Kinesin motor protein inhibition and the development and use of two-photon sensitive caging groups for biological use
Reddie, Khalilah G.
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Adociasulfate-2 (AS-2) inhibits the ATPase activity of kinesin motors by mimicking microtubules, the natural substrate of the protein. The mechanism of inhibition of kinesin by AS-2 was investigated with fluorescence spectroscopy, enzyme kinetics, dynamic light scattering and electron microscopy. The results suggest that AS-2 inhibits kinesin motor function by forming inhibitory aggregates. The AS-2 aggregates formed associate with kinesin at its microtubule-binding site and mimic the structure and electrostatics of microtubules in a competitive manner. Kinesins, therefore, preferentially associate with AS-2 instead of microtubules, resulting in inhibition of activity. The quinolines, DMAQ-OAc, DMAQ-Cl-OAc and TQ-OAc were synthesized and investigated as biologically applicable photochemical cages for carboxylates. The UV-Visible absorption spectra, fluorescence emission spectra, aqueous dark stability, and quantum efficiencies were obtained for each compound. DMAQ-OAc, DMAQ-Cl-OAc and TQ-OAc were determined to have quantum efficiencies (and dark hydrolysis time constants) of 0.046 (31 h), 0.071 (34 h) and 0.063 (29 h), respectively. Anisomycin is an inhibitor of protein synthesis. Anisomycin was caged (inactivated) through conjugation with Bhc, a two-photon sensitive caging group. Caged Bhc-anisomycin was used to investigate two-photon induced control of protein synthesis. The fluorescence of destabilized GFP in HEK 293 cells was determined by confocal microscopy and used to monitor changes in protein synthesis following two-photon excitation of caged Bhc-anisomycin. The data revealed no change in the total fluorescence but instead reflected equilibrium between synthesis and degradation of GFP in the cells. Bhc-diol caged mannosamine (Bhc-ManLev) was synthesized and investigated as a tool to introduce caged ketones to mammalian cell surfaces. Metabolic utilization of the fluorescent compound was determined with confocal microscopy. The membranes of cells incubated with Bhc-ManLev, imaged with confocal microscopy, were fluorescent.