1998 Fiscal Year Final Research Report Summary
Studies on the Photoreactions and Tertiary Structure of Yellow Protein by Use of the Site-Directed Mutagenesis
| Project/Area Number |
09833004
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
光生物学
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| Research Institution | Osaka University |
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Principal Investigator |
IMAMOTO Yasushi Osaka University Graduate school of Science Research Associate, 大学院・理学研究科, 助手 (80263200)
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| Co-Investigator(Kenkyū-buntansha) |
TOKUNAGA Fumio Osaka University Graduate school of Science Professor, 大学院・理学研究科, 教授 (80025452)
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| Project Period (FY) |
1997 – 1998
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| Keywords | Protein Structure / Absorption Spectrum / Fourier Transform Infrared Spectroscopy / Isomerization / Photocycle / Proton Movement / Intermediate / Photoreaction |
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| Research Abstract |
Photoactive yellow protein (PYP) is a water-soluble photoreceptor protein found in the purple photosynthetic bacterium, Ectothiorhodospira halophila.Because its tertiary structure has been elucidated by X-ray crystallography and NMR, it is considered to be one of the most suitable target to understand the mechanisms of photoreception and signal transduction in the atomic level.In this project, the photoreaction of PYP was studied using the PYP mutants prepared by the methods we developed.
1. The chromophore of PYP together with Tyr-42, Glu-46 and Thr5O forms the hydrogen-bonding network, which is stabilized by Arg-52.The mutants in which these amino acids were replaced were prepared and the lifetimes of their M intermediates were studied.The M intermediates of Y42A and E46Q decayed faster than that of wild type whereas those of E46A, T5OA, T5OV and R52Q decayed slower, indicating that not only the strength of the hydrogen-bond but also the bulk of the residues regulates the behavior of the M intermediate.
2. Changes in the hydrogen bonding network around the chromophore was studied by ETIR.The hydrogen-bond of Glu-46 was stronger in B, H, and L intermediates than that of PYP, whereas Glu-46 was protonated in M intermediate.It suggests that the tertiary structure of PYP is remarkably altered in M intermediate.
3. The vibrational modes characteristic for cis-p-coumaric acid were identified by irradiation of trans-p-coumaric acid.Then the difference FTIR spectra between PYP and its intermediates were analyzed.The vibrational modes characteristic for cis form were found in all the intermediates but they were not in PYP.It indicates that the primary photochemical event in PYP is the isomerization of the chromophore like retinal proteins.
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