Ordered Structure Formation of Bacteriorhodopsin-hDHFR in a

The gene encoding hDHFR from Haloferax volcanii (H. volcanii) was conjugated to that encoding bR (bop) from Halobacterium salinarum (H. salinarum)...
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Langmuir 1999, 15, 214-220

Ordered Structure Formation of Bacteriorhodopsin-hDHFR in a Plasma Membrane S. Nomura,† N. Kajimura,† K. Matoba,† T. Miyata,† R. Ortenberg,‡ M. Mevarech,‡ H. Kamikubo,§ M. Kataoka,§ and Y. Harada*,† Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan, Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Tel-Aviv 69978, Israel, and Department of Earth and Space Science, Osaka University, Toyonaka 560-0043, Japan Received June 24, 1998. In Final Form: November 2, 1998 This paper describes a novel surface-processing technique aimed at the in vivo formation of ordered functionalized structures on surfaces. The essential feature of this technique is the utilization of an intrinsic and stable two-dimensional crystal of bacteriorhodopsin (bR) as a template. A simple technique to form a functional chimera of the halophilic enzyme dihydrofolate reductase (hDHFR) with bR is demonstrated. The gene encoding hDHFR from Haloferax volcanii (H. volcanii) was conjugated to that encoding bR (bop) from Halobacterium salinarum (H. salinarum). This chimera was expressed in a bop-deficient strain of H. salinarum. The novel bifunctional fusion protein bR-hDHFR was localized in the plasma membrane of H. salinarum and retained the intrinsic characteristics of each component. Microscopic patches composed of ordered bR-hDHFR molecules were observed on the plasma membrane by electron microscopy. The hDHFR portion of the chimera was detected on the cytoplasmic side of each patch, which confirms that the molecular orientation of the fused proteins was vectorially controlled. The molecular packing of the fusion protein closely resembled the ordered structure of the wild-type bR in the “purple membrane”, which forms a two-dimensional crystal. This technique for the immobilization of functional proteins on a surface is applicable to a wide range of proteins for organizing ordered supramolecular surfaces on the nanometer scale.

Introduction During the last dozen years, various methods for organizing functional protein molecules into ordered structures on the nanometer scale have been investigated.1 The objectives of these investigations were the construction of functional supramolecular surface structures that could be used as biosensors and bioreactors. These objectives were partially fulfilled by recombinant DNA techniques, which produced histidine-tagged proteins or cysteineintroduced proteins immobilized on substrata.2,3 Our basic concept for the construction of ordered protein structures was the utilization of the intrinsic twodimensional crystal of bacteriorhodopsin (bR, 26.7 kDa) as a template. Bacteriorhodopsin, isolated from Halobacterium salinarum (H. salinarum), is a retinal-containing protein composed of seven trans-membrane helices and functions as a light-energy-driven proton pump.4 This protein shows an exceptionally strong tendency to form a self-assembled ordered structure in vivo and in vitro, called the “purple membrane” (PM).5-7 The application of self-assembled proteins as templates has been previously * Corresponding author. † Biomolecular Engineering Research Institute. ‡ Tel-Aviv University. § Osaka University. (1) Beyer, D.; Bohanon, T. M.; Knoll, W.; Ringsdorf, H.; Elender, G.; Sackmann, E. Langmuir 1996, 12, 2514. (2) Dietrich, C.; Boscheinen, O.; Scharf, K.-D.; Schnitt, J.; Tampe´, R. Biochemistry 1996, 35, 1100. (3) Vigmond, S. J.; Iwakura, M.; Mizutani, F.; Katsura, T. Langmuir 1994, 10, 2860. (4) Lanyi, J. K. Nature 1995, 375, 461. (5) Fisher, K. A.; Stoeckenius, W. Science 1977, 197, 72. (6) Usukura, J.; Yamada, E.; Tokunaga, F.; Yoshizawa, T. J. Ultrastruct. Res. 1980, 70, 204. (7) Henderson, R.; Baldwin, J. M.; Ceska, T. A.; Zemlin, F.; Beckman, E.; Downing, K. H. J. Mol. Biol. 1990, 213, 899.

