紫膜与溶剂的相互作用

本文研究了溶剂正己烷,正十六烷,甲苯和二甲基甲酰胺(DMF dimethyl formamide)与紫膜的相互作用.吸收光谱,园二色谱和紫膜光循环中间产物M412的动力学过程的测量表明,在不同条件下,溶剂与紫膜能相互作用而影响到紫膜的光谱特性和光化学循环动力学过程.结果说明,在制作紫膜LB膜时,正己烷和正十六烷是合适的,使用二甲基甲酰胺时必须防止强光照射,甲苯则不能采用.     

Synthetic pigment analogues of the purple membrane protein.

Nonphysiological analogues of retinal have been shown to form pigments in reactions with the apoprotein of the purple membrane of Halobacterium halobium. Both the all-trans and 13-cis isomers of a retinal analogue, having an elongated chain with an extra double bond, formed pigments. Unlike the native all-trans and 13-cis retinal1-based pigments, the new pigments were not interconvertible with each other and were unstable against hydroxylamine. When incorporated into phospholipid vesicles, they showed no proton pumping activity upon illumination. The ability of the extended-length retinal to form pigments contrasts with its nonreactivity with opsin (apoprotein of rhodopsin), suggesting a less stringent binding site for the purple membrane chromophore. All-trans retinal2 also combined with bleached purple membrane to form a blue pigment absorbing at ca. 590 nm. Like the native purple membrane, the blu membrane showed proton pumping activity upon illumination in phospholipid vesicles.     

Fluorescence energy transfer studies of transmembrane location of retinal in purple membrane

A diffusion-enhanced energy transfer technique was employed for the determination of transmembrane location of the retinal chromophore in the purple membrane. Theoretical considerations showed that the rate of energy transfer from an energy donor embedded within a membrane to acceptors dissolved in solvent could be described by an analytical function of the distance a of closest approach between the donor and acceptor, if the "rapid-diffusion limit" was attained. The criterion for this limit was given by the relation: (RO)6 much less than 20D tau Da4, where RO is the characteristic distance of energy transfer, D is the diffusion coefficient of the acceptor and tau D is the fluorescence lifetime of the donor in the absence of acceptor. By photo-reduction of the purple membrane with sodium borohydride, the retinal chromophore was converted to a highly fluorescent derivative, which showed a broad emission band in the visible region. From analysis of the fluorescence decay curves of the photo-reduced purple membrane in the presence of various concentrations of cobalt-ethylenediamine tetraacetate (Co-EDTA: energy acceptor), the depth of the chromophore from the membrane surface was estimated to be 8 (+/-3) A. This result was supported by investigations of energy transfer processes in a system where the native purple membranes and the photo-reduced membranes were stacked in parallel: the energy acceptor in this system was the native retinal chromophore.     

Photochemical and chemical studies on the chromophore of bacteriorhodopsin.

The chromophore (purple complex) of bacteriorhodopsin is reduced by sodium borohydride upon illumination to RPhv with a three-peaked absorption band at 360 nm. Treatment of this reduction product with ultraviolet light or acid yields a modified product from which retro-retinyllysine can be obtained by alkaline hydrolysis. No reduction of the 412 nm complex was found. Under specific conditions the purple complex equilibrates with a photochemically active 460 nm form that can be reduced by borohydride in the dark. This reduction product RP460 behaves idential to RPHV. Reconstitution of the purple complex from chromophore-free membrane (apomembrane) and retinal occurs via intermediates. The first (lambdamax 400nm) shows a three-peaked absorption band and is reduced to RP400 without a change of the three-peaked absorption (lambdamax 360 nm). The same product is obtained from apomembrane and retinol. Detergents shift the absorption band to 330 nm in all cases. From the experiments described no participation of retro-retinal structures during the photochemical cycle can be concluded but stereospecific interaction of the retinal moiety with the protein resulting in a specific retinal conformation os omdocated by the spectral changes observed.     

Interpretation of the absorption and circular dichroic spectra of oriented purple membrane films.

The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.     

Immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate: solvent effect

The immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate was studied in twelve different solvents in order to deduce the solvent effect through an attempt to correlate the highest yield with such solvent properties as hydrophobicity (log P), dielectric constant (epsilon), and Hildebrand solubility parameter (delta). The results showed that the conversion of glycerol trioleate and yield of oleic acid methyl ester were quite dependent on the solvent. The catalyst lipase in various solvents also needed different optimum amount of water to keep its maximum activity, and generally this lipase in more hydrophobic solvents required more water. The correlation between the highest yield and log P value was found to be reasonable except deviation of data points of certain solvents, while no obvious correlation existed between the other two parameters, dielectric constant (epsilon) and Hildebrand solubility parameter (delta), and the enzyme activity. The study revealed that more hydrophobic solvents such as n-hexane or cyclohexane were more suitable solvents for Candida sp. 99-125 catalyzed transesterification of glycerol trioleate to oleic acid methyl ester.     

Optimization of organic solvent in multiphase biocatalysis

The microbial epoxidation of propene and 1-butene was used to study some fundamental aspects of two-liquid-phase biocatalytic conversions. Introduction of a water-immiscible organic solvent phase in a free-cell suspension gave rise to a series of undesired phenomena, e.g., inactivation by the solvent, clotting of biomass, and aggregation of cells at the liquid-liquid interface. Immobilization of the cells in hydrophilic gels, e.g., calcium alginate, prevented direct cell-organic solvent contact and the related clotting and aggregation of biomass. However, the gel entrapment did not seem to provide additional protection against the organic solvent. The influence of various organic solvents on the retention of immobilized-cell activity was related to solvent properties like the polarity (as expressed by the Hildebrand solubility parameter) and the molecular size (as expressed by the molecular weight or molar volume). High activity retention was favored by a low polarity in combination with a high molecular weight. The solubility parameter also proved useful to describe the capacity of various organic solvents for oxygen and alkene oxides. This facilitated the optimization of the solvent polarity.     

Altered protein-chromophore interaction in dicyclohexylcarbodiimide-modified purple membrane sheets

Previous studies of N,N'-dicyclohexylcarbodiimide (DCCD)-modified bacteriorhodopsin (Renthal, R. et al. (1985) Biochemistry 24, 4275-4279) used reaction conditions (detergent micelles) that are not optimal for subsequent physical studies. The present work describes new conditions for reaction of bacteriorhodopsin with DCCD in intact purple membrane sheets in the presence of 4.5% (v/v) diethylether and light. Like the detergent reaction system, the reaction is light induced, incorporates approximately 1 mol [14C]DCCD per mol bacteriorhodospin, and results in a bleached chromophore. Peptide mapping indicates that the likely site of modification in intact membranes is identical to the site in the detergent reaction system: Asp 115. The retinal chromophore of DCCD-modified purple membrane has an absorbance maximum at 390 nm and very little induced circular dichroism. The retinal is easily extracted in hexane, yielding a 3:1 ratio of all-trans to 13-cis retinal. Borohydride reduces the retinal onto the protein within the 1-71 region of the amino acid sequence. These results suggest that Asp-115 is near the retinal binding cavity of bacteriorhodopsin. When DCCD reacts with Asp 115, retinal is displaced from its binding site.     

Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity.

Specific lipids of the purple membrane of Halobacteria are required for normal bacteriorhodopsin structure, function, and photocycle kinetics [Hendler, R.W. & Dracheva, S. (2001) Biochemistry (Moscow)66, 1623-1627]. The decay of the M-fast intermediate through a path including the O intermediate requires the presence of a hydrophobic environment near four charged aspartic acid residues within the cytoplasmic loop region of the protein (R. W. Hendler & S. Bose, unpublished results). On the basis of the unique ability of squalene, the most hydrophobic purple membrane lipid, to induce recovery of M-fast activity in Triton-treated purple membrane, we proposed that this uncharged lipid modulates an electrostatic repulsion between the membrane surface of the inner trimer space and the nearby charged aspartic acids of the cytoplasmic loop region to promote transmembrane alpha-helical mobility with a concomitant increase in the speed of the photocycle. We examined Triton-treated purple membranes in various stages of reconstitution with native lipid suspensions using infrared spectroscopic techniques. We demonstrate a correlation between the vibrational half-width parameter of the protein alpha-helical amide I mode at 1660 cm-1, reflecting the motional characteristics of the transmembrane helices, and the lipid-induced recovery of native bacteriorhodopsin properties in terms of the visible absorbance maxima of ground state bacteriorhodopsin and the mean decay times of the photocycle M-state intermediates.     

