1989 Fiscal Year Final Research Report Summary
Elucidation of Dynamical Structures of Proteins by Ultraviolet Resonance Raman Spectroscopy
| Project/Area Number |
63470141
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Institute for Molecular Science Okazaki National Research Institutes |
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Principal Investigator |
KITAGAWA Teizo Professor, Institute for Molecular Science, 分子科学研究所, 教授 (40029955)
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| Co-Investigator(Kenkyū-buntansha) |
OGURA Takashi Research Associate, Institute for Molecular Science, 分子科学研究所, 助手 (70183770)
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| Project Period (FY) |
1988 – 1989
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| Keywords | Resonance Raman / UV Resonance Raman / Hemoglobin / Time Resolved Raman / Quaternary Structure |
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| Research Abstract |
Dynamical structures of proteins is substantial to understand a structure-function relationship of proteins. Since vibrational spectroscopy is sensitive to a geometrical structure of molecules and its time response is sufficiently fast, we adopted ultraviolet resonance Raman technique to probe selectively the vibrations of aromatic residues with absorption bands around 200-240 nm. First, an experimental system was settled. The fourth harmonic of Nd:YAG laser was brought into a 1 m hydrogen Raman shifter, and the 218 nm radiation was isolated. Raman scattering was detected with a solar blind photomultiplier attached to a 1.26 m single monochomator. A grating with 2400 gr/mm was used in the second order. With this system dynamical features of quaternary structure of carbonmonoxy hemoglobin (COHb) after photolysis were pursued by observing resonance-enhanced Raman bands of the aromatic residues. The Raman scattering was excited by 10 ns pulses at 218 nm, which were obtained from the secon
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d H_2 Raman shift of the fourth harmonic of a Nd:YAG laser, while CO was photodissociated by 10-ns pulses at 419 nm generated by a nitrogen-laser pumped dye laser or by pulses at 436 nm generated from the first H_2 Raman shift of the second harmonic of the Nd:YAG laser The delay time (DELTAt_d) from the photolysis pulse to the Raman probe pulse was varied from -100 to 500 mus. The observed spectra obtained for DELTAt_d =-100 mus and 10 ns were the same as each other and as that obtained without the pump beam, contrary to expectations based on the reported 7-ns change of the protein structure, but the spectral pattern did change when DELTAt_d changed from 10 to 20 mus; the bands at 1613 and 1011 cm^<-1> decreased in intensity, and the band at 878 cm^<-1> shifted to 883 cm^<-1>. These spectral changes appeared as a smooth monotonous function of time, suggesting the absence of an intermediate around DELTA_d = 10 mus. A similar spectral change was also observed upon addition of an effector (inositol hexaphosphate) to metHbF. Accordingly, the observed changes of the UV resonance Raman spectra were attributed to a quaternary structure change, presumably to a status change of beta37-Trp and alpha42-Tyr at the alpha1-beta2 subunit interface. Less
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