Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: Dimerization, complexation, and dissociation

Abstract

The investigation of protein quaternary structure, protein-cofactor, and protein-ligand interactions by mass spectrometry is often limited by the fragility of such interactions under experimental conditions. To develop more gentle conditions of perhaps general use, we used as a model for study the oxygenase domain of murine inducible nitric oxide synthase (iNOS), which is homodimeric, binds heme and tetrahydrobiopterin H 4 B cofactors, and the substrate L-arginine. The energetics of the collisions in q2 and in the lens region of the mass spectrometer were manipulated for varying the degree of solvation around the non-covalently bound ions. Furthermore, the number of low-energy collisions in the collision cell of the instrument was varied, focusing and dampening the ion beam. Under gentle source collision conditions, and using multiple low-energy collisions in the collision cell of the mass spectrometer, dimers of the iNOS oxygenase domain containing heme, H 4 B, and arginine were observed intact after electrospraying at pH values near neutrality; a mutant of this protein (Trp188 3 Phe) was monomeric and did not bind cofactors. The pH dependence of the iNOS oxygenase domain under acidic conditions was also studied; while heme remained bound to the protein between pH 2.5 and 4.0, the dimeric structure was disrupted. Our findings confirm that non-covalently bound macromolecular complexes are retained and observable using electrospray mass spectrometry under the appropriate experimental conditions. (J Am Soc Mass Spectrom 2004, 15, 629 -638)

Kinetic Unfolding Mechanism of the Inducible Nitric Oxide Synthase Oxygenase Domain Determined by Time-Resolved Electrospray Mass Spectrometry

“…The deconvoluted ESI mass spectrum of the apoprotein (obtained after acidification to pH 2.8, Figure 1) shows two peaks of roughly equal intensity, corresponding to masses of 50 425 and 50 337 Da. It seems likely that this phenomenon is related to N-terminal truncation (19). An acceptable match with the two measured masses is obtained by postulating that both forms of the protein carry an 80 Da covalent adduct.…”

“…A number of studies strongly suggest that Biochemistry 2005, 44 (17,18). Recently, Smith et al (19) used ESI-MS for studying quaternary interactions, cofactor binding, and protein conformation in iNOS COD as a function of pH under equilibrium conditions. In near-neutral solutions, the intact iNOS COD dimer was observed together with tightly folded heme-bound monomers.…”

“…Heme-bound monomers in semi-unfolded conformations were the dominant species at pH 3.5. Further acidification induced unfolding of these monomers, along with loss of the heme (19).…”

Stalking metal-linked dimers

“…In 1993, the method was used to detect non-covalently associated leucine zipper dimers [39]. Since then, ESI-MS has been used to detect a multitude of other protein dimers [40][41][42], including the oxygenase domain of nitric oxide synthase [43]. Although the ionization process involves the transfer of a protein from an aqueous to a gaseous phase, some dimeric species are able to survive the process [44][45][46].…”

“…The ability of non-covalent protein complexes to survive the ionization process is aided by using conditions of low temperature and low electrospray voltage [39,[41][42][43][44]47,48]. Also important to the process is the difference between the cone and extractor voltages, referred to as the declustering voltage, DCS.…”