The microtubule cytoskeleton is an effective and validated target for cancer chemotherapy. This is clearly evident by the clinical effectiveness of Taxol that has been approved by the FDA for the treatment of several human malignancies, including breast and ovarian cancers. Taxol is an antimitotic agent that has the capacity to stabilize microtubules against depolymerization. Insight into how Taxol stabilizes microtubules and influences microtubule dynamics is important in the design of new drugs directed at altering the dynamics of microtubule assembly/disassembly and in the development of strategies to prevent or overcome drug resistance. Although structural models for both the alpha/beta-tubulin dimer and microtubules complexed with Taxol are available, the resolution is not sufficient to reveal how tubulin/protofilament structure is altered by drug binding. Hydrogen/Deuterium exchange (HDX)-Mass Spectrometry (MS) has emerged as a rapid and powerful experimental tool to investigate many aspects of protein architecture/dynamics. This technology provides us with an avenue to obtain crucial knowledge of tubulin structure/dynamics that is not available from electron crystallography, and is not likely to be in the near future. In this application, we propose to develop HDX-MS technologies for analysis of the altered solvent accessibility of human tubulin in microtubules as a result of polymerization. Alterations in solvent accessibility within each tubulin monomer will be localized to specific peptide regions of each subunit and potentially to individual residues. In combination with the existing electron crystallography data on tubulin/microtubules, this technology will localize changes in tubulin structure upon (a) polymerization in the presence or absence of Taxol or a nonhydrolyzable GTP analog (b) stabilization of microtubules by the epothilones, discodermolide or laulimalide and (c) the introduction of different tubulin isotype compositions or single point mutations.
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