2-Hydroxy-5-nitrobenzyl bromide as a specific reagent for tryptophan residues in membrane proteins: bacteriorhodopsin as an example

The use of 2-hydroxy-5-nitrobenzyl bromide for the modification of tryptophan residues in integral membrane proteins is exemplified by its application to bacteriorhodopsin from Halobacterium halobium. Complete elimination of the unreacted reagent requires delipidation of the sample with detergents and posterior chromatography. This method also allows separation of the modified from the unmodified bacteriorhodopsin molecules. Modified molecules have lost the retinal, and are thus bleached, whereas the unmodified molecules appear to retain all the characteristics of solubilized native bacteriorhodopsin.     

Interaction of tyrosine residues with the chromophore in bacteriorhodopsin

We previously reported that the absorption spectrum at low temperatures of iodinated bacteriorhodopsin can be separated into four components with maxima at shorter wavelengths than in native bacteriorhodopsin. In this study, the time course of the formation of each spectral component after iodination was analyzed, revealing that these four components correspond to four different iodinated states of tyrosine residues interacting with the retinal chromophore of bacteriorhodopsin. Therefore at least two tyrosine residues interact with the chromophore of bacteriorhodopsin.     

High-resolution 13C NMR study of the topography and dynamics of methionine residues in detergent-solubilized bacteriorhodopsin

The proton transport membrane protein bacteriorhodopsin has been biosynthetically labeled with [methyl-13C]methionine and studied by high-resolution 13C NMR after solubilization in the detergent Triton X-100. The nine methionine residues of bacteriorhodopsin give rise to four well-resolved 13C resonances, two of which are shifted upfield or downfield due to nearby aromatic residues. Methionine residues located on the hydrophilic surfaces, on the hydrophobic surface, and in the interior of the protein could be discriminated by studying the effects of papain proteolysis, glycerol-induced viscosity increase, and paramagnetic broadening by spin-labels on NMR spectra. Such data were used to evaluate current models of the bacteriorhodopsin transmembrane folding and tertiary structure. T2 and NOE measurements were performed to study the local dynamics of methionine residues in bacteriorhodopsin. For the detergent-solubilized protein, hydrophilic and hydrophobic external residues undergo a relatively large extent of side chain wobbling motion while most internal residues are less mobile. In the native purple membrane and in reconstituted bacteriorhodopsin liposomes, almost all methionine residues have their wobbling motion severely restricted, indicating a large effect of the membrane environment on the protein internal dynamics.     

Archaerhodopsin-2, from Halobacterium sp. aus-2 further reveals essential amino acid residues for light-driven proton pumps.

We have isolated a retinal protein which differs from bacteriorhodopsin and archaerhodopsin and pumps out as many protons in the light as those proton pumps. We tentatively named it archaerhodopsin-2. We have cloned and sequenced the gene that encodes archaerhodopsin-2. The gene consists of 780-bp nucleotides for 259 amino acids with a molecular mass of 27,937 Da. The amino acid sequence of archaerhodopsin-2 is 56% identical to bacteriorhodopsin and 88% to archaerhodopsin, with a few gaps of a few amino acids in both cases. Although the amino acid sequence of archaerhodopsin has revealed 157 conserved residues common to bacteriorhodopsin, the sequence of archaerhodopsin-2 reduces that number to 133. Of these, 38 amino acids are also common to chloride pumps and 24 to all bacterial retinal proteins known to date.     

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.     

Immunological probes for bacteriorhodopsin. Identification of three distinct antigenic sites on the cytoplasmic surface

We have prepared site-specific immunological reagents to study the orientation and surface topography of the integral membrane protein bacteriorhodopsin. Monoclonal and polyclonal antibodies with strong affinity for antigenic determinants on proteolytic and cyanogen bromide fragments of bacteriorhodopsin have been isolated and characterized. Three distinct antibody binding sites have been identified on the cytoplasmic surface of bacteriorhodopsin. The first due is readily accessible in native bacteriorhodopsin and lies close to the COOH terminus. This binding site is lost when only three amino acid residues are removed from the COOH terminus. The second site, which is also near the COOH terminus, is located approximately within the 17 COOH terminal amino acid residues. The third site is in the fragment that comprises Tyr-83 to Met-118 and is probably contained in the short loop connecting the third and fourth helices. The use of COOH terminus-specific antibodies in determination of the orientation of bacteriorhodopsin molecules in the Halobacterium halobium membrane confirms the earlier conclusion that the COOH terminus is on the cytoplasmic side.     

