Yasushi Imamoto

RECONSTITUTION PHOTOACTIVE YELLOW PROTEIN FROM APOPROTEIN AND P-COUMARIC ACID DERIVATIVES

We report reconstitution of photoactive yellow protein (PYP) from apoPYP and p‐coumaric acid derivatives. The addition of p‐coumaric acid to the apoPYP sample did not result in the recovery of PYP. In contrast, yellow products were obtained by the addition of p‐coumaryl thiophenyl ester or p‐coumaric anhydride to the apoPYP sample, the absorption spectra of which were indistinguishable from the spectrum of intact PYP. Our findings provide strong evidence that PYP has the p‐coumaryl chromophore. This reconstitution technique opens the way for further biophysical studies of PYP using artificial chromophore analogs.

Femtosecond-picosecond fluorescence studies on excited state dynamics of photoactive yellow protein from Ectothiorhodospira halophila

Abstract Dynamics of photochemical processes of photoactive yellow protein (PYP) which functions as a photoreceptor for a negative phototactic response of the bacteria has been studied by fs-ps fluorescence up-conversion technique. Fluorescence decay curvs were nonexponential and initial main components have time constants of several hundreds fs to a few ps. The decay processes were interpreted as due to the twisting around vinyl of p -coumaric acid chromophore leading to trans/cis isomerization and were similar to those of bacteriorhodopsin despite the quite different chromophore, except that the reaction is somewhat slower in PYP. Reaction mechanisms were discussed in comparison with those of rhodopsins.

Evidence for Proton Transfer from Glu-46 to the Chromophore during the Photocycle of Photoactive Yellow Protein

Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at −40u2009°C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995)Biochemistry 34, 12669–12672) were observed at 1482, 1437, and 1163 cm−1 for PYP. However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm−1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM. A 1736 cm−1 band was observed for PYP, but the corresponding band for PYPM was not. Because it disappeared in the Glu-46 → Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM.

Femtosecond fluorescence studies on ultrafast reaction dynamics of photoactive proteins

A femtosecond fluorescence study on ultrafast reaction dynamics of photoactive proteins has been performed.

Analysis of the temperature dependence of femtosecond excited state dynamics of bacteriorhodopsin by spin-boson model

Abstract The spin-boson model was applied to analyze the temperature dependence of excited state dynamics for the cis-trans photoisomerization of the chromophore in bacteriorhodopsin which was obtained by the Fourier transform of the optical absorption spectrum. The results indicate that the model is valid in the short time region less than about 30 fs and that the excited state dynamics in the time region larger than 30 fs is dominated by the non-harmonic slow vibrational motion, which is temperature independent. It is suggested that this specific vibrational motions might reflect the ultrafast cis-trans isomerization of the chromophore as well as conformation change of the protein environment.

Femtosecond-Picosecond Fluorescence Studies on Excited State Dynamics of Photoactive Yellow Protein (PYP) and It’s Site — Directed Mutants

Femtosecond fluorescence studies on PYP, it’s chromophore and four site-directed mutants are performed. Effects of the protein environment on the photoisomerization of the chromophore are discussed.