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. Author manuscript; available in PMC: 2019 Jun 1.
Published in final edited form as: Front Biosci (Landmark Ed). 2018 Jun 1;23:2133–2145. doi: 10.2741/4694

Figure 4.

Figure 4.

(Left) Steady-state fluorescence of CaM-T34C-AF594 with eNOS-WT vs. Ca2+ concentration. Fluorescence of AF594 (λex = 594 nm, λem = 615 nm) and Fluo-4 (λex = 494 nm, λem = 515 nm) was detected following additions of buffered Ca2+, and Ca2+ concentrations were calculated from Fluo-4 fluorescence. (Right) Amplitudes of time-resolved fluorescence populations as a percentage of total population for CaM-T34C-AF488 (200 nM) with eNOS- S1179D (800 nM) vs. Ca2+ concentration. The Ca2+ concentration was determined in similarly prepared solutions containing the Ca2+ indicator Quin-2. Red lines show fits to the steady-state (left) and time-resolved (right) data of a sequential binding model including contributions from free CaM (black), Ca2CaM-eNOS (blue), and Ca4CaM-eNOS (dark yellow). Fits show that the 0.1.-ns component (bottom panel) forms only with four Ca2+ bound to CaM. Data in right panel were replotted from ref (40).