The site of attachment of retinal in bacteriorhodopsin. The epsilon-amino group in Lys-41 is not required for proton translocation

Chymotryptic fragments C-1 (amino acids 72-248) and C-2 (amino acids 1-71) of bacteriorhodopsin have been shown previously to reassociate so as to regenerate the native bacteriorhodopsin chromophore in lipid/detergent mixtures and to form functional proton-translocating vesicles. The fragment C-2 has now been selectively methylated with formaldehyde and sodium cyanoborohydride to give the epsilon-dimethylamino derivatives of Lys-30, 40, and 41 in 96-99% average yield. The methylated and unmethylated C-2 fragments were identical in their ability to reassociate with fragment C-1 and retinal to regenerate the bacteriorhodopsin chromophore and to form functional proton-translocating vesicles. In contrast, dimethylation of the lysine residues of the C-1 fragment gave a derivative which did not form an active complex with unmethylated C-2. We conclude that the epsilon-amino group in Lys-41 is not required for Schiff's base formation with retinal at any step in the light-driven proton-translocation cycle.     

1H-15N-NMR studies of bacteriorhodopsin Halobacterium halobium. Conformational dynamics of the four-helical bundle.

Series of uniformly and selectively 15N-labeled bacteriorhodopsins of Halobacterium halobium (strain ET 1001) were obtained and a 1H-15N-NMR study was performed in methanol/chloroform (1:1) and 0.1 M NH4CHOO, medium which mimics that in the membrane in vivo. Less than half of the cross-peaks expected from the amino acid sequence of uniformly 15N-labeled bacteriorhodopsin were observed, using heteronuclear 1H-15N coherence spectroscopy. In order to assign the observed cross-peaks, a selective 15N-labeling of amino acid residues (Tyr, Phe, Trp, Lys, Gly, Leu, Val or Ile) was carried out and 1H-15N-NMR spectra of bacteriorhodopsin and its fragments C1 (residues (72-231), C2 (residues 1-71), B1 (residues 1-155) and BP2 (residues 163-231) were investigated. By this procedure, all observed 1H-15N cross-peaks of the entire bacteriorhodopsin were found to belong to the transmembrane segments A, B and G. The cross-peaks from four (C, D, E and F) helical bundles (79-189 residues) were missed. These results clearly indicate that dynamic processes occur in the four helice bundle. The significance of this, in respect to bacteriorhodopsin functioning, is discussed.     

Re-Introduction of Transmembrane Serine Residues Reduce the Minimum Pore Diameter of Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2.     

Conformational change in bacterio-opsin on binding to retinal.

The detailed mechanism of retinal binding to bacterio-opsin is important to understanding retinal pigment formation as well as to the process of membrane protein folding. We have measured the temperature dependence of bacteriorhodopsin formation from bacterio-opsin and all-trans retinal. An Arrhenius plot of the apparent second-order rate constants gives an activation energy of 11.6 +/- 0.7 kcal/mol and an activation entropy of -4 +/- 2 cal/mol deg. Comparison of the activation entropy to model compound reactions suggests that chromophore formation in bacteriorhodopsin involves a substantial protein conformational change. Cleavage of the polypeptide chain between residues 71 and 72 has little effect on the activation energy or entropy, indicating that the connecting loop between helices B and C is not involved in this conformational change.     

Effect of the removal of the COOH-terminal region of bacteriorhodopsin on its light-induced H+ changes.

