title: Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin.
abstract: Using interferometry-based biosensors the binding and release of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) from calmodulin (CaM) was measured. In both isoforms, binding to CaM is diffusion limited and within approximately three orders of magnitude of the Smoluchowski limit imposed by orientation-independent collisions. This suggests that the orientation of CaM is facilitated by the charge arrays on the CaM-binding site and the complementary surface on CaM. Protein kinase C phosphorylation of eNOS T495, adjacent to the CaM-binding site, abolishes or greatly slows CaM binding. Kinases which increase the activity of eNOS did not stimulate the binding of CaM, which is already diffusion limited. The coupling of Ca(2+) binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca(2+) dependence of CaM binding. Hence, changes in the Ca(2+) sensitivity of CaM binding always imply changes in the NOS-CaM affinity. It is possible, however, that in some regimes binding and activation are not synonymous, so that Ca(2+) sensitivity need not be tightly linked to CaM sensitivity of activation. This study is being extended using mutants to probe the roles of individual structural elements in binding and release.
authors: Department of Chemistry & Biochemistry, Kennesaw State University, Kennesaw, GA 30144, USA.
authors: Jonathan L
authors: JL
authors: McMurry
authors: Carol A
authors: CA
authors: Chrestensen
authors: Israel M
authors: IM
authors: Scott
authors: Elijah W
authors: EW
authors: Lee
authors: Aaron M
authors: AM
authors: Rahn
authors: Allan M
authors: AM
authors: Johansen
authors: Brian J
authors: BJ
authors: Forsberg
authors: Kyle D
authors: KD
authors: Harris
authors: John C
authors: JC
authors: Salerno
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism
chemical_list: D002147:Calmodulin; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C; D002118:Calcium