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Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C*

Open AccessPublished:May 05, 2000DOI:https://doi.org/10.1074/jbc.275.18.13431
      Evidence is presented for long range interactions between the extracellular and cytoplasmic parts of the heptahelical membrane protein bacteriorhodopsin in the mutant R82A and its second site revertant R82A/G231C. (i) In the double mutants R82A/G72C and R82A/A160C, with the cysteine mutation on the extracellular or cytoplasmic surface, respectively, the photocycle is the same as in the single mutant R82A with an accelerated deprotonation of the Schiff base and a reversed order of proton release and uptake. Proton release and uptake kinetics were measured directly at either surface by using the unique cysteine residue as attachment site for the pH indicator fluorescein. Whereas in wild type proton uptake on the cytoplasmic surface occurs during the M-decay (τ ∼ 8 ms), in R82A it occurs already during the first phase of the M-rise (τ < 1 μs). (ii) The introduction of a second mutation at the cytoplasmic surface in position 231 (helix G) restores wild type ground state absorption properties, kinetics of photocycle and of proton release, and uptake in the mutant R82A/G231C. In addition, kinetic H/D isotope effects provide evidence that the proton release mechanism in R82A/G231C and in wild type is similar. These results suggest the existence of long range interactions between the cytoplasmic and extracellular surface domains of bacteriorhodopsin mediated by salt bridges and hydrogen-bonded networks between helices C (Arg-82) and G (Asp-212 and Gly-231). Such long range interactions are expected to be of functional significance for activation and signal transduction in heptahelical G-protein-coupled receptors.
      bR
      bacteriorhodopsin
      wt
      wild type
      DMPC
      1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine
      BMF
      5-(bromomethyl)fluorescein
      CHAPS
      3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate
      MOPS
      4-morpholinepropanesulfonic acid
      A160C (G72C
      G231C), mutant with alanine (glycine) in position 160 (72, 231) replaced by cysteine
      R82A
      mutant with arginine in position 82 replaced by alanine
      A160C-MF(AF) (G72C-MF(AF) or G231C-MF(AF))
      mutant A160C (G72C or G231C) with (methyl)- or (acetamido)fluorescein, respectively, bound to cysteine
      Allosteric interactions are well known and of great importance for water soluble enzymes, where ligand or substrate binding to a specific site alters the conformation and changes the affinity at a different site of the protein (for reviews see Refs.
      • Fersht A.
      and
      • Kraut J.
      ). For membrane-bound G-protein-coupled receptors analogous effects are expected to be of considerable functional significance. For this class of proteins ligand binding occurs mostly on the extracellular surface resulting in the active form of the receptor with binding and activation of the G-protein at the opposite cytoplasmic surface of the protein. The membrane protein bacteriorhodopsin (bR)1 shares the heptahelical bundle motif with G-protein-coupled receptors. In this report, we present evidence for long range interactions between the extracellular and cytoplasmic parts of bR based on evidence from the mutant R82A and its second site revertant R82A/G231C.
      Bacteriorhodopsin acts as a light-driven proton pump in the plasma membrane of the archaebacterium Halobacterium salinarium. A retinylidene chromophore is bound via a protonated Schiff base linkage to Lys-216. Upon flash excitation bR undergoes a cyclic photoreaction with distinct spectroscopic intermediates and proton translocation from one side of the membrane to the other (for reviews see Refs.
      • Lanyi J.K.
      ,
      • Ebrey T.G.
      ,
      • Rothschild K.J.
      ). In the first half of this photocycle, the Schiff base becomes deprotonated during the transition to the M-intermediate, and in the same time range a proton is released at the extracellular surface (
      • Alexiev U.
      • Marti T.
      • Heyn M.P.
      • Khorana H.G.
      • Scherrer P.
      ). During the second half of the photocycle, the Schiff base is reprotonated from Asp-96 (
      • Otto H.
      • Marti T.
      • Holz M.
