Roles of amino acid residues near the chromophore of photoactive yellow protein.
To investigate the roles of amino acid residues around the chromophore in photoactive yellow protein (PYP), new mutants, Y42A, E46A, and T50A were prepared. Their spectroscopic properties were compared with those of wild-type, Y42F, E46Q, T50V, R52Q, and E46Q/T50V, which were previously prepared and specified. The absorption maxima of Y42A, E46A, and T50A were observed at 438, 469, and 454 nm, respectively. The results of pH titration for the chromophore demonstrated that the chromophore of PYP mutant, like the wild-type, was protonated and bleached under acidic conditions. The red-shifts of the absorption maxima in mutants tended toward a pK(a) increase. Mutation at Glu46 induced remarkable shifts in the absorption maxima and pK(a). The extinction coefficients were increased in proportion to the absorption maxima, whereas the oscillator strengths were constant. PYP mutants that conserved Tyr42 were in the pH-dependent equilibrium between two states (yellow and colorless forms). However, Y42A and Y42F were in the pH-independent equilibrium between additional intermediate state(s) at around neutral pH, in which yellow form was dominant in Y42F whereas the other was dominant in Y42A. These findings suggest that Tyr42 acts as the hinge of the protein, and the bulk as well as the hydroxyl group of Tyr42 controls the protein conformation. In all mutants, absorbance at 450 nm was decreased upon flash irradiation and afterwards recovered on a millisecond time scale. However, absorbance at 340--370 nm was increased vice versa, indicating that the long-lived near-UV intermediates are formed from mutants, as in the case of wild-type. The lifetime changes with mutation suggest the regulation of proton movement through a hydrogen-bonding network.
Imamoto
Y
Y
Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan. imamoto@ms.aist-nara.ac.jp
Koshimizu
H
H
Mihara
K
K
Hisatomi
O
O
Mizukami
T
T
Tsujimoto
K
K
Kataoka
M
M
Tokunaga
F
F
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D020607:Halorhodospira halophila / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D010782:Photolysis; D020283:Photoreceptors, Microbial / Q000737:chemistry; D011522:Protons; D013053:Spectrophotometry; D013912:Threonine / Q000235:genetics; D017186:Titrimetry; D014443:Tyrosine / Q000235:genetics
D000596:Amino Acids; D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D011522:Protons; C050162:photoactive yellow protein, Bacteria; D013912:Threonine; D018698:Glutamic Acid; D014443:Tyrosine; D010649:Phenylalanine