title: Water structural changes involved in the activation process of photoactive yellow protein.
abstract: Fourier transform infrared (FTIR) spectroscopy was applied to the blue-light photoreceptor photoactive yellow protein (PYP) to investigate water structural changes possibly involved in the photocycle of PYP. Photointermediates were stabilized at low temperature, and difference IR spectra were obtained between intermediate states and the original state of PYP (pG). Water structural changes were never observed in the >3570 cm(-)(1) region for the intermediates stabilized at 77-250 K, such as the red-shifted pR and blue-shifted pB intermediates. In contrast, a negative band was observed at 3658 cm(-)(1) in the pB minus pG spectrum at 295 K, which shifts to 3648 cm(-)(1) upon hydration with H(2)(18)O. The high frequency of the O-H stretch of water indicates that the water O-H group does not form hydrogen bonds in pG, and newly forms these upon pB formation at 295 K, but not at 250 K. Among 92 water molecules in the crystal structure of PYP, only 1 water molecule, water-200, is present in a hydrophobic core inside the protein. The amide N-H of Gly-7 and the imidazole nitrogen atom of His-108 are its possible hydrogen-bonding partners, indicating that one O-H group of water-200 is free to form an additional hydrogen bond. The water band at 3658 cm(-)(1) was indeed diminished in the H108F protein, which strongly suggests that the water band originates from water-200. Structural changes of amide bands in pB were much greater in the wild-type protein at 295 K than at 250 K or in the H108F protein at 295 K. The position of water-200 is >15 A remote from the chromophore. Virtually no structural changes were reported for regions larger than a few angstroms away from the chromophore, in the time-resolved X-ray crystallography experiments on pB. On the basis of the present results, as well as other spectroscopic observations, we conclude that water-200 (buried in a hydrophobic core in pG) is exposed to the aqueous phase upon formation of pB in solution. In neither crystalline PYP nor at low temperature is this structural transition observed, presumably because of the restrictions on global structural changes in the protein under these conditions.
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. kandori@photo2.biophys.kyoto-u.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Kandori
authors.forename: T
authors.initials: T
authors.lastname: Iwata
authors.forename: J
authors.initials: J
authors.lastname: Hendriks
authors.forename: A
authors.initials: A
authors.lastname: Maeda
authors.forename: K J
authors.initials: KJ
authors.lastname: Hellingwerf
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D020283:Photoreceptors, Microbial; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D011994:Recombinant Proteins; C050162:photoactive yellow protein, Bacteria; D014867:Water