Studies On Bacteriorhodopsin Of Purple And Blue Membrane By Fluorescence And Absorption Spectroscopy

Purple membrane is the membrane fragment found in Halobacterium halobium. Each purple membrane fragment contains more than hundred thousand molecules of bacteriorhodopsin(BR), the only integrated membrane protein. BR contains 248 amino acids which comprise of seven transmembraneα-helixes and have a molecular weight of 26KD. BR contains 8 tryptophan and 11 tyrosine residues. BR functions as light-driven proton pump, is able to photocycles and photon-electronic reaction. At pH less than 2 or after the removal of surface bivalent cations(mainly Mg²⁺ and Ca²⁺) the color of purple membrane becomes blue(so called blue membrane). Blue membrane does not undergo photocycles and has no light-driven proton pump function. As to date, we have already had a deep understanding of the structure, photocycles and photochoromic effect of purple membrane and are considering the possible applications of bacteriorhodopsin in photo-electronic detect, neural network and biochip. In this study, we used fluorescence and absorption spectra to compare the conformation difference of BR protein of blue and purple membrane and to discuss the effect of temperature on conformational changes at room temperature. The results are showed as following:1. The absorption spectra studies indicate that the characteristic absorption band of blue membrane retinal is at 605nm at room temperature, characteristic absorption band of light-adaption purple membrane retinal is at 570nm and characteristic absorption band of dark-adaptation purple membrane retinal is at 560nm. The characteristic absorption band of blue membrane retinal is different with purple membrane retinal. Fourth derivative UV absorption spectra indicated that the characteristic peak of blue membrane BR protein shifted slightly to blue comparatively with purple membrane. More interestingly, the absorption spectral studies indicated that as temperature increased, the characteristic absorption peak of bacteriorhodopsin in blue membrane at 605 nm decreased and a new absorption peak at 380 nm formed. The transition occurred at a narrow temperature range(65℃-70℃). The new absorption peak at 380 nm indicates that the confirmation of the retinal choromophore in blue membrane is temperature-dependent, which has not been reported up to now. Moreover, it should be noted that the effect of temperature on blue membrane is irreversible. The characteristic absorption band at 570 nm decreases with the increasing temperature, which is different with blue membrane.2. The endogenous fluorescence spectra studies indicate that the maximum excitation wavelengths of bacteriorhodopsin fluorescence in blue and purple membrane at room temperature are both 280 nm. The maximum emission wavelengths of tryptophan fluorescence in blue and purple membrane proteins at room temperature are 340 nm, and the fluorescence quantum yield of blue membrane is about 1.4 fold higher than that of purple membrane. The maximum emission wavelengths of bacteriorhodopsin fluorescence in blue and purple membrane at 280 nm are 331 nm and 326 nm respectively. The endogenous fluorescence is came from the dedication of tryptophan and tyrosine together and the fluorescence is mainly came from the action of tyrosine in purple membrane. As temperature increases, the tryptophan intrinsic fluorescence of blue membrane decreases, while the tryptophan fluorescence of purple membrane increases obviously. We also found that, as temperature increases, the tryptophan intrinsic fluorescence of both blue and purple membrane slightly shifts to red. At 100℃, the maximum emission only shifts to 346 nm from 340nm, which suggests that BR proteins in blue and purple membrane only partially unfold and own highly thermal stability.3. The studies of extrinsic fluorescent probe bis-ANS indicates that the BR protein can be bond only to blue purple membrane, but not to purple membrane. Dissociation equilibrium constant of bis-ANS bound to blue membrane is 12.21μmol/L at room temperature, which suggests that the hydrophobic surface of bacteriorhodopsin in blue membrane is different with purple membrane. As temperature increases, the fluorescence of bis-ANS after binding to blue membrane markedly increases and shifts towards blue(especially obviously at 65℃-70℃). At 25℃, 65℃, 80℃and 90℃, dissociation equilibrium constant of bis-ANS bound to blue membrane are 12.21μmol/L, 5.22μmol/L, 6.42μmol/L and 7.09μmol/L respectively. The binding of bis-ANS bound to blue membrane at different temperature are only one binding sites.4. The research results of fluorescence, absorption spectra and stopped flow method used to track the process of transition from acidifying purple membrane to blue membrane indicate that the transition of the characteristic absorption band of retinal(from 570 to 605 nm) and the maximum excitation wavelengths of bacteriorhodopsin(from 326 to 331 nm) occurres at a narrow concentration range of HCl(11.5-13.3 mmol/L). The transition process performs within 0 to 2s.5. The results from high hydrostatic pressure test indicated that, when the pressure increases to 260MPa, the fluorescence spectra of bacteriorhodopsin in blue and purple membrane will not change. When the concentration of GuaHCl solution adding to blue and purple membrane increases to 8 mol/L, the denaturation of fluorescence spectra of bacteriorhodopsin do not occurred, and the changes of fluorescence spectra of bacteriorhodopsin will be observed when the hydrostatic pressure to this mixture solutions is 260MPa. The test results by high hydrostatic pressure and denaturant indicate that the tolerance of bacteriorhodopsin in blue and purple membrane are very satisfying.The intrinsic fluorescence of bacteriorhodopsin, bis-ANS binding and absorption spectra were used to study the conformational change and the temperature effects of bacteriorhodopsin in blue and purple membranes. The conformation of bacteriorhodopsin in purple and blue membranes is quite different. The process of the removal of bivalent cations on purple membranes surface of bacteriorhodopsin results in that the purple membrance becomes blue membrane, which not only influences the state of the chromophore retinal, but also induces the changes of the local environments of tryptophan and tyrosine residues and induces the change of hydrophobicity of bacteriorhodopsin surface. In other words, the fundamental changes of bacteriorhodopsin conformations will occurre along with the transition from purple membrane to blue membrane.