Browsing by Author "Hirota, Shun"

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A Single Spherical Assembly of Protein Amyloid Fibrils Formed by Laser Trapping
(2017-06-06)
Yuyama, Ken-ichi
;
Ueda, Mariko
;
Nagao, Satoshi
;
Hirota, Shun
;
Sugiyama, Teruki
Protein amyloids have received much attention owing to their correlation with serious diseases and to their promising mechanical and optical properties as future materials. Amyloid formation has been conducted by tuning temperature and chemical conditions, so that its nucleation and the following growth are analyzed as ensemble dynamics. A single spherical assembly of amyloid fibrils of cytochrome c domain-swapped dimer was successfully generated upon laser trapping. The amyloid fibrillar structure was confirmed by fluorescence characterization and electron microscopy. The prepared spheres were further manipulated individually in solution to fabricate a three-dimensional microstructure and a line pattern. Amyloid formation dynamics and amyloid-based microstructure fabrication are demonstrated based on direct observation of a single spherical assembly, which foresees a new approach in amyloid studies.
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
;
Sekiguchi, Yutaka
;
Matsumura, Hiroyoshi
;
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.