Removal of the COOH-terminal region of bacteriorhodopsin by digestion with trypsin or papain reduces the yield of light-induced H+ release by 50-70%. The rate of H+ release is not affected significantly, but the half time of H+ uptake increases almost twofold. However, there is no effect on the photocycle of bacteriorhodopsin as judged by the yield and decay kinetics of the M412 photointermediate. The H+:M ratio in enzyme-digested membranes is approximately 0.4-0.8, whereas untreated membranes have a H+:M ratio of approximately 2. Purple membrane sheets stored in distilled water at 4 degrees C for prolonged periods also have a low H+:M ratio, probably due to protease activity associated with bacterial contamination. Electrophoresis on sodium dodecylsulfate-polyacrylamide gels showed that both the enzyme-treated and the stored purple membrane samples have a higher electrophoretic mobility compared to the fresh preparation. The reduction in molecular weight can be accounted for by the loss of several residues from the COOH-terminal portion of the bacteriorhodopsin. We propose that the COOH-terminal region is partially responsible for the high yield of H+ release by the purple membrane.     

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.     

Fourier transform infrared difference spectroscopy of bacteriorhodopsin and its photoproducts regenerated with deuterated tyrosine

Fourier transform infrared (FTIR) difference spectroscopy has been used to detect the vibrational modes due to tyrosine residues in the protein that change in position or intensity between light-adapted bacteriorhodopsin (LA) and other species, namely, the K and M intermediates and dark-adapted bacteriorhodopsin (DA). To aid in the identification of the bands that change in these various species, the FTIR spectra of the free amino acids Tyr-d0, Tyr-d2 (2H at positions ortho to OH), and Tyr-d4 (2H at positions ortho and meta to OH) were measured in H2O and D2O at low and high pH. The characteristic frequencies of the Tyr species obtained in this manner were then used to identify the changes in protonation state of the tyrosine residues in the various bacteriorhodopsin species. The two diagnostically most useful bands were the approximately 1480-cm-1 band of Tyr(OH)-d2 and the approximately 1277-cm-1 band of Tyr(O-)-d0. Mainly by observing the appearance or disappearance of these bands in the difference spectra of pigments incorporating the tyrosine isotopes, it was possible to identify the following: in LA, one tyrosine and one tyrosinate; in the K intermediate, two tyrosines; in the M intermediate, one tyrosine and one tyrosinate; and in DA, two tyrosines. Since these residues were observed in the difference spectra K/LA, M/LA, and DA/LA, they represent the tyrosine or tyrosinate groups that most likely undergo changes in protonation state due to the conversions. These changes are most likely linked to the proton translocation process of bacteriorhodopsin.     

Membrane proteins as light-energy transducers

Monoclonal antibodies to different parts of bacteriorhodopsin were raised to define its topography in the membrane. It is shown that the amino acid residue Glu 194 is a part of an antigenic determinant and should be located on the membrane surface. We found that the removal of the C-terminal 17 amino acid sequence does not affect the efficiency of the proton transport in bacteriorhodopsin. From a combination of proteolysis and secondary structure prediction methods an experimentally testable structural model for bovine rhodopsin is presented. The complete amino acid sequence of the transducin γ-subunit consisting of 69 residues was determined.     

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.     

Structure-function studies on bacteriorhodopsin. X. Individual substitutions of arginine residues by glutamine affect chromophore formation, photocycle, and proton translocation

We have individually replaced all 7 of the arginine residues in bacteriorhodopsin by glutamine. The mutants with substitutions at positions 7, 164, 175, and 225 showed essentially the wild-type phenotype in regard to chromophore regeneration, chromophore lambda max, and proton pumping, although the mutant Arg-175----Gln showed decreased rate of chromophore regeneration. Glutamine substitutions of Arg-82, -134, and -227 affected proton pumping ability, and caused specific alterations in the bacteriorhodopsin photocycle. Finally, electrostatic interactions are proposed between Arg-82 and -227, and specific carboxylic acid residues in helices C and G, which regulate the purple to blue transition and proton transfers during the photocycle.     

High-resolution solid state 13C NMR of bacteriorhodopsin: characterization of [4-13C]Asp resonances.

Solid state 13C nuclear magnetic resonance measurements of bacteriorhodopsin labeled with [4-13C]Asp show that resonances of single amino acids can be resolved. In order to assign and characterize the resonances of specific Asp residues, three different approaches were used. (1) Determination of the chemical shift anisotropy from side-band intensities provides information about the protonation state of Asp residues. (2) Relaxation studies and T1 filtering allow one to discriminate between resonances with different mobility. (3) A comparison of the spectra of light- and dark-adapted bacteriorhodopsin provides evidence for resonances from aspartic acid residues in close neighborhood of the chromophore. In agreement with other investigations, four resonances are assigned to internal residues. Two of them are protonated in the ground state up to pH 10 (Asp96 and Asp115). All other detected resonances, including Asp85 and Asp212, are due to deprotonated aspartic acid. Two lines due to the two internal deprotonated groups change upon dark and light adaptation, whereas the protonated Asp residues are unaffected.     