demonstrated in the process of fixation of chemically modified PM onto an inert base8 and the in situ nucleation of CdS onto bacterial S-layers.9 These attempts, however, had the common drawback of stoichiometry: namely, the molecular ratio of the proteins that form the substratum to the overlaid materials could not be controlled. We propose here a novel and strictly stoichiometric method of exploiting two-dimensionally ordered protein arrays by a model system composed of a soluble monomeric protein: halophilic dihydrofolate reductase (hDHFR, 19.3 kDa) from Haloferax volcanii (H. volcanii) fused to bR.10 We show for the first time that hDHFR can be stoichiometrically immobilized on the PM by fusion to bR, which functions as an anchor, as schematically shown in Figure 1. Materials and Methods Bacterial Strains, Vectors, and Chemicals. E. coli DH5R for plasmid preparation was purchased from Takara Shuzo (Otsu, Japan). It was cultivated in an LB medium. The bR-deficient strain of H. salinarum L33, provided by Prof. Lanyi (University of California, Irvine), was grown according to the published procedure.11 The expression vector for H. salinarum pUCNov∆bop (Figure 2), which lacks the gene for bacterioopsin (bop) and carries the novobiocin resistance gene, was obtained from Dr. Ihara (Nagoya University, Japan). Novobiocin (Sigma), dissolved in ethanol, was added to the medium at a final concentration of 1 mg/L for selection. Trimethoprim (2,4-diamino-5-[3,4,5-trimethoxybenzyl]pyrimidine (Sigma)) was used as a selection (8) Harada, Y.; Yasuda, K.; Nomura, S.; Kajimura, N.; Sasaki, Y. C. Langmuir 1998, 14, 1829. (9) Shenton, W.; Dietmar, P.; Sleytr, U. B.; Mann, S. Nature 1997, 389, 585. (10) Zusman, T.; Rosenshine, I.; Boehm, G.; Jaenicke, R.; Leskiw, B.; Mevarech, M. J. Biol. Chem. 1989, 264, 18878. (11) Oesterhelt, D.; Stoeckenius, W. Methods in Enzymology; Academic Press: New York, 1974; Vol. 31, p 667.

10.1021/la980742u CCC: $18.00 © 1999 American Chemical Society Published on Web 12/12/1998

Structure Formation of Bacteriorhodopsin-hDHFR

Figure 1. Schematic drawing of the predicted two-dimensionally ordered structure of the bacteriorhodopsin fusion protein within the plasma membrane. In the native purple membrane, bacteriorhodopsin molecules form islands within a lipid sea.

Figure 2. Construction of the vector pUCBLD44, expressing the fusion protein bR-hDHFR. The black region in pUCNov∆bop was derived from pUC18, and the white region from H. volcanii. AmpR, ampicillin resistance gene; NovR, novobiocin resistance gene derived from H. volcanii; E. coli ori, the pUC18 vector replication origin. This vector does not contain an H. salinarum replication origin. Stable transformants were produced by integration of the plasmid into the H. salinarum chromosome. marker for screening bR-hDHFR overexpressing clones. Dihydrofolic acid (FH2) and NADPH were purchased from Sigma. All salts employed were of analytical grade. Rabbit antiserum for bR detection was provided by Prof. Sugiyama (Nagoya University, Japan). Cloning and Expression of bR-Fused hDHFR. The recombinant plasmid for the expression of the bR-hDHFR fusion protein was constructed as described in Figure 2. A fusion gene, BLD44 (1710 bp), was first constructed. The coding sequence of bop (744 bp, 248 aa) along with its upstream promoter Pbop was fused at the 3′ end to the H. volcanii gene encoding the hDHFR (486 bp, 162 aa) via a linker composed of the nucleotide sequence encoding the first 31 amino acids of the H. salinarum ferredoxin gene.12 The plasmid pUCBLD44 was constructed by introducing BLD44 into the BamH I-Xba I sites of pUCNov∆bop. After H. salinarum L33 was transformed with pUCBLD44, according to the reported transformation procedure,13 recombinant clones were selected with medium containing 1 mg/L novobiocin and 1 mg/L trimethoprim. H. salinarum S9, a strain overexpressing the wild-type bR, and the transformants of H. salinarum L33 were grown by shaking under limited aeration at 110 rpm and (12) Pfeifer, F.; Griffig, J.; Oesterhelt, D. Mol. Gen. Genet. 1993, 239, 66. (13) Cline, S. W.; Lam, W. L.; Charlebois, R. L.; Schalkwyk, L. C.; Doolittle, W. F. Can. J. Microbiol. 1989, 35, 148.

