Group-directed modification of bacteriorhodopsin by arylisothiocyanates. Labeling, identification of the binding site and topology

Group-directed hydrophobic modification of membrane-integrated protein segments by arylisothiocyanates is applied to bacteriorhodopsin. Labeling of purple membrane with phenylisothiocyanate and 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate results in covalent modification of a unique lysine epsilon-amino group of bacteriorhodopsin. Lysine residue 41, located in the amino-terminal chymotryptic fragment, has been identified as the arylisothiocyanate binding site by established sequencing techniques. The phenylisothiocyanate binding site is not accessible for the aqueously soluble analog p-sulfophenylisothiocyanate. Furthermore, the acid-induced bathochromic shift of the bound chromophore reagent is not observed following acidification of 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate-labeled purple membrane. The modification thus occurs in the hydrophobic membrane domain, providing further evidence for intramembraneous disposition of the modified protein segment. Light-induced proton translocation is preserved in reconstituted vesicles containing either phenylisothiocyanate-modified or 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate-modified bacteriorhodopsin.     

Structural determinants of purple membrane assembly

The purple membrane is a two-dimensional crystalline lattice formed by bacteriorhodopsin and lipid molecules in the cytoplasmic membrane of Halobacterium salinarum. High-resolution structural studies, in conjunction with detailed knowledge of the lipid composition, make the purple membrane one of the best models for elucidating the forces that are responsible for the assembly and stability of integral membrane protein complexes. In this review, recent mutational efforts to identify the structural features of bacteriorhodopsin that determine its assembly in the purple membrane are discussed in the context of structural, calorimetric and reconstitution studies. Quantitative evidence is presented that interactions between transmembrane helices of neighboring bacteriorhodopsin molecules contribute to purple membrane assembly. However, other specific interactions, particularly between bacteriorhodopsin and lipid molecules, may provide the major driving force for assembly. Elucidating the molecular basis of protein-protein and protein-lipid interactions in the purple membrane may provide insights into the formation of integral membrane protein complexes in other systems.     

Effect of fluorescamine modification of purple membranes on exciton coupling and light-to-dark adaptation

Purple membranes of $\text{Halobacterium}\text{,}\text{halobium}$ were modified with fluorescamine. At pH 8.8, with a molar ratio of fluorescamine to bacteriorhodopsin of 170, about 6 residues of lysine were modified while the arginines were not affected at all. Except for the appearance of the fluorescamine peak at 394 nm and some broadening of the chromophore peak at 570 nm, the absorption spectrum of bacteriorhodopsin was not significantly changed after modification. After fluorescamine modification, circular dichroism studies indicated loss of exciton coupling between bacteriorhodopsin molecules in the purple membrane. Rotational diffusion studies suggested enhanced mobility of the chromophore after modification. However, the spectral changes accompanying the light-to-dark adaptation of purple membranes were not prevented by fluorescamine modification. The implications of these findings are that exciton coupling between neighboring bacteriorhodopsin molecules in the purple membrane is not required for light-to-dark adaptation.     

Lipidic cubic phases as matrices for membrane protein crystallization

This review provides detailed procedures for the crystallization of membrane proteins via the lipidic cubic phase method. Bacteriorhodopsin-specific, hands-on protocols are given for (i) the preparation of bacteriohordopsin from purple membrane by monomerization in octylglucoside and gel filtration chromatography or by selective extraction after pre-treatment with dodecyl-trimethylammonium bromide, (ii) the incorporation of bacteriorhodopsin into lipidic cubic phases by mixing in vials or within coupled syringes and, (iii) the crystallization of bacteriorhodopsin in the lipidic matrix by adding a solid salt or an overlaying with a solution. References for further useful procedures and materials are listed in order to provide biochemists and crystallographers with all information that is necessary to grow crystals of the membrane protein bacteriorhodopsin.     

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)     

Electrooptical analysis of blue and of cation-regenerated bacteriorhodopsin

Blue bacteriorhodopsin was prepared by electrodialysis, cation-exchange chromatography and acidification. The electrooptical properties of these preparations compared to those of the native purple bacteriorhodopsin suggest that the blue bacteriorhodopsin has a smaller induced dipole moment than the native purple bacteriorhodopsin and that bound cations in the native bacteriorhodopsin stabilize the protein conformation in the membrane.Purple bacteriorhodopsin was regenerated by addition of potassium, magnesium or ferric ions to blue bacteriorhodopsin. Both spectrscopically and electrooptically the potassium- and ferric-regenerated samples are different from the native purple state. Although the magnesium-regenerated sample is spectroscopically similar to the native purple bacteriorhodopsin, the electrooptical properties are rather similar to those of the cation-depleted blue sample, suggesting that it is very difficult to re-stabilize protein structures once cations are depleted.     

