Structure and thermal stability of monomeric bacteriorhodopsin in mixed phospholipid/detergent micelles.
Thermal unfolding experiments on bacteriorhodopsin in mixed phospholipid/detergent micelles were performed. Bacteriorhodopsin was extracted from the purple membrane in a denatured state and then renatured in the micellar system. The purpose of this study was to compare the changes, if any, in the structure and stability of a membrane protein that has folded in a nonnative environment with results obtained on the native system, i.e., the purple membrane. The purple membrane crystalline lattice is an added factor that may influence the structural stability of bacteriorhodopsin. Micelles containing bacteriorhodopsin are uniformly sized disks 105 +/- 13 A in diameter (by electron microscopy) and have an estimated molecular mass of 210 kDa (by gel filtration HPLC). The near-UV CD spectra (which is indicative of tertiary structure) for micellar bacteriorhodopsin and the purple membrane are very similar. In the visible CD region of retinal absorption, the double band seen in the spectrum of the purple membrane is replaced with a broad positive band for micellar bacteriorhodopsin, indicating that in micelles, bacteriorhodopsin is monomeric. The plot of denaturational temperature vs. pH for micellar bacteriorhodopsin is displaced downward on the temperature axis, illustrating the lower thermal stability of micellar bacteriorhodopsin when compared to the purple membrane at the same pH. Even though micellar bacteriorhodopsin is less stable, similar changes in response to pH and temperature are seen in the visible absorption spectra of micellar bacteriorhodopsin and the purple membrane. This demonstrates that changes in the protonation state or temperature have a similar affect on the local environment of the chromophore and the protein conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
Brouillette
C G
CG
Department of Medicine, University of Alabama, Birmingham 35294.
McMichens
R B
RB
Stern
L J
LJ
Khorana
H G
HG
D001436:Bacteriorhodopsins / Q000032:analysis*; D002152:Calorimetry, Differential Scanning; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003124:Colorimetry; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D008823:Micelles; D008854:Microscopy, Electron; D010743:Phospholipids / Q000032:analysis*; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet
D003902:Detergents; D008823:Micelles; D010743:Phospholipids; D001436:Bacteriorhodopsins