Electric field effects in bacteriorhodopsin.

Exposure of aqueous suspensions of fragments of the purple membrane of Halobacterium halobium to electric field pulses leads to transient linear dichroism phenomena. The effects are interpreted in terms of field-induced alignments of the bacteriorhodopsin chromophore. Two observed relaxation times (tau) are attributed to rotation of the whole membrane fragments (tau s approximately 100 ms), and to a much faster reorientation of the chromophore within membrane (tau f approximately 260 microns).     

Location and orientation of the chromophore in bacteriorhodopsin: Analysis by fluorescence energy transfer

The spatial location and orientation of the retinal chromophore in bacteriorhodopsin were estimated from a fluorescence energy transfer study. The energy donor used in this study was a fluorescent retinal derivative, which was obtained by partial reduction of the purple membrane with sodium borohydride, and the energy acceptor was the native chromophore remaining in the same membrane. Since bacteriorhodopsin forms a two-dimensional crystal with P₃ symmetry in the purple membrane, and the membrane structure is maintained after the reduction, the rate of energy transfer from a donor to any acceptor existing in the same membrane can be calculated as a function of the location and orientation of the chromophores in the unit cell. Quantitative analyses of the fluorescence decay curve and the quantum yield, with various extents of reduction, enabled us to determine the most probable location and orientation. The result suggested that the chromophore was situated near the centre of the protein in such an orientation that the dipole-dipole interaction with neighbouring chromophores was close to minimum.     

Fluorescence properties of methyl 8-(2-anthroyl) octanoate, a solvatochromic lipophilic probe.

Fluorescence excitation and emission spectra, relative fluorescence quantum yield phi r and fluorescence lifetime tau of methyl 8-(2-anthroyl)-octanoate have been studied in a set of organic solvents covering a large scale of polarity and in the presence of water. In this probe, the 2-anthroyl chromophore exhibits quite remarkable and unique fluorescence properties. Thus, when going from n-hexane to methanol, the maximum emission wavelength lambda em max shifts from 404 nm to 492 nm while phi r and tau increase from 1 to 17.7 and from 0.91 ns to 13.5 ns, respectively. These increments are still more accentuated in the presence of water with estimated values of 526 nm for lambda em max, 27 for phi r and 20 ns for tau in this solvent. Because of the presence of a keto group which is a hydrogen bond acceptor and which can conjugate with the aromatic ring so as to provide the chromophore with a high dipole moment, the fluorescence properties of the probe strongly depend on the polarity of the surrounding medium. They can be accounted for in terms of general solvent effects (dipolar solute/solvent interactions) in the presence of aprotic solvents and in terms of specific solvent effects (hydrogen bonding) in protic solvents. Such properties of solvatochromism make the 2-anthroyl chromophore, after 8-(2-anthroyl)octanoic acid has been attached to phospholipids (E. Perochon and J.F. Tocanne (1991) Chem. Phys. Lipids 58, 7-17) a potential tool for studying microenvironmental polarity in biological membranes.     

Interpretation of the dissolution of insoluble peptide sequences based on the acid-base properties of the solvent

The dissolution process of model insoluble peptide sequences was investigated in view of the electron acceptor (AN) and electron donor (DN) solvent properties. The Alzheimer's disease-inducing (1-42) Abeta-amyloid peptide and its (1-21) fragment, the (66-97) transmembrane bradykinin B2 receptor sequence, and the strongly aggregated VVLGAAIV were selected as models of insoluble peptides. Solvents presenting similar AN and DN values failed, despite their polarities, to dissociate peptide chains (free in solution or bound to a polymer). The maximum solubility of these aggregated sequences was attained in solvents presenting the highest possible (AN-DN) values (in positive or negative mode). The AN-DN values ranged from approximately -20 to +80 and, notably, the lowest dissociation power was ascribed to solvents presenting values of approximately +40. The strong hydrogen bond donor water is located in this region, indicating that, for dissociation of specific insoluble segments, the solvent should appropriately combine its acid/base strength with the potential for van der Waals interactions. We also observed a sequence-dependent pH effect on peptide solubility confirmed through circular dichroism spectroscopy. This approach also revealed a complex but, in many cases, consistent influence of peptide conformation on its solubility degree, even when structure-inducing solvents were added. In conclusion, the random method of selecting solvents to dissolve insoluble and intractable peptide sequences, still in use by some, could be partially supplanted by the strategy described herein, which may be also applicable to other solute dissociation processes.     