Langmuir, Vol. 15, No. 1, 1999 215 42 °C for 1 week. At the end of the logarithmic growth phase, the aeration was further limited by reducing the shaking speed to 90 rpm, and the cultures were illuminated for one night to induce the bop promoter. Isolation of Membrane Fraction. Cultured cells were harvested by centrifugation at the end of the logarithmic growth phase. The PM and the membrane fractions containing the fusion protein were prepared from cell pellets according to the reported procedure.11 Isolation of the membrane fraction was performed by sucrose density gradient ultracentrifugation at 25 000 rpm for more than 16 h, in a SW41 or SW28 rotor (Beckman), at 4 °C with an initial density gradient range of 34-68% (w/v). The purple bands (membrane fraction) were collected with syringes and were concentrated by ultracentrifugation. The pellets were washed three times with a low salt buffer, 10 mM HEPES (pH 7.0) and 100 mM NaCl, by ultracentrifugation, using a 45Ti rotor (Beckman) at 25 000 rpm for 30 min at 4 °C. The protein content was determined by SDS-polyacrylamide gel electrophoresis (PAGE). Protein bands were visualized with Coomassie Brilliant Blue staining and/or by subsequent western blotting using a poly(vinylidene difluoride) membrane (Immobilon P, Millipore Ltd.). Blotting was done in a buffer of 25 mM Tris-HCl (pH 7.5), 200 mM glycine, and 20% methanol. The bands were detected with rabbit anti-bR and anti-hDHFR sera and were visualized by an enzyme-linked immunosorbent assay (ELISA) using the VECTASTAIN ABC kit (Amersham). Enzymatic Activity Assay. The hDHFR enzymatic activity was measured in 25 mM potassium phosphate-citrate buffer (pH 6.0), containing 3 M KCl, 0.05 mM FH2, and 0.08 mM NADPH.10 The samples and the NADPH were pre-equilibrated at 25 °C, and the reaction was initiated by the addition of substrate (FH2). The initial velocities of NADPH (340 ) 6200 M-1 cm-1) oxidation coupled with FH2 reduction were measured by monitoring the change in absorbance at 340 nm that accompanies the reaction.14 At high substrate concentrations, the initial velocities were measured at 340-380 nm, using the appropriate extinction coefficients. In all assays, the nonenzymatic decomposition of the substrates was measured by excluding the enzyme, and its effect was subtracted from the enzyme-catalyzed reaction. Kinetic constants were calculated from the Lineweaver-Burke plots. The concentrations of substrate and inhibitor (methotrexate) were estimated from the absorbance, where the extinction coefficients were 28 000 M-1 cm-1 for FH2 at 282 nm, pH 7.4, and 22 100 M-1 cm-1 at 302 nm for methotrexate in 0.1 M KOH.14 Spectroscopy. The UV-vis absorption and circular dichroism (CD) spectra at 25 °C were measured with a DU640 spectrometer (Beckman) and a J-720 spectropolarimeter (Japan Spectroscopic Co., Ltd, Hachioji, Japan), respectively. The scanning speed for the CD spectra was 200 nm/min, and the spectra were integrated 60 times. The membrane fractions, dispersed in 10 mM HEPES (pH 7.0) and 100 mM NaCl, were gently suspended to minimize light scattering. Flash photolytic experiments with the native and the derivatized PM were done at room temperature in 10 mM HEPES (pH 7.0) and 100 mM NaCl. The sample droplets on slide glasses were excited with a 300 mW YAG laser at 532 nm. The absorption at 410 nm was monitored, and the timeresolved absorbance decays were recorded on a digital oscillograph. Electron Microscopy. The samples for the electron microscopic observation were prepared by four procedures: two immunochemical labeling methods for the membrane fractions and the ultrathin frozen sections of the cells, the conventional negative staining procedure for the membrane fractions, and the freeze-fracture replica method for the plasma membranes. (1) Immunolabeling of the membrane fractions was performed as follows. Purified specimens were deposited on the ionized carbon-coated grids. After two rinses with phosphate-buffered saline (PBS, pH 7.4) and subsequent blocking with PBS containing 1% bovine serum albumin (BSA) (1% BSA-PBS) for 5 min, the specimens were decorated for 1 h at room temperature with either anti-bR serum or anti-hDHFR serum appropriately diluted in 1% BSA-PBS. After three PBS rinses, the specimens were incubated in a protein A-gold (10 nm) conjugate (British BioCell, (14) Basran, J.; Casarotto, M. G.; Basaran, A.; Roberts, G. C. K. Protein Eng. 1997, 10 (7), 815.

