Fig. 3.

(A) Fluorescence intensity of NS1 bound to various proteins upon two-photon excitation (normalized by the maximum intensity value corresponding to eNOS). The experiments shown in Fig. 2C and D were repeated with NOS isoforms and proteins with ferredoxin reductase, glucose-6-phosphate dehydrogenase (G6PDH), or without [lens epithelium–derived growth factor (LEDGF), fatty acid–binding protein (FABP), myoglobin, and E. Coli RecQ helicase] a NADPH-binding site; 5 μM of protein was mixed with increasing concentrations of NS1 until the fluorescence intensity reached a plateau. The relative fluorescence intensity was calculated based on the plateau value. The apparent dissociation constants of NS1 from the NOS isoforms determined using 2-PE fluorescence display similar affinities for NS1, K_d = 4–5 μM. (Inset) Two-photon fluorescence emission of NS1 (ex 940 nm) bound to eNOS or free in DMSO. (B) Zoom on the solvation near the nitro terminal of NS1 chromophore bound to iNOS reductase domain. Three water molecules located at 3.5 Å from the NO₂ extremity of NS1 are shown by purple spheres. The white surfaces are solvent molecules at 3.5 Å from D1123 and (variable) Y1077, the latter being part with F986, F989, and F964 to the hydrophobic patch at the edge of NS1-binding site; contribution of water from iNOS dimeric interface (small cyan spheres) is observed; (C) The distance to the conserved D decreases while the distance to F (F998, F1234, F964) and the number of water surrounding D (D1157, D1393, D1123) increases in the order e/n/iNOS (SI Appendix, Fig. S8).