Investigation of Dynamical Properties of Proteins by Hole-Burning Spectroscopy

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1. Holes were burned into visible absorption bands of myoglobin substituted with a Zn ion or two hydrogen ions. Observed phonon sideband profiles were almost identical for myoglobin solution and freeze-dryed myoglobin. Thus it is confirmed that the phonon sideband profiles reflect not the vibration of the solvent but the vibration of the protein.2. In order to investigate the hole-filling process across the barriers between the initial and the final state of the hole-burning process, a heat cycle experiment was carried out. It was concluded that the barrier height distribution obtained from the experiment on Zn-substituted myoglobin reflects the energy structure of protein itself and shows no remarkable peak structure while the distribution obtained from the H_2 - substituted myoglobin corresponds to the barrier between the tautomers of the central hydrogens in porphyrin and shows sharp peak structure.3. Phonon sideband profiles were calculated on the basis of a normal mode analysis of the internal motion of myoglobin using linear approximation for electron-phonon interaction. The results were compared with experimental results, and they agree well in the region above 20 wave numbers.4. We tried the hole-burning experiments on UV absorption bands of proteins which have no visible absorption bands. As a result, holes were burned into the absorption bands of tyrosine, and phonon-sidebands were observed. But it was difficult to obtain accurate profile of the phononーsideband because of saturation effect.