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Figure 3. (A) Isolation by sucrose density gradient centrifugation of the wild-type bR fraction (purple membrane) from H. salinarum S9, the bR-hDHFR fraction from a recombinant H. salinarum L33, and a mixture of the bR and bR-hDHFR fractions. The two bands (arrowheads) corresponding to bR and bR-hDHFR are purple. (B) Electrophoretic fractionation on 12.5% polyacrylamide gels of the plasma membrane fractions of H. salinarum S9 and H. salinarum L33 overexpressing bR-hDHFR: (1) Coomassiestained gel; (2) western blot probed with antiserum against bR; (3) western blot probed with antiserum against hDHFR. The fusion protein bR-hDHFR was the dominant component produced by recombinant H. salinarum L33. Cardiff, U.K.) in 1% BSA-PBS for 1 h at room temperature. After successive rinses with PBS and pure water, the specimens were stained with 1% uranyl acetate and were air-dried. (2) Immunolabeling of the ultrathin sections of H. salinarum cells was essentially performed according to the reported procedure,15 with a slight modification for adaptation to the halophilic characteristics of H. salinarum. Cell pellets were washed with a basal salt buffer13 and then were fixed with a solution containing 0.2% glutaraldehyde (Wako, Osaka, Japan), 4% paraformaldehyde (Merck), 25% NaCl, and 1% CaCl2, for 2 h at room temperature. After centrifugation, the cell pellets were resuspended in 4% paraformaldehyde, 25% NaCl, and 1% CaCl2, for 2 days at room temperature. After a wash with 25% NaCl and 1% CaCl2, the specimens were embedded in gelatin gels by adding an excess of 10% gelatin in PBS, prewarmed at 55 °C. After centrifugation at room temperature, the cell pellets embedded in the gelatin were solidified to gels on ice. Sucrose was then infused into tiny blocks of the gel pellets by immersion in an excess volume of 1.6 M sucrose and 25% poly(vinylpyrrolidone) (MW 10 000, Sigma) in PBS for 2 h or overnight. After the gels were mounted onto the sample carrier, they were quickly frozen by immersion in liquid nitrogen and were sliced with a Leica cryosectioning system (Leica) at -80 °C to prepare the 85 nm thick ultrathin frozen sections. The ultrathin sections were then transferred from the knife edge to the surfaces of droplets of 2 M sucrose in PBS including 0.75% gelatin, suspended on a fine platinum wire, transferred to the supporting films of Formvar on the grids coated with carbon, and ionized. The specimens were washed with PBS to remove the salt and the sucrose and were then immunochemically labeled. The grids were immersed in the blocking solution of 1% BSA-PBS and subsequently in the first antibody (anti-hDHFR rabbit serum) in 1% BSA-PBS overnight at 4 °C. After they were washed with PBS, the grids were reacted with the second antibody, the 5 nm gold-labeled goat anti-rabbit IgG (Auro Probe EM GAR IgG G5, Amersham LIFE SCIENCE), and were washed with PBS. The specimens were then refixed with 2% glutaraldehyde in PBS for 10 min, washed with PBS, and stained with 1% osmium (VIII) oxide and 1% uranyl acetate. The specimens, dehydrated by successive immersions in 50%, 70%, 90%, and 100% ethanol, were solidified by immersion in 50% LR White, medium grade (London Resin Co. Ltd., Berkshire, England), for 5 min and twice in 100% LR White for 5 min. After the excess LR White was removed by aspiration, the specimens were incubated overnight at 60 °C. (3) The purified membrane fractions on the carbon-coated grids were also negatively stained with 1% uranyl acetate by the conven(15) Tokuyasu, K. T. Histochem. J. 1980, 12 (4), 381.

