FRET Conformational Analysis of Calmodulin Binding to Nitric Oxide Synthase Peptides and Enzymes

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• 作者：Donald E. Spratt ; Valentina Taiakina ; Michael Palmer ; J. Guy Guillemette
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：November 18, 2008
• 年：2008
• 卷：47
• 期：46
• 页码：12006-12017
• 全文大小：348K
• 年卷期：v.47,no.46(November 18, 2008)
• ISSN：1520-4995

文摘

Calmodulin (CaM) is a ubiquitous Ca²⁺-sensor protein that binds and activates the nitric oxide synthase (NOS) enzymes. We have used fluorescence resonance energy transfer (FRET) to examine the conformational transitions of CaM induced by its binding to synthetic nitric oxide synthase (NOS) CaM-binding domain peptides and full length heme-free constitutive NOS (cNOS) enzymes over a range of physiologically relevant free Ca²⁺ concentrations. We demonstrate for the first time that the domains of CaM collapse when associated with Ca²⁺-independent inducible NOS CaM-binding domain, similar to the previously solved crystal structures of CaM bound to the Ca²⁺-dependent cNOS peptides. We show that the association of CaM is not detectable with the cNOS peptides at low free Ca²⁺ concentrations (<40 nM). In contrast, we demonstrate that CaM associates with the cNOS holo-enzymes in the absence of Ca²⁺ and that the Ca²⁺-dependent transition occurs at a lower free Ca²⁺ concentration with the cNOS holo-enzymes. Our results suggest that other regions outside of the CaM-binding domain in the cNOS enzymes are involved in the recruitment and binding of CaM. We also demonstrate that CaM binds to the cNOS enzymes in a sequential manner with the Ca²⁺-replete C-lobe binding first followed by the Ca²⁺-replete N-lobe. This novel FRET study helps to clarify some of the observed similarities and differences between the Ca²⁺-dependent/independent interaction between CaM and the NOS isozymes.