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Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin

Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary... 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 Ca2+ binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca2+ dependence of CaM binding. Hence, changes in the Ca2+ 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 Ca2+ 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Febs Journal Wiley

Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin

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References (41)

Publisher
Wiley
Copyright
Copyright © 2011 Wiley Subscription Services, Inc., A Wiley Company
ISSN
1742-464X
eISSN
1742-4658
DOI
10.1111/j.1742-4658.2011.08395.x
pmid
22004458
Publisher site
See Article on Publisher Site

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 Ca2+ binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca2+ dependence of CaM binding. Hence, changes in the Ca2+ 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 Ca2+ 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.

Journal

The Febs JournalWiley

Published: Dec 1, 2011

Keywords: ; ; ; ;

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