The influence of a transmembrane pH gradient on protonation probabilities of bacteriorhodopsin: the structural basis of the back-pressure effect

Bacteriorhodopsin pumps protons across a membrane using the energy of light. The proton pumping is inhibited when the transmembrane proton gradient that the protein generates becomes larger than four pH units. This phenomenon is known as the back-pressure effect. Here, we investigate the structural basis of this effect by predicting the influence of a transmembrane pH gradient on the titration behavior of bacteriorhodopsin. For this purpose we introduce a method that accounts for a pH gradient in protonation probability calculations. The method considers that in a transmembrane protein, which is exposed to two different aqueous phases, each titratable residue is accessible for protons from one side of the membrane depending on its hydrogen-bond pattern. This method is applied to several ground-state structures of bacteriorhodopsin, which residues already present complicated titration behaviors in the absence of a proton gradient. Our calculations show that a pH gradient across the membrane influences in a non-trivial manner the protonation probabilities of six titratable residues which are known to participate in the proton transfer: D85, D96, D115, E194, E204, and the Schiff base. The residues connected to one side of the membrane are influenced by the pH on the other side because of their long-range electrostatic interactions within the protein. In particular, D115 senses the pH at the cytoplasmic side of the membrane and transmits this information to D85 and the Schiff base. We propose that the strong electrostatic interactions found between D85, D115, and the Schiff base as well as the interplay of their respective protonation states under the influence of a transmembrane pH gradient are responsible for the back-pressure effect on bacteriorhodopsin.     

Photoreaction of tyrosin-iodinated bacteriorhodopsin at low temperature

To elucidate the role of tyrosine residues in the shift of max and the light-driven proton pump of bacteriorhodopsin~ the photochemical reaction of tyrosine-iodinated bacteriorhodopsin (tyr-mod-bR) was investigated by low-temperature spectrophotometry. After 4–5 of 11 tyrosine residues of bacteriorhodopsin were iodinated, the meta-intermediate of tyr-mod-bR in 75% glycerol solution became so stable that its decay could be observed even at room temperature and i t was stable in the dark for several hours at –65°C.Four batho-intermediates were formed by irradiation with green light (500 nm) at –170°C. Like native bacteriorhodopsin, these batho-intermediates were photoreversible at –170°C. Four corresponding meta-intermediates were also formed by irradiation at –60°C. Using the difference spectra between meta-intermediates and tyr-mod-bR, the absorption spectra of four kinds of tyr-mod-bRs, batho-intermediates, and meta-intermediates were estimated. Each was at shorter wavelengths than that of its corresponding type in native bacteriorhodopsin. The results indicate that two or more tyrosine residues have some role in determining color in native bacteriorhodopsin.     

A new double-labelling procedure for determination of amino acid composition: application to bacteriorhodopsin

A new double-labelling procedure for amino acid analysis which requires only routine chromatographic equipment is described. When 1-fluoro-2,4-dinitro[3H]benzene is reacted with a mixture of 14C-labelled amino acids followed by reaction with the same 14C-labelled amino acid mixture diluted with an unlabelled sample of amino acids, the 3H:14C ratio in the resulting 2,4-dinitrophenyl (DNP) amino acid derivatives of the diluted sample will be increased in proportion to the quantity of unlabelled amino acid in the diluted sample. This procedure gave reliable results when applied to the known proteins insulin and lysozyme. The procedure is most advantageous when applied to amino acids which are unstable during acid hydrolysis or present in low molar fractions. When applied to the analysis of the bacteriorhodopsin in Halobacterium cutirubrum, this procedure showed the presence of one histidine residue and four tryptophan residues per mole protein but no cystine or cysteine; in general, the analyses obtained were consistent with those originally reported by Oesterhelt, D. and Stoeckenius, W. (1971) (Nature (London) New Biol. 233, 149-152) for bacteriorhodopsin of H. halobium.