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Open Access Publications from the University of California

Scholarly Works (2 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2017)

The bacterium Clostridium difficile or C. diff is the cause of Clostridium difficile-associated infection (CDI). CDI is a growing health concern in hospitals and health clinics. Proteins of pathogenic strains of C. diff have the potential of being pharmaceutical drug targets as there are currently no effective treatment or drug combination that eliminate infection with high efficacy. Incidences of

relapse for CDI are as high as 20% for first recurrence and increases with subsequent relapses [17, 36, 49, 71].

Though some companies and hospitals have experimented with fecal transplant as a treatment, this procedure is still classified as an Investigational New Drug and is considered by most insurance companies to be an uncovered experimental treatment [17]. While almost all transcription factors that lie within the pathogenicity locus as well as the proteins they encode have been well-characterized, the TcdE protein remains less understood though sequence homology studies suggest it may function is a holin-like protein to allow the selective release of toxins TcdA and TcdB during pathogenesis [8, 37, 39, 40, 45, 69]. Elucidating the structure of the protein may provide clues as to how C. diff pathogenicity progresses and may offer a new target for therapeutic drugs. The structure of membrane proteins of similar size (21kDa) and transmembrane helices have been successfully studied and solved using nuclear magnetic spectroscopy (NMR). As with structural studies of membrane proteins by NMR, the lipid environment chosen is important in guiding protein folding and its local dynamics. Past studies have shown that micellar environments can distort membrane protein structures while bicelles and bilayers provide environments that more closely recapitulate native environments.

All proteins behave differently in different lipid environments and not all proteins are suitable for studies by NMR as it requires the protein to be over-expressed, pure, monomeric and to fold and behave in lipids. Here, a proof-of-

concept is demonstrated that TcdE can be successfully over-expressed and reconstituted in bicelles for studies by solution- and solid-state NMR.

    Cover page: Over-expression and Purification of full-length TcdE and Met27 TcdE Isoform for Structural Studies by NMR Spectroscopy
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2012)
    CXCR1 is one of two high-affinity receptors for the CXC chemokine interleukin-8 (IL-8), a major mediator of immune and inflammatory responses implicated in many disorders, including tumour growth. IL-8, released in response to inflammatory stimuli, binds to the extracellular side of CXCR1. The ligand-activated intracellular signalling pathways result in neutrophil migration to the site of inflammation. CXCR1 is a class A, rhodopsin-like G-protein-coupled receptor (GPCR), the largest class of integral membrane proteins responsible for cellular signal transduction and targeted as drug receptors. Despite its importance, the molecular mechanism of CXCR1 signal transduction is poorly understood owing to the limited structural information available. Recent structural determination of GPCRs has advanced by modifying the receptors with stabilizing mutations, insertion of the protein T4 lysozyme and truncations of their amino acid sequences, as well as addition of stabilizing antibodies and small molecules that facilitate crystallization in cubic phase monoolein mixtures. The intracellular loops of GPCRs are crucial for G-protein interactions, and activation of CXCR1 involves both amino-terminal residues and extracellular loops. Our previous nuclear magnetic resonance studies indicate that IL-8 binding to the N-terminal residues is mediated by the membrane, underscoring the importance of the phospholipid bilayer for physiological activity. Here we report the three-dimensional structure of human CXCR1 determined by NMR spectroscopy. The receptor is in liquid crystalline phospholipid bilayers, without modification of its amino acid sequence and under physiological conditions. Features important for intracellular G-protein activation and signal transduction are revealed. The structure of human CXCR1 in a lipid bilayer should help to facilitate the discovery of new compounds that interact with GPCRs and combat diseases such as breast cancer.
      Cover page: Structure of the chemokine receptor CXCR1 in phospholipid bilayers
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