X-ray scattering studies on structural changes of rhodopsin following light absorption.

Research Project

Project/Area Number	61460249
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	生物物性学
Research Institution	Osaka University
Principal Investigator	HAMANAKA Toshiaki Research Associate, Fac. of Engn. Sci., Lsaka Univ., 基礎工学部, 助手 (60093449)
Project Period (FY)	1986 – 1987
Project Status	Completed (Fiscal Year 1987)
Budget Amount *help	¥6,000,000 (Direct Cost: ¥6,000,000) Fiscal Year 1987: ¥600,000 (Direct Cost: ¥600,000) Fiscal Year 1986: ¥5,400,000 (Direct Cost: ¥5,400,000)
Keywords	Bovine rhodopsin / Squid rhodopsin / X-ray small-angle scattering / Low temperature measurement / Photoproduct / 構造変化 / ロドプシン / X線溶液散乱 / 光中間体 / コントラスト変化法
Research Abstract	The measurements of x-ray small-angle scattering have been done on the bovine and squid (Euprymna) rhodopsins and their photoproducts which were solubilized by sucrose laurate. In the case of bovine rhodopsin, the experiment was also made at the low temperature that the photo-intermediate, metarhodopsin II, was stable. The highly purified and concentrated rhodopsin solutions for x-ray measurement could be prepared by the affinity chromatography with Concanavaline A-Sepharose column and using polybuffer exchanger gel. The amount of free detergent micelles in the sample could also be controlled by this procedure. The x-ray scattering profile showed that the bleached bovine rhodopsin has formed the aggregates at room temperature. The significant change in the x-ray scattering was not observed between 0゜C and -29゜C upon light illumination. However, it was found that the large change of x-ray scattering profile has taken place following the freezing of solvent. This change was reversible and might not be due to the segregation of rhodopsins from the domain of frozen solvent of being formed. The x-ray experiment has been done on the Euprymna rhodopsin and its photoproduct, acid metarhodopsin, at 12 ゜C. There was the significant difference between the scattering profile of rhodopsin and acid metarhodopsin. Upon light illumination, more than 3 % change in scattering intensity was observed around the secondary peak, the profile of which depended strongly on the internal structure of the rhodopsin-detergent complex. In the future, the more quantitative analysis will be made on the change in rhodopsin structure following the light absorption, by doing the contrast variation measurement.