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dc.contributor.authorDeNunzio, Nicholas Josephen_US
dc.date.accessioned2015-08-04T18:21:56Z
dc.date.available2015-08-04T18:21:56Z
dc.date.issued2012
dc.date.submitted2012
dc.identifier.other(ALMA)contemp
dc.identifier.urihttps://hdl.handle.net/2144/12343
dc.descriptionThesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.en_US
dc.description.abstractProtein-protein interactions are integral to myriad molecular biological processes as they transfer information between molecules to effect a variety of goals. These include complex formation to activate gene transcription, serial interactions in signal transduction cascades, and one-time receptor-ligand or enzyme-substrate binding. Better understanding these interactions can therefore inform our view of larger biological frameworks and may be accomplished via direct visualization studies using structural and microscopy-based platforms. Using x-ray crystallography, the complex formed by the catalytic light chain of the Clostridium botulinum Serotype A neurotoxin (BoNT/A-LC) and its physiological substrate, synaptosomal-associated protein 25 (SNAP-25), has been examined to aid future inhibitor development. While small molecule and peptidic active site directed inhibitors have been published for this zinc-dependent protease, additional binding sites outside this region may exist given the extensive binding interface between these proteins. To identify these putative sites we conducted a computational analysis of the hydration structure of BoNT/A-LC across crystal structures to identify water molecules that are highly conserved as well as those that are more easily displaced, particularly when the BoNT/A-LC:SNAP-25 complex is formed. Results of these analyses are presented, including implications for de novo inhibitor design and extending previously established chemical scaffolds. In contrast, lower-resolution time-resolved luminescence microscopy (TRLM) was employed to develop a novel probe for imaging the receptor-ligand complex formed by interleukin-1 beta (IL1β) or epidermal growth factor (EGF) and their respective receptors. While a plethora of molecular tags exist for cellular localization studies, they often rely on chemically ligating an exogenous fluorophore to a protein target or endogenously expressing large protein-based tags (e.g. green fluorescent protein) in tandem with the protein to be tracked. We aimed to develop a compact genetically encoded tag that may be detected via in vitro and in cellula visualization studies using TRLM to measure long- lived luminescence while bypassing the epifluorescence that can be observed when irradiating biological specimens. Provided a sufficient concentration of LBT-tagged ligand is localized along a surface, the luminescent signal can be detected exclusive of epifluorescence. Future efforts to improve the physicochemical properties of the LBT and the imaging platform characteristics are discussed.en_US
dc.language.isoen_US
dc.publisherBoston Universityen_US
dc.titleBiophysical methods for analyzing protein-protein interactionsen_US
dc.typeThesis/Dissertationen_US
etd.degree.nameDoctor of Philosophyen_US
etd.degree.leveldoctoralen_US
etd.degree.disciplineBiophysicsen_US
etd.degree.grantorBoston Universityen_US


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