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Journal of Biochemistry Advance Access originally published online on July 15, 2009
Journal of Biochemistry 2009 146(4):581-590; doi:10.1093/jb/mvp107
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Photocontrol of Calmodulin Interaction with Target Peptides using Azobenzene Derivative

Hideki Shishido, Masafumi D. Yamada, Kazunori Kondo and Shinsaku Maruta*

Division of Bioinformatics, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan

*To whom correspondence should be addressed. Tel: +81-426-91-9443, Fax: +81-426-91-9312, E-mail: maruta{at}soka.ac.jp

Received March 31, 2009; Accepted June 22, 2009


  Abstract

Calmodulin (CaM), a physiologically important Ca2+-binding protein, participates in numerous cellular regulatory processes. It is dumbbell shaped and contains two globular domains connected by a short {alpha}-helix. Each of the globular domains has two Ca2+-binding sites, the EF hands. CaM undergoes a conformational change upon binding to Ca2+, which enables it to bind to specific proteins for specific responses. Here, we successfully photocontrolled CaM binding to its target peptide using the photochromic compound N-(4-phenylazophenyl) maleimide (PAM), which reversibly undergoes cis–trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. In order to specifically incorporate PAM, CaM mutants having reactive cysteine residues in the functional region were prepared; PAM was stoichiometrically incorporated into the cysteine residues in these mutants. Further, we prepared the target peptide, M13, fused with yellow fluorescent protein (YFP) to monitor the CaM–M13 peptide interaction. The binding of the PAM–CaM mutants, N60C, D64C and M124C, to M13–YFP was reversibly photocontrolled upon UV–VIS light irradiation at appropriate Ca2+ concentrations.

Key Words: azobenzene, calmodulin, nanomachine, photochromic molecule, photo-regulation

Abbreviations: ABDM, 4,4'-azobenzene-dimaleimide; CaM, calmodulin; Ca2+/CaM, CaM in a Ca2+-dependent manner; DMF, N,N'-dimethylformamide; NMR, nuclear magnetic resonance; PAM, N-(4-phenylazophenyl) maleimide; PCR, polymerase chain reaction; RLCs, regulatory light chains; SEC–HPLC, size-exclusion chromatography combined with high-performance liquid chromatography; smMLCK, smooth muscle myosin light chain kinase; skMLCK, skeletal muscle myosin light chain kinase; S1, subfragment-1; UV, ultraviolet; VIS, visible; YFP, yellow fluorescent protein; WT, wild-type


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