Effects of modification of the tyrosine residues of bacteriorhodopsin with tetranitromethane.

Treatment of the purple membrane of Halobacterium halobium with tetranitromethane led to modification of tyrosine residues. Modification of more than 3-4 tyrosine residues per bacteriorhodopsin monomer caused a decrease in the light-induced proton-pumping ability of purple membrane in synthetic lipid vesicles, loss of the sharp X-ray-diffraction patterns characteristic of the crystal lattice, loss of the absorbance maximum at 560 nm, and change in the buoyant density of the membrane. No modification of lipid was detected. These changes were interpreted as a gradual denaturation of the protein component such that when 8-9 tyrosine residues are modified, no proton pumping is observed. Modification of less than 3-4 tyrosine residues with tetranitromethane caused an increse in light-induced proton pumping. It was possible to generate partly modified purple membrane which had completely lost the property of diffracting X-rays into the sharp pattern observed with native purple membrane, but which still retained the ability to pump protons in a vectorial manner. Retention of crystal lattice is not essential for proton pumping.     

生物素结合对紫膜结构和功能的影响

为了提高细菌视紫红质（ＢＲ）的可组装怀,使之使用于生物传感器等生物器件,利用生物素对紫膜进行修饰,使之可以被亲和素识别,从而可以定向的固定于固体支撑物表面。实验结果表明：生物素可以修饰紫膜表面的赖氨酸,修饰的程序依修饰的时间不同而有所不同,但即使被修饰２４小时的紫膜,其表面的赖氨酸仍然没有被完全修饰。同时,生物素的修饰不会影响紫膜的结构和功能,但是不同的结合位点对Ｍ４１２的衰减会产生不同的影响。这     

Reversible loss of crystallinity on photobleaching purple membrane in the presence of hydroxylamine

Structural changes of purple membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the purple membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact purple membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of purple membrane. The data provide novel insights into factors triggering purple membrane formation and structure.     

脱脂菌紫质与天然紫膜水合变化的比较

本实验通过不同水合度下天然紫膜、脱脂菌紫质吸附等温线分析、红外光谱对比,讨论了天然紫膜小磷脂、蛋白质、水三者作用关系,认为磷脂对天然紫膜中蛋白质表而一些极性基团的分布及水合有重要作用,这些位点的水合对蛋白质进一步水合变化起重要作用.     

Purple-to-blue transition of bacteriorhodopsin in a neutral lipid environment.

The red shift in the absorption maximum of native purple membrane suspensions caused by deionization is missing in lipid-depleted purple membrane, and the pK of the acid-induced transition is down-shifted to pH approximately 1.4 and has become independent of cation concentration (Szundi, I., and W. Stoeckenius. 1987. Proc. Natl. Acad. Sci. USA. 84:3681-3684). However, the proton pumping function cannot be demonstrated in these membranes. When native acidic lipids of purple membrane are exchanged for egg phosphatidylcholine or digalactosyldiglyceride, bacteriorhodopsin is functionally active in the modified membrane. It shows spectral shifts upon light-dark adaptation, a photocycle with M-intermediate and complex decay kinetics; when reconstituted into vesicles with the same neutral lipids, it pumps protons. Unlike native purple membrane, lipid-substituted modified membranes do not show a shift of the absorption maximum to longer wavelength upon deionization. A partial shift can be induced by titration with HCl; it has a pK near 1.5 and no significant salt dependence. Titration with HNO3 and H2SO4, which causes a complete transition in the lipid-depleted membranes, i.e., it changes their colors from purple to blue, does not cause the complete transition in the lipid-substituted preparations. These results show that the purple color of bacteriorhodopsin is independent of cations and their role in the purple-to-blue transition of native membranes is indirect. The purple and blue colors of bacteriorhodopsin are interpreted as two conformational states of the protein, rather than different protonation states of a counterion to the protonated Schiff base.     

The preparation of lipid-depleted bacteriorhodopsin

Bacteriorhodopsin, the protein of the purple membrane of Halobacterium halobium, was freed to the extent of 90–95% from the natural membrane lipids without loss of function. The residual lipid corresponded to less than 1 mol/mol of bacteriorhodopsin. Delipidation was achieved by treatment of the purple membrane with a mixture of the detergent dimethyldodecylamine oxide and sodium chloride. The detergent was removed by dialysis or by sucrose density gradient centrifugation. Analysis of the lipids removed and those still bound to bacteriorhodopsin was facilitated by the use of purple membrane preparations labelled with ³⁵S, ³²P, or ¹⁴C. The composition of the residual lipids associated with bacteriorhodopsin was similar to that of the total lipid in the purple membrane.     

