Fluorescence quenching studies of structure and dynamics in calmodulin–eNOS complexes

• Published in: FEBS letters Vol. 589; no. 11; pp. 1173 - 1178
• Main Authors: Arnett, David C., Persechini, Anthony, Tran, Quang-Kim, Black, D.J., Johnson, Carey K.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 08.05.2015
• Subjects: AF488; AF594; Alexa fluor 488; Alexa fluor 594; Animals; bovine serum albumin; BSA; Calmodulin; Calmodulin - chemistry; CaM; Cattle; EGTA; electron microscopy; endothelial nitric oxide synthase; eNOS; ethylene glycol tetraacetic acid; FAD; flavin adenine dinucleotide; flavin mononucleotide; Fluorescence decay; Fluorescence Recovery After Photobleaching; Fluorometry; FMN; FRET; Förster resonance energy transfer; inducible nitric oxide synthase; iNOS; Models, Molecular; Multiprotein Complexes - chemistry; NADPH; neuronal nitric oxide synthase; nicotinamide adenine dinucleotide phosphate; Nitric oxide synthase; Nitric Oxide Synthase Type III - chemistry; nNOS; Protein Binding; Protein Structure, Quaternary; Protein Structure, Tertiary; Single-molecule fluorescence; TCSPC; time-correlated single photon counting; TRIS; tris(hydroxymethyl)aminomethane; NADPH; Förster resonance energy transfer; eNOS; FMN; EM; FAD; TCSPC; Fluorescence decay; CaM; EGTA; TRIS; BSA; Single-molecule fluorescence; nNOS; iNOS; Nitric oxide synthase; FRET; AF488; Calmodulin; AF594
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2015.03.035

•Fluorescence decays of labeled CaM bound to eNOS show four quenching states.•A highly quenched state can be assigned to CaM docked to the oxygenase domain of eNOS.•Single-molecule fluorescence trajectories show transitions between states.•The kinetics of the presumptive docked state suggest that its formation or dissociation is rate limiting. Activation of endothelial nitric oxide synthase (eNOS) by calmodulin (CaM) facilitates formation of a sequence of conformational states that is not well understood. Fluorescence decays of fluorescently labeled CaM bound to eNOS reveal four distinct conformational states and single-molecule fluorescence trajectories show multiple fluorescence states with transitions between states occurring on time scales of milliseconds to seconds. A model is proposed relating fluorescence quenching states to enzyme conformations. Specifically, we propose that the most highly quenched state corresponds to CaM docked to an oxygenase domain of the enzyme. In single-molecule trajectories, this state occurs with time lags consistent with the oxygenase activity of the enzyme.