Daptomycin (Dap) is a cyclic lipodepsipeptide antibiotic that is currently used in the treatment of bacterial infections caused by Staphylococcus aureus including the strains that are methicillin-resistant (MRSA), as well as vancomycin-resistant (VRSA). Although it is known that daptomycin inserts into the membrane of the bacteria causing cell death very few details of its specific mechanism of action have been elucidated. Studies on Dap-resistant bacteria suggest that Dap may interact with specific proteins as well as specific lipids. It is anticipated that a photoaffinity label (PAL) of daptomycin (Dap-PAL) will be useful in determining what bacterial membrane components interact with Dap. The objective of this proposal is to prepare a photoaffinity label of daptomycin. Starting from 4,4’-dimethylbenzophenone, a benzophenone derivative was prepared that contained a 4-aldehyde moiety and a 4’-alkyl tail which contained a terminal alkyne group. This derivative was attached to the Orn residue in Dap via reductive amination to give the desired Dap-PAL. The minimal inhibitory concentration (MIC) of the Dap-PAL with Bacillus subtillus was determined to be 1.5 μg/mL which is only two times greater than the MIC of Dap.

No description available.

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Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 169-175).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [173]-180).

Follicle stimulating hormone (FSH) comprises an ?? subunit and a ?? subunit,whereas the FSH receptor consists of two halves with distinct functions, the N-terminalextracellular exodomain and C-terminal membrane associated endodomain. FSH initiallybinds to exodomain, and the resulting FSH/exodomain complex modulates the endodomainand generates signal. However, it has been difficult to determine which subunit of FSHcontacts the exodomain or endodomain, and in what orientation FSH interacts with them.To address these crucial issues, the receptor was Ala-scanned and the hormone subunitswere probed with photoaffinity labeling with receptor peptides corresponding to the Nterminalregion of the exodomain and exoloop 3 of the endodomain. The results show thatboth regions of the receptors are important for hormone binding and signal generation. Inaddition, the FSH ?? subunit is specifically labeled with the N-terminal peptide, whereas the?? subunit is labeled with the exoloop 3 peptide. These contrasting results show that the FSH?? subunit is close to the N-terminal region and the ?? subunit is projected toward exoloop 3in the endodomain. The results raise the fundamental question whether the ?? subunit,common among the glycoprotein hormones, plays a major role in generating the hormonesignal common to all glycoprotein hormones.

Glycoproteins have diverse and essential roles within biological systems. They are formed by enzymatic addition of saccharides to proteins during, or shortly after, translation. However, saccharides can also react with proteins non-enzymatically, a process termed glycation, which can cause impaired function and improper folding. Glycated proteins further react to form advanced glycation end-products, which have been implicated in the pathogenesis and progress of many diseases. Due to this pathological effect, glycation has been studied as a potential biomarker of these diseases. Photoaffinity labelling is a technique that is used to investigate the structure, and presence, of biological molecules; a precedent exists for its use in the study of carbohydrates in biological systems. Chapter 1 outlines the background of this thesis exploring previous studies of glycation, its effects, and methods used in recognition and photoaffinity labelling. Chapter 2 details the design and synthesis of a novel photoaffinity probe, and the optimisation of this synthesis. The target molecule was successfully produced and simpler alternatives to the initial synthetic route with similar yields are discussed. In Chapter 3 the use of the photoaffinity probe is studied. Labelling trials were performed on three proteins: human serum albumin (HSA), macrophage migration inhibitory factor (MIF), and casein. Mass spectrometry showed that the experiments with both HSA and MIF were successful, while the procedure appeared to lead to degradation of casein. Additionally, our work into developing techniques for identifying labelled samples is detailed. A diol-doped electrophoresis gel was not successful created, however, staining protein samples in polyacrylamide gel electrophoresis with curcumin showed promise. Chapter 4 explores the electrochemistry of the photoaffinity probe and details the use of the probe in functionalising a fluorine doped tin oxide (FTO) glass electrode. Cyclic voltammograms of Alizarin Red S (ARS), obtained using a treated electrode, suggest that surface functionalisation was successful.