tional procedure. (4) Freeze-fracture replicas were prepared as follows: Cells suspended in 30% glycerol containing 25% NaCl were mounted on a specimen holder, quickly frozen by immersion in liquid nitrogen, and fractured by a FR-7000 unit (Hitachi, Ltd., Japan). The fractured surfaces were immediately shadowed with platinum, followed by carbon, to make replicas. Electron micrographs were obtained with a transmission electron microscope 100CX (JEOL, Japan) with an accelerating voltage of 100 kV. The micrographs were screened with optical diffraction equipment to detect the regions in which the electron micrograph showed two-dimensional periodicity. X-ray Diffraction Experiments. A 20 µL drop of a 60-200 µM PM suspended in 10 mM HEPES (pH 7.0) and 100 mM NaCl was dried in room air on pieces of aluminum foil for about half a day, and the procedure was repeated several times. The samples were equilibrated for 1 or 2 days at 92% relative humidity. The X-ray diffraction experiments were carried out with the MUSCLE diffractometer16 installed at BL-15A in the Photon Factory of the National Laboratory for High Energy Physics at Tsukuba, Japan. The diffraction profiles were recorded with a onedimensional position-sensitive counter. The background of each diffraction profile was estimated by spline interpolation of the data points where diffraction of PM did not contribute. After Lorentz correction, the background was subtracted.

Results Fusion Protein Expression and Purification. Sucrose gradient ultracentrifugation of the wild-type bR (PM) and the bR-hDHFR resulted in two distinct purple bands with different densities, as shown in Figure 3A. The densities of the bands were found to be 1.18 g/cm3 for the PM, as reported,11 and 1.19 g/cm3 for the bR-hDHFR, by measuring the weights of appropriate volumes of each fraction. These results mean that the membrane fractions of the wild-type bR and the bR-hDHFR were homogeneous, from the point of view of molecular packing. The fact that the density of the bR-hDHFR was greater than that of the wild-type bR implies that the protein content of the membrane fraction of bR-hDHFR was greater than that of the native PM, which is expected from their molecular structures and the fact that the density of protein is greater than that of lipid in general. (16) Amemiya, Y.; Wakabayashi, K.; Hamanaka, T.; Wakabayashi, T.; Matsushita, T.; Hashizume, H. Nucl. Instrum. Methods 1983, 208, 471.

Structure Formation of Bacteriorhodopsin-hDHFR

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Figure 5. Enzymatic activity of bR-hDHFR as DHFR. The activity was measured at 5 min past the start of the reaction by monitoring A340, indicating consumption of NADPH in reaction aliquots.

Figure 4. (A) Absorption spectra of the bR-hDHFR (solid line) and the wild-type bR (broken line) at room temperature. The ordinate scale is arbitrary, due to normalization of the absorbance at 568 nm. (B) Flash photolytic experiment with the bR-hDHFR and the wild-type bR of 10A568 at room temperature. Two spectra with a couple of slide coordinates were overlaid. (C) Circular dichroism spectra at 25 °C of 2.0A568 samples of bR-hDHFR (solid line) and wild-type bR (broken line). The two spectra are almost identical.

The protein component of the purple band fractions was examined by SDS-PAGE, western blotting, and subsequent ELISA. As shown in Figure 3B(1), dominant (>90%) bands at 28 kDa (26.7 kDa from calculation) for the wildtype bR and at 47 kDa (48.4 kDa from calculation) for the bR-hDHFR fusion protein were detected. The major band at 47 kDa was confirmed to be bR-hDHFR by ELISA using anti-bR serum (Figure 3B(2)) and anti-hDHFR serum (Figure 3B(3)). To investigate the yield and the purity of bR-hDHFR, and the conformation of the bR portion, the UV-vis spectra were measured (Figure 4A). The membrane fraction containing bR-hDHFR showed a specific absorption maximum at 568 nm, which is identical to that for the wild-type PM. As shown in Figure 4B, flash photolytic analyses at 410 nm of the PM and bR-hDHFR suspensions showed lifetimes of approximately 5 ms, which are almost equivalent to the reported value for the M

