Partial dehydration of the retinal binding pocket and proof for photochemical deprotonation of the retinal Schiff base in bicelle bacteriorhodopsin crystals.
In bicelle bacteriorhodopsin (bcbR) crystals, the protein has a different structure from both native bacteriorhodopsin (bR) and in-cubo bR (cbR) crystals. Recently, we studied the ability of bcbR crystals to undergo the photocycle upon laser excitation, characterized by the appearance of the M intermediate by single crystal resonance Raman spectroscopy. Calculation of the M lifetime by flash photolysis experiments demonstrated that in our bcbR crystals, the M rise time is much faster than in the native or cbR crystals, with a decay time that is much slower than these other two forms. Although it is now known that the bcbR crystals are capable of photochemical deprotonation, it is not known whether photochemical deprotonation is the only way to create the deprotonated Schiff base in the bcbR crystals. We measured both the visible and Raman spectra of crystals dried under ambient lighting and dried in the dark in order to determine whether the retinal Schiff base is able to thermally deprotonate in the dark. In addition, changes in the visible spectrum of single bcbR crystals under varying degrees of hydration and light exposure were examined to better understand the retinal binding environment.
Sanii
Laurie S
LS
Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400, USA.
El-Sayed
Mostafa A
MA
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000528:radiation effects; D001665:Binding Sites / Q000528:radiation effects; D002460:Cell Line; D003460:Crystallization; D003890:Desiccation; D019617:Halobacterium salinarum / Q000378:metabolism; D006358:Hot Temperature; D008027:Light; D010777:Photochemistry; D011522:Protons; D012172:Retinaldehyde / Q000737:chemistry*; D012545:Schiff Bases / Q000737:chemistry* / Q000528:radiation effects; D013059:Spectrum Analysis, Raman
D011522:Protons; D012545:Schiff Bases; D001436:Bacteriorhodopsins; D012172:Retinaldehyde