• Bulletin of the Korean Chemical Society
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• v.25 no.11
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• pp.1676-1680
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• 2004

We demonstrate that wide-angle X-ray scattering pattern from photoactive yellow protein (PYP) in solution using a high flux third generation synchrotron X-ray source reflects not only the overall structure, but also fine structures of the protein. X-ray scattering data from PYP in solution have been collected in q ranges from 0.02 ${\AA}^{-1}$ to 2.8 ${\AA}^{-1}$. These data are sensitive to the protein structure and consistent with the calculation based on known crystallographic atomic coordinates. Theoretical scattering patterns were also calculated for the intermediates during the photocycle of PYP to estimate the feasibility of time-resolved wide-angle X-ray scattering experiments on such proteins. These results demonstrate the possibility of using the wide-angle solution X-ray scattering as a quantitative monitor of photo-induced structural changes in PYP.

• Journal of Photoscience
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• v.9 no.2
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• pp.1-4
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• 2002

Hula-twist is a volume-conserving photoisomerization reaction mechanism postulated in 1985 to account for the rapid photoisomerization of the retinyl chromophore in rhodopsin. The requisite stereochemical consequence of simultaneous isomerization of a double bond and an adjacent single bond has recently been demonstrated in isomerization of pre-vitamin D in an organic glass and by many other examples of organic systems already reported in the literature This paper reports the consequence in applying the mechanism to the primary photochemical process of several photosensitive biopigments: bilirubin, photoactive yellow protein, bacteriorhodopsin and rhodopsin. It is shown that the anchored nature of the chromophores must first be taken into consideration.

• Journal of Photoscience
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• v.9 no.2
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• pp.134-137
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• 2002

PYP consists of a water-soluble apoprotein and 4-hydroxycinnamyl chromophore bound to Cys69 via thiolester linkage, Upon absorption of a photon, the photocycle is initiated, leading to formation of several photo-intermediates. Among them, M intermediate is important to understand the signal transduction mechanism of PYP, because it is a putative signaling state. As well known, the dynamics of a protein is closely correlated with the occurrence of its function. Here we report the results of IO ns molecular dynamics (MD) simulation for the M intermediate in aqueous solution and discuss the characteristic feature of this state from a viewpoint of structural fluctuation.

• Journal of Photoscience
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• v.9 no.2
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• pp.130-133
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• 2002

The last step in the photocycle of photoactive yellow protein (PYP) is a spontaneous recovery of the dark state from the active state in which the p-coumaric acid chromophore is thermally isomerized, concomitantly with the deprotona- tion of the chtomophore and the refolding of the protein moicty. For the purpose of understanding the mechanism of the thermal back-isomerization, we have investigated the rate-determining step by analyzing mutant PYPs of Met100, which was previously shown to play a major role in facilitating the reaction (1). The mutation to Lys, Leu, Ala, or Glu decelerated the dark state recovery by 1 to 3 three orders of magnitude. By evaluating temperature-dependence and pH-dependence of the kinetics of the dark state recovery, it was found that the retardation by mutations resulted from elevation of the activation enthalpy ( H$\^$┿/) and that the pKa of the chromophore, which was affected by the mutation, is in a linier correlation with the amplitude of the rate constants. It was, therefore, deduced from the correlation that the free energy for crossing the activated state in the dark recovery process is proportional to the free energy for the deprotonation of the chromophore, identifying the rate-determining step as the deprotonation of the chromophore. (1) Devanathan, S. Genick, U. K. Canestrelli, I. L. Meyer, T. E. Cusanovich, M. A. Getzoff, E. D. Tollin, G., Biochemistry 1998, 37, 11563 - 11568

• Journal of Photoscience
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• v.9 no.2
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• pp.126-129
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• 2002

The chromophore/protein interactions in the photocycle intermediates of photoactive yel- low protein (PYP) were probed by site-directed mutagenesis. The absorption spectra of L- intermediates produced from E46Q, T50V, and R52Q mutants were calculated using the absorption spectra of dark states and difference absorption spectra between L-intermediates and dark states, and compared with that of PYP$\_$L/. The absorption spectrum of R52Q$\_$L/ agreed with that of PYP$\_$L/, but those of E46Q$\_$L/ and T50V$\_$L/ were red-shifted. The effect of these mutations on the absorption spectrum for L-intermediate was comparable to that for the dark state, suggesting that the interaction around the phe-nolic oxygen of the chromophore is conserved in PYP$\_$L/ unlike the crystal structure. On the other hand, we have reported that the absorption spectra of Y 42F$\_$M/, T50V $\_$M/, and R52Q$\_$M/ agreed with that of PYP$\_$M/, but that of E46Q$\_$M/ was red-shifted, suggesting that the hydrogen bond of the chromophore with Glu46 is conserved but that with Tyr42 is broken in PYP$\_$M/. These results suggest that the chromophore inter-acts with Glu46 throughout the photocycle, but never directly interacts with Arg52. This model con- flicts with some of the structural model of PYP intermediates proposed based on the high-resolution X -ray crystallography.

• Journal of Photoscience
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• v.9 no.2
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• pp.122-125
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• 2002

Coherent oscillations in is fluorescence dynamics of W.-t. PYP and its site-directed mutants have been observed. Two oscillatory modes coupled with the ultrafast fluorescence due to the twisting of the excited chromophore were identified, a high ftequency mode (∼135 cm$\^$-1/) with ∼550 is damping time and a low frequency overdamped mode (-45 cm$\^$-1/) with ∼250 is damping time, respectively. Both modes disappear in the fluorescence dynamics of denatured PYP emphasizing the important role of the protein nanospace as the environment for photoreaction. The qualitative picture of fluorescence dynamics in site-directed mutants was rather similar to that in W.-t. PYP, i.e., similar oscillatory modes (∼130-140 cm$\^$-1/ and ∼40-70 cm$\^$-1/) have been observed. This indicates that the vibrational modes and electron-vibration couplings do not change dramatically due to the mutation though the damping time of low frequency mode a little decreases as the protein nanospace structure becomes looser and more disordered by mutation. On the other hand, in the case of some PYP analogues, the qualitative picture of fluorescence dynamics changes, showing the familiar picture of solvation effect whereas the oscillations are almost damped. Comparative analyses of these observations are presented.