intermediate of bR.17 It is known that the absorption resulting from M intermediate formation is strongly affected by conformational changes of the residues involved in the photoreaction. These results suggest that the bR portion of the bR-hDHFR is properly folded and retains its proton-pumping activity. The yield of bR-hDHFR in H. salinarum was estimated to be 5-10 mg/L (culture) by using the reported relationship between the wild-type bR and the absorbance (1.0A568 ) 410 µg/mL).18 By comparing the A280/A560 values of the wild-type bR (1.8)11 and the bR-hDHFR (3.9), the protein purity of the bR-hDHFR in the fraction was estimated to be approximately 76% ()1.8 × the calculated 280 of bR-hDHFR/(3.9 × the calculated 280 of bR), where the 280 values were estimated to be 92 400 and 56 100 for bR-hDHFR and bR, respectively19), which seems to be lower than the value estimated from the SDS-PAGE result (Figure 3B(1)). As bR-hDHFR has a greater domain contribution to A280 than bR, the higher A280/A560 value for bR-hDHFR is reasonable. Conservation of Enzymatic Activity. The kinetic constants of the DHFR activity of the membrane fraction of bR-hDHFR were obtained from experiments with various substrate and inhibitor concentrations. From the Lineweaver-Burke plots (Figure 5), the kinetic constants were calculated as Km ) 13 (( 2) × 10-5 M for bR-hDHFR and kcat ) 1.1 ((0.2) s-1. These values are slightly lower than those previously reported (Km ) 8 × 10-5 M and kcat ) 3 s-1) for the recombinant hDHFR expressed in E. coli.20 Since most enzymes from halobacteria are active and stable at high salt concentrations and become inactive under monovalent salt concentrations below 2 M,21 the monovalent salt concentration dependence of the intrinsic DHFR activity of the fractionated purple band, including bR-hDHFR, was examined. The KCl concentration dependence of the bR-hDHFR was similar to that of the native halophilic DHFR (data not shown).20 Thus, the purple membrane fraction was confirmed to retain the intrinsic enzymatic activities of DHFR, even though it was attached to bR. Namely, the gene encoding the bifunctional fusion protein bR-hDHFR was successfully expressed in H. salinarum. Localization and Orientation of bR-hDHFR in the Plasma Membrane. Figure 6 shows the staining of (17) Soppa, J.; Otomo, J.; Straub, J.; Tittor, J.; Meessen, S.; Oesterhelt, D. J. Biol. Chem. 1989, 264 (22), 13049. (18) Rehorek, M.; Heyn, M. P. Biochemistry 1979, 18, 4977. (19) Schellman, J. A.; Schellman, C. In The Proteins. 2md ed.; Neurath, H. Ed.; Academic Press: New York, 1964; Vol. 2, p 1. (20) Blecher, O.; Goldman, S.; Mevarech, M. Eur. J. Biochem. 1993, 216, 199. (21) Jaenicke, R. Annu. Rev. Biophys. Bioeng. 1981, 10, 1.

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Figure 6. Electron micrographs of the immunostained membrane fractions purified from H. salinarum S9 overproducing bR and recombinant H. salinarum L33 producing bR-hDHFR, detected by either anti-bR serum or anti-hDHFR serum and emphasized by gold-labeled protein A. Scale bars represent 300 nm for A-D. The membranes purified from S9 were almost uniformly stained with the anti-bR (A) but not with the anti-hDHFR (B). The membranes purified from recombinant H. salinarum L33 producing bR-hDHFR formed rough fringes in comparison with those of S9 and were slightly stained by the anti-bR serum (C). There were two distinct types of membranes decorated by the gold clusters (10 nm) and those not decorated, in the case of the anti-hDHFR detection (D). Electron micrographs of an immunostained, ultrathin section of the recombinant H. salinarum L33 producing bR-hDHFR (E). Scale bars represent 1 µm for the main photograph and 200 nm for the inset. Gold clusters (5 nm) are mainly located on the cytoplasmic side of the demarcated regions of the plasma membrane.

