Two processes lead to a stable all-trans and 13-cis isomer equilibrium in dark-adapted bacteriorhodopsin; effect of high pressure on bacteriorhodopsin, bacteriorhodopsin mutant D96N and fluoro-bacteriorhodopsin analogues

• Published in: European biophysics journal 2002-12, Vol.31 (7), p.539-548
• Main Authors: Bryl, Krzysztof, Yoshihara, Kazuo
• Format: Article
• Language: English
• Subjects: Absorption spectroscopy; Adaptation, Physiological; Bacteriology; Bacteriorhodopsins - chemistry; Bacteriorhodopsins - genetics; Bacteriorhodopsins - radiation effects; Biophysics; Darkness; Halobacterium salinarum - chemistry; Halobacterium salinarum - metabolism; High pressure; Light; Mutation; Pressure; Protein Conformation; Proteins; Spectrophotometry, Atomic - methods; Stereoisomerism
• ISSN: 0175-7571; 1432-1017
• DOI: 10.1007/s00249-002-0250-2

The combination of absorption spectroscopy and extraction techniques was applied to study the effect of high pressure on the dark-adapted state of bacteriorhodopsin, 14-(12-,10-)fluoro-bacteriorhodopsin, a D96N bacteriorhodopsin mutant, and 14-(12-,10-)fluoro-D96N. Evidence is presented that, at high pressure, the isomers' equilibrium is shifted from all- trans isomers towards the 13-cis isomers. Two groups of values for calculated molar volume changes indicate that there are at least two different processes leading to a stable all-trans and 13-cis isomers' equilibrium called the dark-adapted bacteriorhodopsin. The first process may be attributed to changes in the distances and rearrangement of functionally important residues and a retinal Schiff base. It is suggested that the moved residues (probably Asp-212 with the contribution of Tyr-185 and/or Asp-85) closer to the chromophore could catalyse its trans-cis isomerization. These changes require smaller pressure changes and induce larger volume changes (large-volume-change process). The second process may be attributed to the formation of the three hydrogen bonds that additionally decrease the volume and strengthen further stabilization of the 13-cis isomer. To induce these changes, larger changes of pressure are required and the final molar volume changes are smaller (small-volume-change process). The total molar volume change between all-trans bacteriorhodopsin and 13-cis bacteriorhodopsin in the dark-adapted state of native bacteriorhodopsin was found to be about -28 mL/mol, which is much higher than the value of about -7 mL/mol obtained previously (Tsuda and Ebrey 1980, Schulte and Bradley 1995). The data provide a novel insight into factors leading to stable isomer equilibrium in dark-adapted bacteriorhodopsin.