The transverse location of tryptophan residues in the purple membranes of Halobacterium halobium studied by fluorescence quenching and energy transfer

Fluorescence quenching by a series of spin-labelled fatty acids is used to map the transverse disposition of tryptophan residues in bacteriorhodopsin (the sole protein in the purple membranes of Halobacterium halobium). A new method of data analysis is employed which takes into account differences in the uptake of the quenchers into the membrane. Energy transfer from tryptophan to a set of $\text{n-(9-}\text{anthroyloxy)}$ fatty acids is used as a second technique to confirm the transverse map of tryptophan residues revealed by the quenching experiments. The relative efficiencies of quenching and energy transfer obtained experimentally are compared with those predicted on the basis of current models of bacteriorhodopsin structure. Most of the tryptophan fluorescence is located near the surface of the purple membrane. When the retinal chromophore of bacteriorhodopsin is removed, tryptophan residues deep in the membrane become fluorescent. These results indicate that the deeper residues transfer their energy to retinal in the native membrane. The retinal moiety is therefore located deep within the membrane rather than at the membrane surface.     

Restricted motion of photoexcited bacteriorhodopsin in purple membrane containing ethanol.

The molecular motion of retinal within the purple membrane was investigated by flash-induced absorption anisotropies with or without ethanol. In the absence of ethanol, the measured anisotropies at several wavelengths exhibited almost the same slow decay. This slow decay was attributed to only the rotation of purple membrane sheet itself in the aqueous suspension. In the presence of ethanol, however, we observed the wavelength-dependent anisotropies. The fluidity of the purple membrane, investigated with a fluorescence anisotropy method, was increased by the addition of ethanol. These facts indicated that the characteristic motion of bacteriorhodopsin is induced in perturbed purple membrane with ethanol. The data analysis was performed, taking account of the overlapping of absorption from ground-state bacteriorhodopsin and photointermediates. The results showed that the rotational motion of photointermediates within the membrane was more restricted than that of nonexcited bacteriorhodopsin. The addition of ethanol facilitated the rotation of nonexcited protein, whereas it did not significantly affect the motion of photointermediates. The restricted motion of photointermediates is probably caused by a conformational change in them, which may hinder the rotation of monomer protein and/or induce the interaction between photointermediate and neighboring proteins.     

The antigenic structure and topography of bacteriorhodopsin in purple membranes as determined by interaction with monoclonal antibodies

The spatial organization and the antigenic structure of the bacteriorhodopsin molecule in the purple membrane were studied by immunochemical techniques. Five monoclonal antibodies directed against exposed parts of the protein molecule in the membrane were prepared and characterized. Antigenic determinants were localized in the bacteriorhodopsin polypeptide chain by analysis of the interaction between monoclonal antibodies and protein fragments. The structure of antigenic determinants was revealed by the interaction of monoclonal antibodies with (i) isolated bacteriorhodopsin fragments further modified by sequential Edman degradation and (ii) derivatives of bacteriorhodopsin obtained biosynthetically or by selective chemical modification. Five antigenic determinants were localized in the following parts of bacteriorhodopsin: < Glu'-Met²⁰ involving one of the 3 amino acid residues of the N-terminal part; Gly³³-Met⁵⁶ involving Asp³⁶ and/or Asp³⁸ and Phe⁴²; Phe¹⁵⁶-Met¹⁶³ involving Phe¹⁵⁶; Glu¹⁹⁴-Leu²⁰⁷ involving Glu¹⁹⁴; Pro²⁰⁰-Leu²⁰⁷.     

Effect of iodination of the purple membrane on the photocycle of bacteriorhodopsin

Bacteriorhodopsin in the purple membrane of Halobacterium halobium is coupled to a photocycle that results in the release and uptake of protons. The role of tyrosyl residues in the photocycle of bacteriorhodopsin has been investigated by the technique of chemical modifications of these residues by iodination and nitration. The studies indicate that modification of a tyrosyl residue accelerates M₄₁₂ formation, whereas modification of another type of tyrosine residue(s) accessible from the cytoplasmic surface of the purple membrane inhibits M₄₁₂ decay. The results support the hypothesis that a reversible deprotonation of tyrosine residues prior to and after M₄₁₂ formation in the photocycle are steps in the light-driven pathway of H⁺ translocation by bacteriorhodopsin.     

