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Article

Generation of a Calmodulin-Based EPR Calcium Indicator^†

Junlong Shao, John Cieslak and Adrian Gross*

Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611

Biochemistry, 2009, 48 (3), pp 639–644

DOI: 10.1021/bi8014295

Publication Date (Web): December 30, 2008

†

This work was supported by NIH Grant GM58568 to A.G.

* Correspondence should be addressed to this author. Tel:(312) 503-3375. Fax: (312) 503-5349. E-mail:a-gross@northwestern.edu.

Abstract

Calmodulin is a ubiquitous calcium dependent protein. In the presence of calcium, calmodulin adopts an altered conformation that leads to the generation of downstream cellular calcium signals. Here we describe the introduction of nitroxide EPR probes into calmodulin by means of site-directed spin labeling. These probes sense the calcium-dependent conformational change of calmodulin and, therefore, can serve as calcium indicators. In combination with a light-sensitive calcium chelator, spin-labeled calmodulin can be used to demonstrate calcium release by flash photolysis. These results provide an important step toward describing the molecular dynamics of calcium-induced conformational changes in proteins using EPR spectroscopy.

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History

Published In Issue January 27, 2009
Article ASAPDecember 30, 2008
Received: July 30, 2008
Revised: November 25, 2008

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Article

Generation of a Calmodulin-Based EPR Calcium Indicator^†