Indole ring orientations of Trp189 in the ground and M intermediate states of bacteriorhodopsin as studied by polarized UV resonance Raman spectroscopy

• Published in: Journal of Raman spectroscopy 2006-01, Vol.37 (1-3), p.255-262
• Main Authors: Asakawa, Kazuhiro, Masuda, Satoshi, Takeuchi, Hideo
• Format: Article
• Language: English
• Subjects: Amino acids; Bacteriorhodopsin; Indoles; Orientation; photo-excited intermediate; polarized UV resonance Raman spectroscopy; Proteins; Raman spectroscopy; Residues; side chain orientation; tryptophan; Vibration
• ISSN: 0377-0486; 1097-4555
• DOI: 10.1002/jrs.1463

Polarized resonance Raman spectroscopy provides a means for orientation analysis of proteins in aligned samples. Previously, we developed a Raman linear intensity difference (RLID) method to determine the orientations of aromatic amino acid side chains in flow‐oriented or membrane‐bound proteins. In this study, we have applied the RLID method to Trp189 in bacteriorhodopsin (BR), a transmembrane protein that acts as a light‐driven proton pump. Among the eight Trp residues in BR, the Raman spectrum of Trp189 has been extracted by subtracting the spectrum of the Trp189 → Phe mutant from that of wild‐type BR. By examining the 251.3‐nm‐exited polarized resonance Raman intensities of two indole ring vibrations of Trp189, the directions of the La and Bb transition moments have been determined with respect the membrane normal in the light‐adapted ground state (BR568) and a photo‐excited intermediate (M). Comparison of the orientations of the Trp189 indole ring derived from the La and Bb inclination angles has shown that the indole ring slightly but significantly reorients toward the ionone ring of the retinal chromophore in the M intermediate. The reorientation of Trp189 is consistent with the previous observation that helix F, on which Trp189 is located, undergoes an outward tilt and the hydrophobic interaction of Trp189 increases in the M intermediate. The RLID method combined with 251.3 nm excitation and point mutation is useful for detecting even a small reorientation of a targeted Trp residue. Copyright © 2006 John Wiley & Sons, Ltd.