      • Mogi T.
      • Lindau M.
      • Khorana H.G.
      • Heyn M.P.
      ), which is located in the cytoplasmic part of the protein, and a proton is taken up from the cytoplasm. In the proton release pathway, Asp-85 has been identified as the primary acceptor of the proton from the Schiff base by site-directed mutagenesis and Fourier transform infrared difference spectroscopy (
      • Braiman M.S.
      • Mogi T.
      • Marti T.
      • Stern L.J.
      • Khorana H.G.
      • Rothschild K.J.
      ). Arg-82 is located one helix turn away from Asp-85, facing toward the proton channel (
      • Grigorieff N.
      • Ceska T.A.
      • Downing K.H.
      • Baldwin J.M.
      • Henderson R.
      ,
      • Luecke H.
      • Richter H.-T.
      • Lanyi J.K.
      ). Steady-state and time-resolved UV/Vis absorption (
      • Govindjee R.
      • Misra S.
      • Balashov S.P.
      • Ebrey T.G.
      • Crouch R.K.
      • Menick D.R.
      ,
      • Balashov S.P.
      • Imasheva E.S.
      • Govindjee R.
      • Ebrey T.G.
      ,
      • Richter H.-T.
      • Brown L.S.
      • Needleman R.
      • Lanyi J.K.
      ) and Fourier transform infrared (
      • Hatanaka M.
      • Sasaki J.
      • Kandori H.
      • Ebrey T.G.
      • Needleman R.
      • Lanyi J.K.
      • Maeda A.
      ) spectroscopy results indicate that Asp-85, Glu-204, Glu-194, and Arg-82 interact via a direct or indirect coupling of their pK values. Proton release to the extracellular medium may be facilitated by a hydrogen-bonded network between these residues (
      • Luecke H.
      • Richter H.-T.
      • Lanyi J.K.
      ,
      • Richter H.-T.
      • Brown L.S.
      • Needleman R.
      • Lanyi J.K.
      ,
      • Balashov S.P.
      • Imasheva E.S.
      • Ebrey T.G.
      • Chen N.
      • Menick D.R.
      • Crouch R.K.
      ,
      • Dioumaev A.K.
      • Richter H.-T.
      • Brown L.S.
      • Tanio M.
      • Tuzi S.
      • Saito H.
      • Kimura Y.
      • Needleman R.
      • Lanyi J.K.
      ). In the three-dimensional structure of the unphotolyzed bR, a defined density for the arginine 82 side chain, has recently been resolved by x-ray crystallography at a resolution of 2.5 (
      • Pebay-Peyroula E.
      • Rummel G.
      • Rosenbusch J.P.
      • Landau E.M.
      ) and 2.3 Å (
      • Luecke H.
      • Richter H.-T.
      • Lanyi J.K.
      ). The proposed movement of this side chain toward the extracellular surface upon formation of the M-intermediate, based on computer calculations (
      • Scharnagl C.
      • Fischer S.F.
      ), is not yet established. However, recent time-resolved electrical measurements support this proposal (
      • Dickopf S.
      • Heyn M.P.
      ).
      To further understand the mechanism of proton transport, we have studied the mutant R82A. In R82A an accelerated deprotonation of the Schiff base and a reversed order of proton release and uptake, detected with pH indicator dyes in the aqueous solution, were already observed (
      • Otto H.
      • Marti T.
      • Holz M.
      • Mogi T.
      • Stern L.J.
      • Engel F.
      • Khorana H.G.
      • Heyn M.P.
      ,
      • Balashov S.P.
      • Govindjee R.
      • Kono M.
      • Imasheva E.
      • Lukashev E.
      • Ebrey T.G.
      • Crouch R.K.
      • Menick D.R.
      • Feng Y.
      ). Moreover, in R82A the pK a of Asp-85, which controls the purple to blue transition, is increased by about 5 pH units compared with wild type (wt) (
      • Subramaniam S.
      • Marti T.
