Taro Kimura

Synthesis of a novel fluorescent non-nucleotide ATP analogue and its interaction with myosin ATPase

A novel non-nucleotide fluorescent ATP analogue, N-methylanthraniloylamideethyl triphosphate (MANTTP), was designed and synthesized for kinetic studies with ATPases. The interaction of MANTTP with myosin ATPase was characterized. MANTTP was used as a substrate of myosin ATPase, and acceleration of actin-dependent hydrolysis was observed. The fluorescence property of MANTTP was not greatly affected by its binding to the ATPase site of myosin. In contrast, during MANTTP hydrolysis, significant fluorescence resonance energy transfer (FRET) was observed between MANTTP and intrinsic tryptophan residues in the myosin motor domain. Binding of MANTTP and formation of a ternary complex with a myosin-N-methylanthraniloylamideethyl diphosphate (MANTDP)-Pi analogue, which may mimic ATPase transient states, were monitored by FRET. The kinetic parameters of MANTTP binding to myosin and MANTDP release from the ATPase site were determined using a stopped-flow apparatus and compared with those of other ATP analogues. This novel fluorescent ATP analogue was shown to be applicable for kinetic analysis of ATPases.

Interaction of a Novel Fluorescent Non-Nucleotide ATP Analogue with ATP-Driven Molecular Motors

Fluorescent nucleotide analogues are essential for analysis of nucleotide-binding proteins. Most of fluorescent-labeled ATP analogues are ribose-modified. However, they are known to be 2’ and 3’ isomers mixture. Often these isomers show different properties each other. To avoid isomers, we designed and synthesized non-nucleotide fluorescent ATP-analogue, N-methylanthraniloyl amino ethyl triphosphate (MANTTP) which similar structure to the non-nucleotide ATP analogue 2-[(4-azido-2-nitrophenyl) amino] ethyl triphosphate (NANTP). It is known that NANTP are good substrate for skeletal myosin and induces actin gliding in vitro motility assay. Excitation and emission maximums in the fluorescence spectrum MANTTP were 330nm and 430nm, respectively. MANTTP was hydrolyzed by conventional kinesin and skeletal myosin, and induced dissociation of acto-myosin. The MANTTPase of myosin and kinesin were significantly activated by actin and microtubule, respectively. The ADP form of MANTTP showed the formation of skeletal muscle myosin-MANTDP-BeFn complex which mimic the transient state in ATPase cycle and this complex detaches from actin filament. KSV of MANTDP-S-1-phosphate analogue complex showed significantly smaller value than that of free MANTTP. The results suggested that the fluorophore moiety of MANTDP in the complex is buried deeply in the ATP binding site. The fluorescent intensity of MANTTP itself does not change on binding to myosin ATP binding site, however, MANTTP showed significant FRET between intrinsic tryptophan residue of skeletal muscle myosin and MANTTP. The binding of MANTTP to myosin can be observed as fluorescence increase on the stopped flow system. The second-order rate constant for MANTTP first binding to myosin is 0.15×10−6M−1s−1. It was shown that the novel fluorescent ATP analogue is applicable to the kinetic studies on ATPases.

Synthesis of Photochromic ATP Analogue and its Interaction with Motor Proteins

Azobenzene is one of the photochromic molecules, which undergoes rapid and reversible transitions between the cis isomer and trans isomer by visible and ultra-violet light irradiation. We have been trying to control the activities of motor proteins using the photochromic molecules as photo-regulatory devices. We have recently demonstrated that microtubules dependent ATPase activity of the kinesin modified by azobenzene derivative was regulated by UV-VIS light irradiation. However, it was not so easy to incorporate the photochromic molecules into the functional site of motor proteins without altering the native enzymatic properties.In the present study, we have designed the novel ATP analogues consist of photochromic molecules in order to photo-regulate the motor protein kinesin without their chemical modification. It is expected that the ATP analogues induce the reversible conformational change in the active site by alternate UV-VIS light irradiation. We have synthesized non-nucleotide ATP analogue composed of azobenzene derivative, Phenylazobenzoic-aminoethyl -triphosphate (PABATP). PABATP showed UV/VIS light absorption spectral change accompanied by transition between cis and trans in a similar manner observed in azobenzene. PABATP was hydrolyzed by conventional kinesin and the hydrolysis rate was activated by microtubules. It has been demonstrated that the cis isomer and trans isomer perform differently on a microtubule gliding motility assay. We have also examined the formation of kinesin-PABADP-Pi analogues (BeFn, AlF4-, Vi) which may mimic the transient states in ATPase cycle.