Dansylation of bacteriorhodopsin near the retinal attachment site

The purple membrane of $\text{Halobacterium}\text{halobium}$ was reacted with 5-dimethylaminonaphthalene-l-sulfonyl chloride (dansyl chloride) at pH 8.0. Chromophoric and functional properties of the product appear unaltered. Approximately 2 moles of dansyl group were incorporated per mole of bacteriorhodopsin, part bound to bacteriorhodopsin and part bound to lipids. Purification and fragmentation of the protein showed most of the dansyl modification in a fragment containing residues 33 to 56. Amino acid analysis indicates that the major dansylated site is lysine 40. We conclude that, contrary to published models, 1) bacteriorhodopsin folds in a way that exposes lysine 40 at the membrane surface, and 2) this side chain is not involved in the proton pump mechanism.     

Spontaneous transmembrane insertion of membrane proteins into lipid vesicles facilitated by short-chain lecithins

Functional reconstitution of the membrane protein bacteriorhodopsin into lipid vesicles is achieved by mixing aqueous suspensions of long-chain lecithins and purple membrane with the short-chain lecithin diheptanoylphosphatidylcholine (20 mol % of total lipid). The membrane protein is transmembranously inserted in the lipid bilayer of the vesicle and highly active as a light-energized proton pump. This rapid, easy, and gentle procedure might allow functional reconstitution of other membrane systems and isolated membrane proteins as well.     

The essential role of specific Halobacterium halobium polar lipids in 2D-array formation of bacteriorhodopsin.

The mechanism whereby bacteriorhodopsin (BR), the light driven proton pump from the purple membrane of Halobacterium halobium, arranges in a 2D-hexagonal array, has been studied in bilayers containing the protein, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and various fractions of H. halobium membrane lipids, by freeze fracture electron microscopy and examination of optical diffractograms of the micrographs obtained. Electron micrographs of BR/DMPC complexes containing the entire polar lipid component of H. halobium cell membranes or the total lipid component of the purple membrane, with a protein-to-total lipid molar ratio of less than 1:50 and to which 4 M NaCl had been added, revealed that trimers of BR formed into an hexagonal 2D-array similar to that found in the native purple membrane, suggesting that one or more types of the purple membrane polar lipids are required for array formation. To support this suggestion, bacteriorhodopsin was purified free of endogenous purple membrane lipids and reconstituted into lipid bilayer complexes by detergent dialysis. The lipids used to form these complexes are 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC) as the major lipid and, separately, each of the individual lipid types from the H. halobium cell membranes, namely 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-phosphate (DPhPGP), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-sulphate (DPhPGS), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol (DPhPG) and 2,3-di-O-phytanyl-1-O-[beta-D-Galp-3-sulphate-(1----6)-alpha-D- Manp-(1----2)-alpha-D-Glcp]-sn-glycerol (DPhGLS). When examined by freeze-fracture electron microscopy, only the complexes containing 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol- 1'-phosphate or 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol-1'-sulphate, at high protein density (less than 1:50, bacteriorhodopsin/phospholipid, molar ratio) and to which 4 M NaCl had been added, showed well defined 2D hexagonal arrays of bacteriorhodopsin trimers similar to those observed in the purple membrane of H. halobium.     

Photoreactions of bacteriorhodopsin at acid pH.

It has been known that bacteriorhodopsin, the retinal protein in purple membrane which functions as a light-driven proton pump, undergoes reversible spectroscopic changes at acid pH. The absorption spectra of various bacteriorhodopsin species were estimated from measured spectra of the mixtures that form at low pH, in the presence of sulfate and chloride. The dependency of these on pH and the concentration of Cl- fit a model in which progressive protonation of purple membrane produces "blue membrane", which will bind, with increasing affinity as the pH is lowered, chloride ions to produce "acid purple membrane." Transient spectroscopy with a multichannel analyzer identified the intermediates of the photocycles of these altered pigments, and described their kinetics. Blue membrane produced red-shifted KL-like and L-like products, but no other photointermediates, consistent with earlier suggestions. Unlike others, however, we found that acid purple membrane exhibited a very different photocycle: its first detected intermediate was not like KL in that it was much more red-shifted, and the only other intermediate detectable resembled the O species of the bacteriorhodopsin photocycle. An M-like intermediate, with a deprotonated Schiff base, was not found in either of these photocycles. There are remarkable similarities between the photoreactions of the acid forms of bacteriorhodopsin and the chloride transport system halorhodopsin, where the Schiff base deprotonation seems to be prevented by lack of suitable aspartate residues, rather than by low pH.