      • Khorana H.G.
      ). These dramatic changes are because of the replacement of the positively charged arginine by the neutral alanine.
      We have used time-resolved absorbance spectroscopy in combination with pH-sensitive dyes (pyranine in the aqueous bulk phase and fluorescein derivatives attached at specific sites on the protein surface) to detect the kinetics of the photocycle and of proton release and uptake. To detect proton concentration changes separately on each surface of the protein, the attachment site (single cysteine residue) for the pH indicator dye at the surface of the mutant protein R82A was varied. We have used the single cysteine residue in position 72 at the extracellular surface and in the positions 160 and 231 at the cytoplasmic surface as attachment sites. Fig.1 shows a tertiary structural model of bR based on x-ray crystallographic data (
      • Essen L.-O.
      • Siegert R.
      • Lehmann W.D.
      • Oesterhelt D.
      ). The positions of the residues Gly-72, Arg-82, Ala-160, and Gly-231 are indicated. Unexpectedly, the cysteine mutation in position 231 at the end of helix G on the cytoplasmic surface leads to an almost complete restoration of the photocycle, pK a of Asp-85, and proton pumping in the double mutant R82A/G231C. The cysteine double mutant R82A/A160C, also with a cysteine mutation on the cytoplasmic surface (EF loop), has on the other hand still all the altered properties of the single mutant R82A. The reversion effect observed only at position Gly-231 suggests an interaction between helices C (Arg-82) and G (Gly-231). The second site revertant produced by the replacement of glycine by cysteine at the cytoplasmic surface of the protein far away from the location of the first mutation (R82A) can shed light on the molecular mechanisms that promote long range interactions in 7-helix transmembrane proteins. Such allosteric interactions are expected to be important for ligand binding and signal transduction in G-protein-coupled receptors (
      • Thomas E.A.
      • Carson M.J.
      • Neal M.J.
      • Sutcliffe G.J.
      ).
      Figure thumbnail gr1
      Figure 1Tertiary structure of bacteriorhodopsin.The structure was obtained from x-ray crystallographic data (
      • Essen L.-O.
      • Siegert R.
      • Lehmann W.D.
      • Oesterhelt D.
      ), (1brr in WebLabViewer). The residues Gly-72, Ala-160, Gly-231, and Arg-82 are marked.

      EXPERIMENTAL PROCEDURES

      1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and Tris-(hydroxy-methyl)-aminomethane (Tris) were obtained from Sigma. Ethylenediaminetetraacetic acid and 1,4-dithio-dl-threitol were from Fluka. 8-Hydroxy-1,3,6-pyrene-trisulfonic acid trisodium salt (pyranine) was from Serva. Sephadex G-25 (fine) was from Amersham Pharmacia Biotech, 5-(iodoacetamido)fluorescein and 5-(bromomethyl)fluorescein (BMF) were from Molecular Probes.
      The preparation and expression of the bR mutants A160C and G231C inH. salinarium, in which cysteine replaces Ala-160 and Gly-231, respectively, and the preparation of R82A, in which alanine is substituted for arginine in position 82, has been reported (
      • Alexiev U.
      • Mollaaghababa R.
      • Scherrer P.
      • Khorana H.G.
      • Heyn M.P.
      ,
      • Krebs M.P.
      • Mollaaghababa R.
      • Khorana H.G.
      ). The bR double mutants R82A/A160C and R82A/G231C were prepared by following the described procedures (
      • Krebs M.P.
      • Mollaaghababa R.
      • Khorana H.G.
      ) except that the synthetic restriction fragments BspHI-Psp14061 andSphI-NotI were used, respectively, to construct the mutant genes.
      Solubilization of bR membrane fragments and regeneration of bR in DMPC/CHAPS micelles were performed as described (
      • Liao M.-J.
      • London E.
      • Khorana H.G.
      ). The regeneration procedure was modified according to Ref.
      • Scherrer P.