one side of the plasma membrane with either anti-bR or anti-hDHFR serum, as revealed by immunoelectron microscopy of the isolated membrane fractions randomly immobilized on the carbon-coated grids prior to immunochemical labeling. In the case of the PM, the membrane was almost uniformly labeled with the conjugates of antibR serum and protein A-gold (Figure 6A) but not with the conjugates of anti-hDHFR serum and protein A-gold

(Figure 6B). In the case of the membrane fractions recovered from the bR-hDHFR-producing recombinants, the polyclonal anti-bR reacted faintly but uniformly with the membrane (Figure 6C). When anti-hDHFR antibodies were used for detection, there were two distinct types of membrane domains, those that were decorated by gold clusters and those that were not decorated. Figure 6D shows an area of a doubly folded membrane. These results

Structure Formation of Bacteriorhodopsin-hDHFR

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Figure 7. Electron micrographs of the replicas of the freeze-fractured cells and the negatively stained membrane samples prepared from H. salinarum S9 producing wild-type PM (A and C), and those of the recombinant H. salinarum L33 producing bR-hDHFR (B and D). Scale bars represent 300 nm. Insets are optical diffraction patterns of each electron micrograph.

were interpreted to be random adsorption of both sides of the membrane fraction (the cytoplasmic side of the plasma membrane up or the extracellular side up) on the carboncoated grids and showed that the anti-hDHFR antibody could distinguish the cytoplasmic side from the extracellular side of the plasma membrane. Namely, when the bR-hDHFR-producing membrane was adsorbed on the grids in the cytoplasmic side down orientation, then the anti-hDHFR antibody could not access the antigen, which might be located on the cytoplasmic side of the membrane, while the anti-bR serum could access the antigen from both sides. The cellular localization of bR-hDHFR in the recombinant H. salinarum cells was investigated by immunoelectron microscopy of ultrathin cellular sections with the anti-hDHFR serum. As shown in Figure 6E, the 5 nm gold clusters are mainly located on the cytoplasmic side of the demarcated regions of the plasma membrane, which are apparently dense in the figure. This pattern coincides well with the configuration drawn in Figure 1. These electron microscopic results indicate that the orientation of bR-hDHFR in the plasma membrane is unique. Conservation of Molecular Packing. Figure 4C shows the almost identical CD spectra of the wild-type PM and the membrane fraction of the bR-hDHFR, dispersed in 10 mM HEPES buffer (pH 7.0) and 100 mM NaCl at 25 °C. The CD spectrum of the PM shows asymmetric positive and negative bands around the region of absorption, with maxima near 538 nm for the positive

band and near 602 nm for the negative band, and a crossover near 574 nm. The negative band in the CD spectrum is regarded as a measure of exciton coupling in the molecular assembly of trimeric bacteriorhodopsin.22 As shown in Figure 4C, this phenomenon was also observed in the case of the fusion protein. Figure 7 shows electron micrographs and their optical diffraction patterns of the freeze-fracture replicas of the plasma membrane and the negatively stained membrane fractions prepared from H. salinarum S9 (A and C) and an H. salinarum clone expressing bR-hDHFR (B and D). Roundish patches were detected in the freeze-replica samples of the wildtype PM (A) and the bR-hDHFR (B). Optical diffraction of these areas yielded almost identical hexagonal patterns of the first order, corresponding to a periodicity of approximately 6 nm, which is the lattice size of the reported p3 hexagonal packing of the wild-type PM.23,24 As for the optical diffraction patterns of the negatively stained membrane fractions, diffraction spots of the first order as well as the second order were observed in the case of the native PM (C), while only second-order diffraction spots were observed in the case of the membrane derived from the recombinant H. salinarum producing bR-hDHFR (D). This means that the periodicity was reduced from the reported value of 6.2 nm to 3.6 nm ()6.2/x 3 nm). On the (22) Du-Jeon-Jang; El-Sayed, M. A.; Stern, L. J.; Mogi, T.; Khorana, H. G. J. Mol. Biol. 1990, 121, 283. (23) Blaurock, A. E. J. Mol. Biol. 1975, 93, 139. (24) Henderson, R. J. Mol. Biol. 1975, 93, 123.

