Diverse roles of glycine residues conserved in photoactive yellow proteins.
The role of glycine residues was studied by alanine-scanning mutagenesis using photoactive yellow protein, a structural prototype of PER ARNT SIM domain proteins, as a template. Mutation of glycine located close to the end of beta-strands with dihedral angles disallowed for alanine (Gly-37, Gly-59, Gly-86, and Gly-115) induces destabilization of the protein structure. On the other hand, substitution for Gly-77 and Gly-82, incorporated into the fifth alpha-helix, slows the photocycle by 15-20 times, suggesting that these residues regulate the light-induced structural switch between dark-state structure and signaling-state structure. Most importantly, a significant amount of G29A is in the bleached state and showed a 1000-fold slower photocycle. As O(epsilon2) of the carboxylic acid of Glu-46 is close enough for contact with C(alpha) of Gly-29, alanine mutation perturbs this packing. Fourier transform infrared spectroscopy demonstrated that the C=O(epsilon2) stretching mode of Glu-46 is 6 cm(-1) upshifted in G29A, suggesting that C(alpha) of Gly-29 acts as a proton donor for the C(alpha)-H...O(epsilon2) hydrogen bond with Glu-46, which stabilizes the dark-state structure. During the photocycle, Glu-46 becomes negatively charged by donating a proton to the chromophore, resulting in breakage of this hydrophobic packing and consequent conformational change of the protein.
Imamoto
Yasushi
Y
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan. imamoto@vision-kyoto-u.jp
Tatsumi
Sanae
S
Harigai
Miki
M
Yamazaki
Yoichi
Y
Kamikubo
Hironari
H
Kataoka
Mikio
M
D000042:Absorption; D000409:Alanine; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D017124:Conserved Sequence; D005998:Glycine / Q000378:metabolism*; D020607:Halorhodospira halophila; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010782:Photolysis; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors; D017186:Titrimetry; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria; D000409:Alanine; D005998:Glycine