Chromophore of Bacteriorhodopsin is Closer to the Cytoplasmic Surface of Purple Membrane: Fluorescence Energy Transfer on Oriented Membrane Sheets

Transmembrane location of the retinal chromophore, either native or reduced in situ to a fluorescent derivative, of the purple membrane of Halobacterium halobium was investigated with fluorescence energy transfer techniques. Single sheets of purple membrane, either native or reduced with borohydride, were adsorbed on polylysine-coated glass; the orientation, whether the exposed surfaces were cytoplasmic or extracellular, was controlled by adjusting the pH of the membrane suspension before the adsorption. On the exposed surface of the reduced membrane, a layer of cytochrome c, hemoglobin, or ferritin was deposited. The rate of excitation energy transfer from the fluorescent chromophore in the membrane to the colored protein was greater when the protein was on the cytoplasmic surface of the membrane than when it was on the extracellular surface. Analysis in which uniform distribution of the protein on the surface was assumed showed that the reduced chromophore is situated at a depth of <1.5 nm from the cytoplasmic surface. The location of the native retinal chromophore was examined by depositing a small amount of tris(2,2′-bipyridyl)ruthenium(II) complex on the native membrane adsorbed on the glass. Energy transfer from the luminescent complex to the retinal chromosphore was more efficient on the cytoplasmic surface than on the extracellular surface, suggesting that the native chromophore is also on the cytoplasmic side. From these and previous results we conclude that the chromophore, whether native or reduced, of bacteriorhodopsin is located at a depth of 1.0 ± 0.3 nm from the cytoplasmic surface of purple membrane.     

Measurement of the optical parameters of purple membrane and plant light-harvesting complex films with optical waveguide lightmode spectroscopy

Purple membrane (bacteriorhodopsin) and plant light-harvesting complexes (LHCII) were dried on the optical waveguide sensor with varying thicknesses in a wide range (from 20 to several hundreds of nanometers) and the optical parameters were studied with optical waveguide lightmode spectroscopy. It was found that applying the approximate 4-layer mode equations for the measured effective refractive indices resulted in unacceptable results for the optical parameters: with increasing thickness the refractive index decreased monotonously from 1.5 to 1.1. Therefore an inverse waveguide numerical method was developed and used to obtain reliable results from the experiments. The inverse method yielded an approximately constant (1.53) refractive index independently of the thickness for the purple membrane and LHCII films. Light-induced changes in the optical parameters of the purple membrane and LHCII films were also studied. For purple membrane films the most significant effect is the change in refractive index and absorption. For LHCII films prolonged illumination induced irreversible structural changes, most probably of thermo-optic origin.     

A comparison of the second harmonic generation from light-adapted, dark-adapted, blue, and acid purple membrane.

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.     

Angle of the retinal of bacteriorhodopsin in blue membrane

The electric dichroism of purple and cation-depleted (blue) membrane was measured in a.c. electric fields at saturation. A decrease of 5.5° in the direction of the chromophore transition moment with respect to the membrane normal was found upon removal of cations from purple membrane.     

Electrophoretic mobility of membrane fragments on a sucrose gradient. Application to isolated purple membrane fragments from Halobacterium halobium.

A simple technique for electrophoresis of particles is presented. The technique is based on running charged particles in a vertical tube along a sucrose gradient (20–50%). Purple membrane fragments from Halobacterium halobium were used to demonstrate the method. The migration of the fragments was linear with time in the region of 20 to 40% sucrose. Electrophoresis of purple membrane fragments under illumination, darkness, or darkness interrupted by short periods of illumination showed that at pH 4.5 the dark-adapted form of bacteriorhodopsin is less negative than its light-adapted form. At pH 6.5 and 8.5 no difference between these forms could be detected.     

Improved isolation procedures for the purple membrane of Halobacterium halobium.

Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.3.-3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.     

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)