Masaaki Doi

Universal Processing Technique for Protein Crystals Using Pulsed UV Laser

We demonstrated the laser processing of three types of soft protein crystals using a 193 nm deep-UV pulsed laser. In particular, high-solvent-content crystals, phosphoenolpyruvate carboxylase (PEPC) crystals, were successfully processed without causing significant damage. An unchanged resolution limit between pre- and post-processings in X-ray diffraction measurements showed that laser irradiation has little influence on crystallinity. These results reveal that the processing using a deep-UV laser is effective for various protein crystals and will be very useful in accelerating protein structural analysis.

High-resolution displacement measurement using mode interference in the optical waveguide

Using an optical waveguide, high-resolution displacement measurement is achieved for the first time. The measurement principle is based on the interference between the two (even and odd) modes in the double-mode waveguide. After the light from a laser diode is focused onto the object to be measured, the wavefront gradient of the reflected light is detected using a Ti-indiffused LiNbO/sub 3/ channel waveguide. Although this optical measurement system is very simple, very high resolution (of less than 1 mn) is obtained. Besides, the characteristics of the measurement system do not depend on the surface condition of the object.

Protein Cryocrystallography Using Laser-Processed Crystal

We propose a new procedure in biological cryocrystallography, using the laser-ablation technique. This is the first report on the successful processing of cryo-cooled crystals to create a protein crystal ball that is conducive to X-ray diffraction (XRD) data collection. Pulsed UV laser soft ablation (PULSA) modifies protein crystals into a spherical shape and removes surrounding materials, but does not negatively affect crystallinity and may improve diffraction data quality. Additionally, we demonstrate treatment of problematic samples to make them serviceable for XRD analysis. Isolation of single crystals can be performed after flash cooling with the PULSA technique.

Application of UV-Laser Ablation to Detaching Protein Crystal from Growth Vessel

We propose a new procedure for detaching protein crystals from a growth vessel, which we call harvesting adhered crystals by laser irradiation (HACLI). A single laser shot at a wavelength of 193 nm successfully detaches hen egg-white lysozyme crystals from a fused-silica glass plate or capillary tube. X-ray diffraction measurements demonstrate that laser irradiation has little influence on the crystallinity. The HACLI technique will be a powerful tool for handling fragile protein crystals, especially those grown in a glass capillary tube.

Effect of Laser Irradiation on Enzyme Activity

We previously developed a protein crystallization technique using a femtosecond laser and protein crystal processing and detaching techniques using a pulsed UV laser. In this study, we examine the effect of laser irradiation on protein integrity. After several kinds of laser were irradiated on part of a solution of glycerol-6-phosphate dehydrogenase from Leuconostoc mesenteroides, we measured the enzyme activity. Femtosecond and deep-UV laser irradiations have little influence on the whole enzyme activity, whereas the enzyme lost its activity upon high-power near-infrared laser irradiation at a wavelength of 1547 nm. These results suggest that suitable laser irradiation has no remarkable destructive influence on protein crystallization or crystal processing.

Protein Crystal Growth Using Laser-Processed Seed Crystals

We demonstrate the use of crystals processed by a pulsed UV laser as seeds for protein crystallization. A hen-egg-white lysozyme crystal, which was successfully laser-processed without causing significant damage, was seeded and grown larger than its original size. Polarized light microscopy and X-ray diffraction analysis showed that the crystal grown by seeding was a single crystal of suitable quality for X-ray crystallography. The results support the effectiveness of UV-laser processing for protein crystals. Furthermore, this seeding technique, which we call laser-cut seeding, will be useful for the production of a seed of suitable size and shape in macroseeding, for the elimination of a damaged section in a growing crystal, and for removing useless crystals from polycrystals.

Characteristics of a high-resolution displacement sensor using mode interference in the optical waveguide

A high-resolution optical displacement sensing system using an optical waveguide is fabricated and characterized. The measurement principle is based on interference between an even and odd modes in the double-mode waveguide. After the light from a laser light source is focused onto the object to be measured, a wavefront gradient in the converged reflected light beam from the object gives a phase asymmetry at the entrance of the double-mode (DM) waveguide. A change of the phase asymmetry due to the displacement of the object along the optical axis is detected as a change of light intensity distribution at the exit of the DM waveguide. Although the optical system is very simple, experimental results using Ti-indiffused LiNbO3 waveguide device shows a very high resolution less than 1 nm. Next, a compact-type displacement sensor module using silica waveguide is fabricated and shows an identical high resolution. In conclusion, it will be very useful as a built-in component for various kinds of the industrial equipment.

Novel approach to process protein crystals using deep-UV laser

We propose new application of a pulsed deep-UV laser to processing biological macromolecule crystals. Single crystals of model protein, hen egg white lysozyme, were processed by deep-UV laser irradiation at 193 nm and 266 nm. Desired laser ablation was achieved using the 193-nm light source. A crystallinity of the processed crystals was evaluated by X-ray diffraction pattern recording. The crystal diffracted beyond a 1.9 Å resolution, which was the same as that of the as-grown crystal obtained under identical growth conditions. In addition, we observed that a diffraction pattern from a cracked protein crystal was considerably improved by eliminating the damaged section with the 193-nm laser ablation. A processing tool for protein crystals currently employed, such as a micro needle or knife, suffers from low reproducibility and poor accuracy. The technique using deep-UV laser pulses will be a powerful tool for processing very fragile protein crystals.