Photocontrol of Motor Proteins using Photo-Responsive Calmodulin Dimer

Abstract

SOKA univ., Tokyo, Japan. Calmodulin (CaM) is a physiologically important Ca 2þ -binding protein that participates in numerous cellular regulatory processes. CaM undergoes a conformational change upon binding to Ca 2þ , which enables it to bind to target proteins for specific responses. For example, Ca 2þ /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 crosslinked 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 Ca 2þ , 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.