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Absorption (color) of Several rhodopsins is shown in a picture. Variation of the function and adsorption is provided by the varieties of the amino acid sequence. One of our research goals is description of the physical properties of each rhodopsin using some chemical and biological terms. The scientific questions, such as “What factors determine the function and absorption of each rhodopsin?” and “How diversity of the rhodopsins
contributes in the living organism?”, are still opening.

Spectra from the infrared spectroscopy provide abundant information, such as the molecular vibration, formation of the secondly structure and conformation dynamics. Cryo Fourier Transform Infrared Spectroscopy (Cryo-FTIR) and Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) are available in our laboratory.

Cryo FTIR

Light induced Cryo FTIR measurement can detect dynamics of the hydrogen-bonding networks. By comparing the two absorption spectra -one corresponding to the ground state (before light irradiation) and the other to the intermediate state (after light irradiation)- we obtain and analyze the differences spectrum. Intermediate state of the rhodopsin can be trapped under the cryo condition using cryostat, such as K intermediate at 77 K. The different spectra we obtained provide many information, such as the structural rearrangement between protonated retinal Schiff base (PRSB) and the counter ion, protonation/deprotonation state of the carboxylic acid, and conformational changes of the retinal.

A typical analysis of a bacterial rhodopsin (green proteorhodopsin (GPR)) using the Cryo-FTIR at 77K is shown in the spectrum. To reveal the dynamics of the water molecules, the spectrum the lipid reconstructed rhodopsin sample hydrated with H₂O (red line) or D₂¹⁸O (blue line) was measured and compared. The positive peaks at 2385(+), 2482, 2530, 2634 and 2676 cm^-1 and negative peaks at 2315, 2463, 2667 and 2683 cm ^-1 in the H₂O spectrum are shifted to the lower wavenumbers in the D₂¹⁸O spectrum, showing the dynamics of the water molecules. The positive bands at 2180 and 2198 cm ^-1 and the negative bands at 2094 and 2150 cm ^-1 possess the N-D starching vibrations of the PRSB.

ATR-FTIR

The measurement of the ATR-FTIR method can observe the vibrational signals of the sample from the evanescence field and minimizes contribution from the water background. This technique is powerful tool for the detection of the protein dynamics in the physiological condition. To combine a peristaltic pump with two flow channels systems, titration experiment is available in our ATR-FTIR spectroscopy. Titration of the ion or ligand concentration can reveal the affinity and hill equivalation between our target protein and ion and ligand.

Key terms

Amide I region (1600 ~ 1700 cm^-1): C=O stretching vibration of the protein backbone,
α helix:1648-1657 cm^-1, β sheet:1623-1641 cm^-1, 1674-1695 cm^-1

Amide II region (1500 ~ 1600 cm^-1): N-H deformation and C-N vibration of the protein backbone

Amide A region (3400 cm^-1):　N-H starching vibration

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