Recent improvements in mass spectrometry instrumentation have stimulated the fusion of this technology with protein crosslinking to advance the structural proteomics field. However, analysis of complex datasets from crosslinking experiments remains a bottleneck. The majority of crosslinking studies for structural characterization of protein- protein interactions have been conducted with reagents specific for discrete amino acids. While this approach simplifies data analysis, the requirement for specific functionalities to be present at the interaction interface limits resolution. Herein, we report the application of stapled peptides for the development of photoaffinity reagents for mass spectrometric characterization of BCL-2 family protein interactions. To validate this approach, we synthesized photoreactive stabilized alpha-helices of BH3 domains (pSAHBs) incorporating a benzophenone containing amino acid, and demonstrated that the photo crosslinking specificity of these reagents paralleled the interaction specificity of the native proteins. We show that the standard SEQUEST algorithm is effective at identifying specific amino acids crosslinked by pSAHBs, and that this information can be used to create distance restraints for characterizing interaction interfaces by in silico docking. The pSAHB approach is applied to characterize previously elusive activating interactions between BH3 domains and the proapoptotic proteins BAX and BAK. We demonstrate that full-length BAK requires a direct activation stimulus, and that this involves interaction at a canonical surface groove at the C-terminal face of BAK. We confirmed that initiation of direct BAX activation occurs at a geographically distinct site at the N-terminal face of BAX, but further find that induced release of its C-terminus from the canonical groove exposes these residues for secondary BH3 interaction. These data suggest that BAX may be subject to a two-step activation mechanism within distinct cytosolic and mitochondrial compartments. Finally, we report the structural characterization of an interaction between BAD and glucokinase, the first description of a BH3 domain interaction with a non-BCL-2 family member. We identify the active site region of glucokinase as the BAD interaction site, establishing this region as a novel target for development of glucokinase activators. We conclude that the pSAHB approach represents a rapid and powerful approach to protein interaction site identification that complements conventional structural biology techniques.

The luteinizing hormone / choriogonadotropin receptor (LH/CG-R) is a member of theG protein-coupled receptor family. The LH/CG-R has seven transmembrane helices, threeexoloops, three cytoloops, a C-terminal tail, and an extensive N-terminal exodomain. Theexodomain is capable of binding hormone with high affinity without hormone action. Previousstudies have shown that the amino-terminal region of the LH/CG receptor contacts both subunitsof human chorionic gonadotropin (hCG). In particular, three residues (Leu20, Cys22, and Gly24)were found to be crucial for hormone binding. In this thesis work, benzoylphenylalanine (Bpa),a photoactivatable reagent, was used to continue investigating the interactions of the N-terminalregion of the LH/CG-R with hCG. Bpa has been directly incorporated at a defined position intopeptides representing amino acids 17-36 of the LH/CG-R. These peptides were radiolabeledwith 125I and used in photoaffinity labeling studies to identify and characterize the contact site(s)between the N-terminal region of the LH/CG-R and hCG. Results suggest that Cys22 is theprimary contact residue in this region. Peptide and hormone concentration dependent as well asUV duration dependent photoaffinity labeling experiments confirm that the photolabeling ofhCG by hLHR17-36(C22Bpa) is specific. Competition of labeling studies indicate that the hLHR17-36(C22Bpa) peptide is a good mimic of the wild type N-terminal portion of the receptor. In-geldigestions of photolabeled hCG ?? and photolabeled hCG ?? with CNBr indicate that the Nterminalregions of both hCG ?? and hCG ?? were photoaffinity labeled by hLHR17-36(C22Bpa).Based on the fact that the N-terminal regions of each subunit are located on the convex side ofthe heterodimer, these results provide evidence that the N-terminal portion of the receptor wrapsaround the back of hCG, contacting the convex face of the hormone.

The purpose of this study was to identify the site of interaction of antimycin with the ubiquinone-cytochrome b-c1 oxidoreductase in the photosynthetic bacteria, Rhodopseudomonas sphaeroides. To accomplish this goal, three areas of research were undertaken: the synthesis of a radiolabeled, photoaffinity analog of antimycin, identification of the inhibitory characteristics of this analog, and the photoaffinity labeling of the antimycin binding site. All three areas were accomplished. The major finding of this study was the identification of an 11,000 dalton polypeptide as the predominantly labeled protein. Although this polypeptide was not exclusively labeled, it was consistently labeled and showed competition with antimycin. These results are consistent with a similar study performed by das Gupta and Rieske (1973) with a mitochondrial preparation. These results are not conclusive, but do show several interesting points. First, cytochrome b is not the only site of interaction of antimycin with the ubiquinone~cytochrome b-c1 region of the electron transport chain. Secondly, an 11,000 dalton polypeptide is an important component of this protein complex. The function of this polypeptide is unknown, but should provide interesting research for future studies.