Photoactivated Cross-Linking Mass Spectrometry for Probing Multidomain Protein Dynamics in Inducible Nitric Oxide...

Cross-linking mass spectrometry (XL-MS) with photoactivatable reagents has been increasingly applied to probe protein conformational dynamics; however, its sensitivity to shifts in conformational equilibrium involving transient, sparsely populated docked states within multidomain proteins remains largely unexplored. Mammalian nitric oxide synthase (NOS), a family of modular redox enzymes, relies on large-amplitude FMN domain swinging motions and local, dynamic interdomain FMN-heme docking to function. This makes it a stringent test case for evaluating how sensitive photoactivated XL-MS is to shifts in the docking equilbrium. In our previous work, quantitative XL-MS using the primarily lysine-reactive homobifunctional cross-linker DSBU revealed E546N mutation-induced decreases in specific intersubunit FMN-heme cross-links in an oxygenase/FMN construct of human inducible NOS (iNOS). Here, we extended these XL-MS studies to sulfo-SDA, a heterobifunctional, photoactivatable cross-linker with ultrafast reactivity and broad residue specificity. SDA captured interdomain FMN-heme cross-links at the docking interfaces but, unlike DSBU, did not reveal statistically robust changes in the abundances of these cross-links between wild-type and E546N mutant. SDA produced a higher proportion of dead-end labeling products for iNOS oxyFMN proteins, particularly the E546N mutant, than for the conformationally stable protein BSA. Moreover, the SDA cross-link distance restraints were largely satisfied by a minimal subset of docked states with distinct FMN domain orientations, whereas DSBU cross-links require a combination of docked and undocked conformations for optimal restraint satisfaction. These findings reflect the short effective distance range and near-instantaneous reactivity of SDA, which restrict FMN-heme cross-link formation to a narrow subset of transient docking states. Taken together, these results underscore the importance of selecting appropriate cross-linking reagents and employing complementary cross-linkers to define the conformational landscapes of dynamic multidomain proteins.