Biophysical methods for analyzing protein-protein interactions
DeNunzio, Nicholas Joseph
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Protein-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.
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