ABSTRACT
Multi-domain oxidoreductases are a family of enzymes that catalyze oxidation-reduction reactions through a series of electron transfers. Efficient electron transfer requires a sequence of protein conformations that position electron donor and acceptor domains in close proximity to each other so that electron transfer can occur efficiently. An example is mammalian nitric oxide synthase (NOS), which consists of an N-terminal oxygenase domain containing heme and a C-terminal reductase domain containing NADPH/FAD and FMN subdomains. We describe the use of time-resolved and single-molecule fluorescence to detect and characterize the conformations and conformational dynamics of the neuronal and endothelial isoforms of NOS. Fluorescence signals are provided by a fluorescent dye attached to the Ca2+-signaling protein calmodulin (CaM), which regulates NOS activity. Time-resolved fluorescence decays reveal the presence of at least four underlying conformational states that are differentiated by the extent of fluorescence quenching. Single-molecule fluorescence displays transitions between conformational states on the time scales of milliseconds to seconds. This review describes the type of information available by analysis of time-resolved and single-molecule fluorescence experiments.
Subject(s)
Calmodulin/chemistry , Fluorescent Dyes/chemistry , Nitric Oxide Synthase/chemistry , Protein Conformation , Animals , Calcium/metabolism , Calmodulin/metabolism , Fluorescent Dyes/metabolism , Humans , Isoenzymes/chemistry , Isoenzymes/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase/metabolism , Protein BindingABSTRACT
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.
Subject(s)
Calmodulin/chemistry , Models, Molecular , Multiprotein Complexes/chemistry , Nitric Oxide Synthase Type III/chemistry , Animals , Cattle , Fluorescence Recovery After Photobleaching , Fluorometry , Protein Binding , Protein Structure, Quaternary , Protein Structure, TertiaryABSTRACT
We demonstrate a wavelength tunable optical excitation source for coherent Raman scattering (CRS) spectroscopy based on a single femtosecond fiber laser. Electrically controlled wavelength tuning of Stokes optical pulses was achieved with soliton self frequency shift in an optical fiber, and linear frequency chirping was applied to both the pump and the Stokes waves to significantly improve the spectral resolution. The coherent anti-Stokes Raman scattering (CARS) spectrum of cyclohexane was measured and vibrational resonant Raman peaks separated by 70 cm(-1) were clearly resolved. Single laser-based tunable excitation may greatly simplify CRS measurements and extend the practicality of CRS microscopy.
