIC1. The mechanism by which the TMD binds, inserts and oligomerises in membranes to form a functional chloride channel is unknown. Here the secondary, tertiary and quaternary structural changes of the CLIC1 TMD and several TMD mutants are reported in an attempt to elucidate the membrane insertion mechanism. A synthetic 30-mer peptide comprising the TMD was examined in 2,2,2-trifluoroethanol (TFE), SDS micelles and POPC liposomes using far-UV CD, fluorescence and UV absorbance spectroscopy. The results suggest a fourstep mechanism whereby the TMD, which is unfolded in buffer, refolds into a helix which partitions onto the membrane, followed by insertion and dimerisation to form a membranecompetent protopore complex. These helices associate via a Lys37-mediated cation-π interaction to form weakly active dimers. The complex is then tethered to the membrane by a cationic motif acting as an electrostatic plug. Thus, electrostatic interactions provide both a strong thermodynamic driving force for helix-helix association as well as structural integrity within the membrane. This represents an important step towards understanding how amphitropism occurs in CLIC1 and offers a unique insight into how CLIC1 and other proteins defy the ‘one-sequence one-fold’ hypothesis.

Ph.D, Faculty of Science, University of the Witwatersrand, 2011 / Chloride intracellular channel proteins (CLICs) are able to exist in a soluble or membrane-bound state. The mechanism by which the transition between the two states takes place is yet to be elucidated. It is proposed that structural rearrangements of the N-terminal domain take place when CLICs encounter the lower pH environment of the membrane surface (pH 5.5). This prompts the CLICs to form a soluble membrane-ready state prior to pore formation and membrane transversion. Since the insertion of CLIC1 into membranes occurs at low pH, perhaps protonation and electrostatic effects of key conserved residues at the domain interface situated within the transmembrane region bring about the structural changes necessary for this transition. Structural and sequence alignments revealed that a conserved salt-bridge interaction between conserved residues on helices 1 and 3 of the N-terminal domain is present at the domain interface of CLICs. Therefore, this interaction was proposed to play an important role in maintaining the structural integrity and conformational stability of the N-terminal domain. This hypothesis was tested by mutating conserved CLIC1 residues Arg29 and salt-bridge partner Glu81 to methionine, thus removing the salt-bridge interaction. The conformational stabilities of each mutant at pH 7 (cytosol) and pH 5.5 (membrane surface) in the absence of membranes was then measured and compared to that of the wild type protein. The mutations did not impact upon the structural integrity of the protein. However, removal of the salt-bridge and hydrogen bonding interactions caused a loss in the cooperativity of unfolding from the native to unfolded state that resulted in the formation of an intermediate species. The intermediate species are less stable than the intermediate species of wild type CLIC1 at pH 5.5. Nevertheless, the properties (secondary and tertiary structure, ANS binding and cooperative unfolding (N ↔ U)) of the intermediate species are the same for all mutants and wild type protein. It can be concluded that the salt-bridge and more importantly hydrogen bonding interactions between helices 1 and 3 stabilise the Nterminal domain of CLIC1. It can be hypothesised that in the absence of membranes under acidic conditions, such as those at the surface of the membrane, protonation of acidic amino acid residues at the domain interface cause destabilisation of the Nterminal domain. This causes a reduction in the activation energy barrier for the conversion of soluble CLIC1 to its membrane-insertion conformation.

Yu, Chi Shing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 115-125). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Thesis (M.Sc.)--York University, 2006. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 62-69). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR29595

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiv, 201 p.; also includes graphics (some col.). Includes abstract and vita. Advisor: Stephen A. Osmani, Dept. of Molecular Genetics. Includes bibliographical references (p. 184-201).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 92-112).

Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. In mammals, including humans, oviduct functions as a sperm reservoir which is created by the binding of spermatozoa to the epithelial lining in the oviduct. Interaction between sperm and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. However, the mechanism(s) by which spermatozoa-oviduct interaction producing these beneficial effects is unknown. One possibility is that oviduct protects spermatozoa from oxidative stress. The hypothesis of this project was that oviductal cell membrane proteins interact with spermatozoa to protect them from oxidative damage. Due to the limited availability of human oviductal tissue for research, an immortalized human oviductal epithelial cell line, OE-E6/E7, was used as a study model. The first objective examined the effect of OE-E6/E7 membrane proteins on human spermatozoa. The extracted OE-E6/E7 membrane proteins bound to sperm head and preferentially to uncapacitated sperm. Pretreatment with OE-E6/E7 membrane proteins significantly suppressed ROS-induced adverse effects in sperm motility, membrane integrity, DNA integrity, and intracellular ROS level. OE-E6/E7 membrane proteins also increased the endogenous enzyme activities of sperm superoxide dismutase (SOD) and glutathione peroxidase (GPx). Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human sperm. The second objective investigated the involvement of sFUT5 in sperm-oviduct interaction. Purified sFUT5 bound to OE-E6/E7 cells and anti-FUT5 antibody inhibited this interaction. Pre-absorption of OE-E6/E7 membrane proteins with purified sFUT5 or blocking of sFUT5 on sperm with anti-FUT5 antibody significantly inhibited the protective effects of OE-E6/E7 membrane proteins against ROS-induced damages in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of OE-E6/E7 membrane proteins. Sperm processing in assisted reproductive technology (ART) treatment, including centrifugation and cryopreservation, has shown to induce ROS production and oxidative damage in sperm. The third objective investigated the possible use of OE-E6/E7 membrane proteins or asialofetuin as an antioxidant supplement during centrifugation and cryopreservation. No adverse effect on sperm functions was detected by centrifugation using our centrifugation protocols. OE-E6/E7 membrane proteins or asialofetuin pretreatment suppressed the cryopreservation-induced damage on sperm in terms of motility and DNA fragmentation. The fourth objective aimed to identify the sFUT5-interacting proteins from OE-E6/E7 membrane extracts. By using immuno-affinity chromatography and mass spectrometry analysis, cell adhesion molecule 4 (CADM4) was identified as a potential sFUT5-interacting protein. This result was further supported by co-immunoprecipitation, immunofluorescent staining and immunohistochemistry. CADM4 expression level was shown to be higher at follicular phase when compared to luteal phase of the menstrual cycle. In conclusion, this thesis demonstrated that oviductal epithelial cell membrane proteins bind to the human spermatozoa and protect them from ROS-induced damages in terms of motility, membrane integrity, and DNA integrity. sFUT5 mediates the spermatozoon-oviductal epithelial cell interaction and the beneficial effects of such interaction on the fertilizing ability of spermatozoa. Results from this study provide the potential use of sFUT5-interacting proteins to enhance the fertilization ability of human spermatozoa by protecting them from oxidative stress. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

KefC is a potassium transport system of <i>E.coli</i> that is regulated by glutathione metabolites. An analysis of the KefC protein was undertaken in order to advance towards an understanding of the transport and regulatory processes at a molecular level. KefC-LacZ hybrid proteins were constructed by mini-Mu transposon insertion mutagenesis into <i>kefC</i> plasmids. The distribution of the B-galactosidase activity between membrane and soluble fractions indicated that the KefC moieties of the hybrid proteins were directing the proteins to the membrane, suggesting that KefC is a membrane protein. Sequencing the fusion junctions of the <i>kefC'-'lacZ</i> gene fusions allowed progress to be made towards topological mapping of the KefC protein. Two stable, high activity hybrid proteins confirmed the location of the first cytoplasmic loop and the large, cytoplasmic C-terminal domain proposed on the basis of the deduced amino acid sequence. A knowledge of the gene orientation, derived from restriction mapping of the transposon insertions, enabled kefC to be cloned downstream of a bacteriophage T7 promoter and expressed using the T7 polymerase/promoter system, overcoming initial problems of low expression. This identified KefC as a membrane located protein of apparent molecular mass 55-60 kDa. The oligomerization of KefC was investigated. It was shown that treatment with the cross-linking reagent formaldehyde moved the KefC band to a higher molecular weight and it was suggested that KefC-LacZ hybrid proteins interfered with potassium efflux via KefC. These observations are consistent with KefC functioning as an oligomer. Regimes suitable for solubilization and purification of KefC-LacZ hybrid proteins were developed. Attempts were made to generate antibodies against KefC but encountered difficulties due to contamination with antibodies not specific for KefC.