Browsing by Author "Inoue, Tsuyoshi"

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Excimer Emission Properties on Pyrene-Labeled Protein Surface: Correlation between Emission Spectra, Ring Stacking Modes, and Flexibilities of Pyrene Probes
(2015-03-01)
Fujii, Akira
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Sekiguchi, Yutaka
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Matsumura, Hiroyoshi
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Inoue, Tsuyoshi
;
Chung, Wen-Sheng
The excimer emission of pyrene is popularly employed for investigating the association between pyrene-labeled biomolecules or between pyrene-labeled places in a biomolecule. The property of pyrene excimer emission is affected by the fluctuation in ring stacking modes, which originates from the structural flexibilities of pyrene probes and/or of labeled places. Investigations of the excimer emission in terms of dynamics of pyrene stacking modes provide the detailed spatial information between pyrene-labeled places. In order to evaluate the effects of probe structures and fluctuation in pyrene pyrene association modes on their emission properties on protein surface, three types of pyrene probe with different linker lengths were synthesized and conjugated to two cysteine residues in the A.55C/C77S/V169C mutant of adenylate kinase (Adk), an enzyme that shows a structural transition between OPEN and CLOSED forms. In the CLOSED form of Adk labeled by a pyrene probe with a short linker, excimer emission was found to be predominated by the ground-state association of pyrenes. The pyrene stacking structure on the protein surface was successfully determined by an X-ray crystallographic analysis. However, the emission decay in the protein suggested the existence of several stacking orientations in solution. With the increase in the linker length, the effect of fluctuation in pyrene association modes on the spectral properties distinctly emerged at both ground and excited states. The combination of steady-state and time-resolved spectroscopic analyses is useful for differentiation in the origin of the excimer emission, which is essential for precisely understanding the interaction fashions between pyrene-labeled biomolecules.
Laser ablation for protein crystal nucleation and seeding
(2014)
Yoshikawa, Hiroshi Y.
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Murai, Ryota
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Adachi, Hiroaki
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Sugiyama, Shigeru
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Maruyama, Mihoko
With the recent development in pulsed lasers with ultrashort pulse widths or wavelengths, spatially precise, low- damage processing by femtosecond or deep- UV laser ablation has shown promise for the production of protein single crystals suitable for X- ray crystallography. Femtosecond laser processing of supersaturated solutions can shorten the protein nucleation period or can induce nucleation at low supersaturation, which improves the crystal quality of various proteins including membrane proteins and supra- complexes. In addition to nucleation, processing of protein crystals by femtosecond or deep- UV laser ablation can produce single crystalline micro- or macro- seeds without deterioration of crystal quality. This tutorial review gives an overview of the successful application of laser ablation techniques to nucleation and seeding for the production of protein single crystals, and also describes the advantages from a physico-chemical perspective.
Spatially Precise, Soft Microseeding of Single Protein Crystals by Femtosecond Laser Ablation
(2012-09-01)
Yoshikawa, Hiroshi Y.
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Hosokawa, Yoichiroh
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Murai, Ryota
;
Sazaki, Gen
;
Kitatani, Tomoya
We developed a spatially precise, soft microseeding method for the production of single protein crystals that are suitable for X-ray crystallographic studies. We used focused femtosecond laser pulses to produce, via multiphoton absorption processes, seed crystals from small regions (similar to 1 mu m(2)) of crystals. Hen egg-white lysozyme seed crystals, produced in this manner, grew to be single crystals without any deterioration in their crystallinity. We also validated the technique using polycrystals for the membrane protein, acriflavine resistance protein B, for which single crystals are very difficult to obtain. In addition, we found that the shape of a tetragonal lysozyme crystal prepared from the seed could be controlled by altering the time interval between the initiation of crystallization and laser ablation. We also tried to comprehend the mechanism of femtosecond laser-induced microseeding. We visualized the ablated surfaces of the lysozyme crystals by atomic force microscopy and by laser confocal microscopy combined with differential interference microscopy. The results obtained in this study clearly demonstrate that femtosecond laser ablation of protein crystals is based on a photomechanical process, which ejects crystal fragments with little thermal damage. Femtosecond laser ablation is indeed very promising to produce high quality protein seed crystals from polycrystals or cracked crystals that are not suitable for X-ray diffraction studies.