Takeshi Itaba

Formation of the Motor Protein-Photochromic ADP Analogue-Fluorometal Ternary Complex and Photo-Reversible Transition along the Steps in ATPase Cycle

In the presence of Mg2+ADP, myosin forms stable ternary complexes with phosphate analogues, fluoroberyllate (BeFn), fluoroaluminate (AlF4-) and orthovanadate (Vi), each of which may mimic different transient state along ATPase cycle. It is known that kiensin also forms similar ternary complexes. Our previous studies on the photoaffinity-labelling using photoactive ADP analogue and 19F NMR spectroscopy using 19F-labeled ADP analogue for the ternary complexes revealed that there is some variation in the myosin-nucleotide contacts at the nucleotide base among the ternary complexes. Previously, we have incorporated photochromic molecule, azobenzen derivative into the functional region of ATP driven motor proteins and succeeded to control activities and conformation reversibly by photochromic irradiation. Aim of this study is to induce transition between the different steps in ATPase cycle of motor proteins utilizing photoisomerization on the motor protein-phtochromic ADP analogue-fluorometal ternary complexes. In this study, three types of photochromic ATP analogues composed of azobenznene, spiropyran and fulgimide derivatives were synthesized. The photo-responsive interaction of the photochromic ATP analogues with myosin and kinesin were examined. Photo-dependent conformational changes of the myosin motor domain were monitored by X-ray small angle scattering. And the photo-reversible changes in the velocity of the microtubule glyding for kinesin in the presence of the photochromic ATP analogues were also observed. In the presence of fluorometal, BeFn, photochromic ADP analogues were trapped within the ATPase site of kinesin and myosin resulted in formation of ternary complexes. The conformational changes of the complexes induced by photo irradiation were studied.

Conformational Change of Myosin Head Induced by Photo-Isomerization of Photochromic ATP Analogue

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Previously, we have cross-linked reactive cysteine SH1 and SH2 of skeletal muscle myosin with the sulfhydryl-reactive bifunctional azobenzene derivative, azobenzene-dimaleimide (ABDM) and succeeded to induce lever arm swinging reversibly by photo-irradiation. However, incorporation of the photochromic molecules into the functional regions abolished native enzymatic properties of myosin.In the present study, we have employed photochromic ATP analogues that change its structure reversibly by light irradiation in order to photo-regulate function of myosin. Phenylazobenzoyl-iminoethyl-Tri-Phosphate (PABITP) have been designed and synthesized. In the preliminary experiment cis isomer of PABITP was hydrolyzed by skeletal and smooth muscle myosin in the presence of Mg2+ much faster than trans isomer. The results suggested that the cis isomer and trans isomer perform differently as a substrate of myosin. PABITP induced dissociation of acto-myosin. Myosin-PABIDP-Pi analogues (BeFn) ternary complex that may mimic transient state in ATPase was formed. X-ray solution scattering showed that the radius of gyration values of S1•PABITP(trans) and S1•PABITP(cis) are almost identical to those of S1•ATP and S1•ADP respectively. The results suggested that the photo-isomerization of the PABITP induce conformational change of myosin head, which may reflect energy transduction.