Photocontrol of Motor Proteins using Photo-Responsive Calmodulin Dimer

Abstract

Calmodulin (CaM) is a physiologically important Ca2+-binding protein that participates in numerous cellular regulatory processes. CaM undergoes a conformational change upon binding to Ca2+, which enables it to bind to target proteins for specific responses. For example, Ca2+/CaM regulates function of myosin V, myosin light chain kinase (MLCK) etc. Previously, we succeeded to photocontrol CaM function using photochromic molecule N- (4-phenylazophenyl) maleimide (PAM), which reversibly undergoes cis-trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. The CaM mutants, which have a single cysteine in the functional region of CaM, were prepared and modified with PAM. The binding of PAM-CaM to target peptide M13 was controlled reversibly upon UV and VIS light irradiation. In this study, we prepared the photo responsive dimer CaM by cross-linking of CaM mutants with bifunctional photochromic compound, 4,4′-azobenzene-dimaleimide (ABDM) in order to apply to photo control of motor proteins. For the CaM dimer, it is expected that the relative special configuration of each of CaM cross-linked with ABDM changes by UV-VIS light irradiation. Subsequently, we prepared the fusion protein, K355-M13 composed of kinesin motor domain and M13 peptide. The monomeric K355-M13 formed dimer configuration by CaM dimer. In the presence of Ca2+, two K355-M13 bound to the each site of CaM dimer resulted in formation of kinesin dimer linked by CaM-ABDM-CaM. The phtocontrol of the motor activity and microtubule dependent ATPase activity of the photochromic kinesin dimer was studied. Application of CaM-ABDM-CaM to MLCK and myosin was also performed.