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Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin
Author(s) -
McMurry Jonathan L.,
Chrestensen Carol A.,
Scott Israel M.,
Lee Elijah W.,
Rahn Aaron M.,
Johansen Allan M.,
Forsberg Brian J.,
Harris Kyle D.,
Salerno John C.
Publication year - 2011
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2011.08395.x
Subject(s) - calmodulin , chemistry , enos , biophysics , binding site , nitric oxide synthase type iii , nitric oxide , biochemistry , nitric oxide synthase , biology , enzyme , organic chemistry
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. Structured digital abstract• CaM binds to eNOS by biophysical (View interaction ) • CaM binds to nNOS by biophysical (View interaction ) • eNOS binds to CaM by biophysical (View interaction )