Exploring Fine Structures of Photoactive Yellow Protein in Solution Using Wide-Angle X-ray Scattering

Kim, Tae-Kyu,Zuo, Xiaobing,Tiede, David M.,Ihee, Hyot-Cherl Korean Chemical Society 2004 Bulletin of the Korean Chemical Society Vol.25 No.11

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.

Exploring Fine Structures of Photoactive Yellow Protein in Solution Using Wide-Angle X-ray Scattering

Tae Kyu Kim,Xiaobing Zuo,David M. Tiede,이효철 대한화학회 2004 Bulletin of the Korean Chemical Society Vol.25 No.11

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 Å−1 to 2.8 Å−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.

Regulation of Protein Structural Changes by Incorporation of a Small-Molecule Linker

Kim, Youngmin,Yang, Cheolhee,Kim, Tae Wu,Thamilselvan, Kamatchi,Kim, Yonggwan,Ihee, Hyotcherl MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.12

<P>Proteins have the potential to serve as nanomachines with well-controlled structural movements, and artificial control of their conformational changes is highly desirable for successful applications exploiting their dynamic structural characteristics. Here, we demonstrate an experimental approach for regulating the degree of conformational change in proteins by incorporating a small-molecule linker into a well-known photosensitive protein, photoactive yellow protein (PYP), which is sensitized by blue light and undergoes a photo-induced N-terminal protrusion coupled with chromophore-isomerization-triggered conformational changes. Specifically, we introduced thiol groups into specific sites of PYP through site-directed mutagenesis and then covalently conjugated a small-molecule linker into these sites, with the expectation that the linker is likely to constrain the structural changes associated with the attached positions. To investigate the structural dynamics of PYP incorporated with the small-molecule linker (SML-PYP), we employed the combination of small-angle X-ray scattering (SAXS), transient absorption (TA) spectroscopy and experiment-restrained rigid-body molecular dynamics (MD) simulation. Our results show that SML-PYP exhibits much reduced structural changes during photo-induced signaling as compared to wild-type PYP. This demonstrates that incorporating an external molecular linker can limit photo-induced structural dynamics of the protein and may be used as a strategy for fine control of protein structural dynamics in nanomachines.</P>

Molecular Dynamics of the M intermediate of photoactive yellow protein in solution

Sakurai, Minoru,Shiozawa, Mariko,Arai, Shohei,Inoue, Yoshio,Kamiya, Narutoshi,Higo, Junichi Korean Society of Photoscience 2002 Journal of Photosciences Vol.9 No.2

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.

Kinetics of the E46Q mutant of photoactive yellow protein investigated by transient grating spectroscopy

Yang, Cheolhee,Kim, Tae Wu,Kim, Youngmin,Choi, Jungkweon,Lee, Sang Jin,Ihee, Hyotcherl Elsevier 2017 Chemical physics letters Vol.683 No.-

<P>To elucidate the role of internal proton transfer in the photodynamics of photoactive yellow protein (PYP), the photocycle of the Glu46Gln mutant of PYP (E46Q-PYP) is investigated by transient grating (TG) spectroscopy. Compared with wild-type PYP (wt-PYP), the first spectrally blue-shifted intermediate of E46Q-PYP is formed more slowly, which is consistent with the absence of direct protonation from Glu46 residue, if the parallel kinetic model for wt-PYP is invoked. The smaller conformational change in E46Q-PYP, as manifested by the smaller change in the diffusion coefficient, mainly arises from the relatively larger volume of the ground state E46Q-PYP than wt-PYP rather than from the smaller volume of the pB state. (C) 2017 Elsevier B. V. All rights reserved.</P>

The rate-determining step in the dark state recovery process in the photocycle of PYP

Sasaki, Jun,Kumauchi, Masato,Hamada, Norio,Tokunaga, Fumio Korean Society of Photoscience 2002 Journal of Photosciences Vol.9 No.2

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

Hula-twist, a Supramolecular Photoisomerization Reaction Mechanism in Reactions of Photosensitive Biopigments

Liu, Robert S.H. Korean Society of Photoscience 2002 Journal of Photosciences Vol.9 No.2

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.

The Role of Vibrational Coherency in Ultrafast Reaction Dynamics of PYP

Chosrowjan, Haik,Mataga, Noboru,Taniguchi, Seiji,Shibata, Yutaka,Hamada, Norio,Tokunaga, Fumio,Imamoto, Yasushi,Kataoka, Mikio Korean Society of Photoscience 2002 Journal of Photosciences Vol.9 No.2

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.