Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Abstract

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.

Keywords: FRET; conformational motion; domain–domain interaction; electron transfer; flavoprotein.

Fig. 1.

(A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome c reductase activity of nNOSr proteins in their CaM-bound and CaM-free states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark cyan, E827C/Q1268C CL nNOSr labeled.

Fig. S1.

UV-vis spectra of Cy3 and Cy5 dye-reacted nNOSr proteins. Two molar equivalents of the donor and acceptor dyes (Cy3 and Cy5, respectively) are bound at ∼1:1 ratio. Extinction coefficients are: nNOSr (457 nm) ε = 22.9 mM⁻¹ cm⁻¹, Cy3 (550 nm) ε = 155 mM⁻¹ cm⁻¹, and Cy 5 (655 nm) ε = 250 mM⁻¹ cm⁻¹. The flavin absorbance is overlapped by Cy3 absorbance. Flavin absorbance is calculated based on the known absorbance ratio 280 nm: 457 nm. Proteins were reacted with the indicated dyes and subject to gel-filtration for further use.

Fig. 2.

Single-molecule fluorescence images (10 µm × 10 µm) of Cy3/Cy5-labeled E827C/Q1268C nNOSr, the emission is from the FRET dyes. (A) Donor. (B) Acceptor. (C) A portion of a single-molecule intensity vs. time trajectory, green represents donor, and red represents acceptor. (D) Auto-correlation of donor and acceptor from intensity vs. time trajectory. (E) Cross-correlation of donor and acceptor from intensity vs. time trajectory. (F) Single-molecule FRET efficiency vs. time trajectory. (G) The distribution of the FRET efficiencies.

Fig. 3.

(Upper) Histograms of the of FRET efficiency distribution from the E_FRET vs. time trajectories (each occurrence is the mean value from histograms as in Fig. 2G) without (A) or with (B) CaM. (Lower) Histograms of the of delay time (τ) distribution from autocorrelation of the intensity vs. time trajectories without (C) or with (D) CaM.

Fig. 4.

Impact of CaM binding on the distribution of single nNOSr molecules regarding their conformational fluctuation correlation times (t) and their associated FRET efficiencies (E_FRET). (A and B) Blue and red boxes contain all molecules that lie within 1 and 2 SD from the mean value pair, respectively. (C and D) 3D histograms plotting the molecular pair distributions in A and B. The heights of the peaks and their colors are proportional to the number of molecules, as indicated by the bars.