Laser Pump Diffracted-X-Ray-Tracking (Laser-Pump DXT) for Single Molecule Measurement of Rotational Motion in Reaction with Photodissociated ATP

Abstract

Laser-pump diffracted-X-ray-tracking measurement system has been developed using nanosecond pulse laser at the beamline NW14A, Photon Factory Advanced Ring, KEK, Japan. Generally, diffracted X-ray tracking (DXT) has elucidated rotational motions of single protein molecule in real time [1]. In this work, single protein molecule labeled with gold nanocrystals in reaction with ATP molecule was irradiated by wide-energy band X-ray and nanosecond UV laser pulse. The motion of diffraction spots from gold nanocrystals were tracked in real time. The laser pulse produced the ATP molecules from photodissociation of caged-ATP in solution. The real-time rotational motion of single protein molecule reacting with ATP molecule can be observed using laser pump DXT.Pulse laser system (Powelite8000, Continuum Inc., USA) was synchronized with the diffracted X-ray tracking system. We took 90-120 diffraction images, and time resolution of DXT was from 5 μs/frame to 36 ms/frame. The wavelength, pulse width and laser intensity of single nanosecond laser pulse was 355 nm of third harmonic generation of the Nd:YAG laser, 10 ns and 20 mJ/cm2, respectively. This beamline gave the wideband-energy X-ray in the energy range 13-18 keV using an undulator at a repetition rate of 794 kHz. Delay time of laser pulse in DXT measurement was controlled by delay generator. Laser pulse can be irradiated sample in the arbitrary frames. We compared the motion of diffraction spots from gold nanocrystals attached protein molecule before laser irradiation with that after laser irradiation. The dynamical structural change of protein molecule in capturing ATP molecule will be revealed using laser-pump DXT. The rotational motion of Chaperonin in the presence of ATP is demonstrated as a typical example.[1] H. Shimizu, et al., Cell, 132, 67 (2008).