The production of nitric oxide in endothelium cells plays important roles in regulation of vascular tone, leukocyte adhesion and platelet aggregation. Endothelial nitric oxide synthase (eNOS) is an enzyme that catalyzes the production of NO. This enzyme is activated by calmodulin (CaM), a calcium-modulated protein, which forms the active complex CaM-eNOS in the presence of calcium. Previous studies have shown that the complex adopts several conformational states, yet not much is known about the mechanism. Here, the goal is to better understand the nature of conformational states of CaM-eNOS through analysis of fluorescent decays by time correlated single photon counting (TCSPC). Two mutants of CaM, CaM T34C and CaM K115E, were labeled with Alexa Fluor-488 and used for fluorescent studies. Fluorescence decays of the fluorophore are sensitive to conformation due to energy transfer to the heme cofactor of the enzyme. CaM K115E is expected to have a reduced interaction of CaM with the oxygenase domain of the enzyme. Samples containing 200nM of either mutant with, 800nM – 2,400nM of eNOS, and in high calcium buffer were compared against each other using a pulsed diode laser. The concentration of eNOS was increased until no changes in the relative peaks were observed. Maximum entropy analysis was used to fit the normalized amplitudes as a function of fluorescent lifetimes. The differences in fluorescent lifetimes between these two mutants will allow us to further investigate the population of each conformational state in the complex.

Fluorescence lifetime studies of calmodulin-eNOS complexes

255th National Meeting (2018)

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