      • Alexiev U.
      • Marti T.
      • Khorana H.G.
      • Heyn M.P.
      . After regeneration the retinal band is shifted back completely to 550 nm, resulting in 96–100% regeneration, using an estimated extinction coefficent of ε550 ≈ 56,000 m−1cm−1. Labeling with 5-(iodoacetamido)- and 5-(bromomethyl)-fluorescein and the determination of the labeling stoichiometry were performed as described (
      • Alexiev U.
      • Mollaaghababa R.
      • Scherrer P.
      • Khorana H.G.
      • Heyn M.P.
      ,
      • Scherrer P.
      • Alexiev U.
      • Marti T.
      • Khorana H.G.
      • Heyn M.P.
      ).
      Flash spectroscopy and data analysis with a sum of exponentials were performed as described elsewhere (
      • Otto H.
      • Marti T.
      • Holz M.
      • Mogi T.
      • Lindau M.
      • Khorana H.G.
      • Heyn M.P.
      ). The excitation was with 10-ns pulses of 3–6 mJ of energy at 590 or 500 nm. Under these conditions about 15% of bR are cycling. Typically 30–50 time traces were averaged for the kinetics of the M-intermediate and 70–100 for the dye kinetics.
      Proton release and uptake were detected in the aqueous bulk medium of the purple membrane suspension, containing 4–15 μm bR in 150 mm KCl, by calculating the difference of the measured flash-induced absorbance changes at 450 nm between samples with and without 45 μm pyranine at pH 7.3 and 22 °C (
      • Alexiev U.
      • Mollaaghababa R.
      • Scherrer P.
      • Khorana H.G.
      • Heyn M.P.
      ,
      • Moltke S.
      • Alexiev U.
      • Heyn M.P.
      ). The light-induced proton concentration changes, detected in samples with fluorescein attached to cysteine residues in bR, were determined by calculating the measured flash-induced absorbance difference at 495 nm between samples with and without 10 mm Tris or MOPS buffer, pH 7.3, in 150 mm KCl at 22 °C (
      • Alexiev U.
      • Mollaaghababa R.
      • Scherrer P.
      • Khorana H.G.
      • Heyn M.P.
      ,
      • Moltke S.
      • Alexiev U.
      • Heyn M.P.
      ).
      Titration experiments and analysis were performed as described in (
      • Alexiev U.
      • Marti T.
      • Heyn M.P.
      • Khorana H.G.
      • Scherrer P.
      ). To obtain the apparent pK a, the measured absorbance changes were fitted with the Henderson-Hasselbalch equation.
      ΔA=ΔAmax/(1+10n(pKapH))
      Equation 1


      ΔA max is the maximal absorbance difference, n is the number of protons involved in the transitions and pK a is the midpoint of the titration.
      The data plotted in Fig. 4 were fitted with the following equation described by Balashov et al. (
      • Balashov S.P.
      • Govindjee R.
      • Kono M.
      • Imasheva E.
      • Lukashev E.
      • Ebrey T.G.
      • Crouch R.K.
      • Menick D.R.
      • Feng Y.
      ),
      ΔA=α/(α+β*γ)
      Equation 2


      with α = 1 + 10(pH − pK3), β = 1 + 10(pH − pK2), and γ = 10(pH − pK1).
      Figure thumbnail gr4
      Figure 4pH titration of the mutant R82A/G231C.Analysis of the pH titration data for the mutant R82A/G231C in 0.1% CHAPS/0.0025% DMPC and 150 mm KCl. The absorbance change at 628 nm, representing the fraction of blue membrane, was plotted as a function of pH. The pK values were analyzed with Equation according to the model proposed in Ref.
      • Balashov S.P.
      • Govindjee R.
      • Kono M.
      • Imasheva E.
      • Lukashev E.
      • Ebrey T.G.
      • Crouch R.K.
      • Menick D.R.
      • Feng Y.
      , which is presented in theinset.

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