Diverse Roles of Glycine Residues Conserved in Photoactive Yellow Proteins

¹ Kyoto University
² Nara Institute of Science and Technology
³ Nara Inst. of Science & Technology

^* To whom correspondence should be addressed. E-mail: imamoto{at}vision-kyoto-u.jp.

Submitted on October 9, 2007
Revised on November 7, 2007
Accepted on 21 December 2007

	Abstract

The role of glycine residues was studied by alanine-scanning mutagenesis using photoactive yellow protein, a structural prototype of PAS domain proteins, as a template. Mutation of glycine located close to the end of -strands with dihedral angles disallowed for alanine (Gly37, Gly59, Gly86, and Gly115) induces destabilization of the protein structure. On the other hand, substitution for Gly77 and Gly82, incorporated into the fifth -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 O₂ of the carboxylic acid of Glu46 is in proximity close enough for contact with C of Gly29, alanine mutation perturbs this packing. Fourier transform infrared spectroscopy demonstrated that the C=O₂ stretching mode of Glu46 is 6 cm^-1 up-shifted in G29A, suggesting that C of Gly29 acts as a proton donor for the C-H···O₂ hydrogen bond with Glu46, that stabilizes the dark-state structure. During the photocycle, Glu46 becomes negatively-charged by donating a proton to the chromophore, resulting in breakage of this hydrophobic packing and consequent conformational change of the protein.

Key Words: CH/O hydrogen bond, photocycle, photoreceptor protein, phototaxis, protein folding, sensory rhodopsin