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Figure 8. Comparison of the X-ray diffraction profile from the membrane fraction containing bR-hDHFR and that from the wild-type purple membrane (bR). The diffraction profile of bR is shifted to the left for 0.001 Å-1.

other hand, almost identical lattice constants and symmetry (p3, a ) 6.2 nm, γ ) 120°) were confirmed by preliminary small-angle X-ray scattering experiments on suspensions of the wild-type PM and the membrane fraction containing the bR-hDHFR. Figure 8 shows a comparison of the two X-ray diffraction spectra from the two membrane fractions. Peak positions of the wild-type PM were almost identical to those of the bR-hDHFR. These diffraction patterns imply that the overall molecular packing of the bR-hDHFR in the plasma membrane was isomorphous with that of the wild-type PM,23 as explained in Figure 1. Discussion A bifunctional recombinant protein, bR-hDHFR, in which the ratio of hDHFR to bR was stoichiometrically predetermined to be 1 (Figure 2), was expressed in H. salinarum (Figure 3). This fusion protein was detected within the plasma membrane by anchoring the bR portion and exposing the hDHFR portion on the cytoplasmic side (Figure 6), and formed a highly packed, two-dimensionally ordered structure similar to that of the wild-type PM (Figure 7), as schematically shown in Figure 1. The bR-hDHFR-containing membrane fraction showed spectroscopic characteristics similar to those of the wildtype PM (Figure 4), and simultaneously, it possessed dihydrofolate reductase activity. However, for the hDHFR portion, a slight decrease in the enzymatic activity was observed. This lower hDHFR enzymatic activity of bRhDHFR, in comparison with the authentic DHFRs, might be caused partly by the immobilization of the hDHFR on the membrane3 and partly by the exposure of the bRhDHFR fraction to a low-salt-concentration buffer in the purification process. These results strongly suggest (1) that the bR and hDHFR portions in the bR-hDHFR, although conjugated in one molecule, were each correctly folded and (2) that the bR portion functions well as a molecular anchor in the plasma membrane. Optical diffraction patterns obtained from the negatively stained membrane fraction and from patches detected on the freeze-fracture replicas of cells (Figure 7), and X-ray diffraction from pellets of membranes (Figure 8) implied that the bR-hDHFR forms an ordered structure that is

Nomura et al.

almost identical to that of the wild-type PM. However, the reduction of the lattice periodicity in the negatively stained membrane fraction derived from the bR-hDHFRproducing cells, from 6.2 to 3.6 nm, implies that the hDHFR domains were packed in a less-ordered manner on the plasma membrane than the bR domains. Namely, from the point of view of the hDHFR packing, the environments around the two distinct 3-fold axes in the p3 symmetry of the native PM became almost identical. However, this disorder is not deleterious to applications of fabricated supramolecular structures to biosensors or bioreactors in general, since the vectorial control of the orientation of the functionalized domain relative to the membrane is most important in these applications. The stoichiometric ratio of the hDHFR portion to the bR portion, from the macroscopic point of view of the SDSPAGE pattern and the spectroscopic data, was not strictly shown to be 1, as predetermined by the chimeric gene structure (Figure 2). However, this does not necessarily deny that the stoichiometric ratio is exactly 1, from the microscopic point of view of the patches on the plasma membrane. Intensive structural studies by electron microscopy, atomic force microscopy, and small-angle X-ray scattering analysis should be done to elucidate the fine structure of the membranes in which the bR-hDHFR protein is incorporated. Proteins such as bR and S-layer proteins, which have strong tendencies toward ordered self-assembly, have served as templates for ordered molecular array formation in the emerging science of nanotechnology. In this study, we demonstrated for the first time that a functional protein can be immobilized by conjugation with bR. We have already demonstrated that membrane-embedded bR can be fixed in a vectorially oriented manner onto an inert base.8 A proper combination of these two techniques can be used for the functionalization of various surfaces and will also contribute to the development of the new field of nanotechnology. Conclusion A bifunctional fusion protein, bR-hDHFR, was expressed in vivo and was localized in the plasma membrane. The bR-hDHFR formed two-dimensional protein arrays in the membrane, in which the hDHFR portion was exposed on a unique side. This method of producing functional chimeric proteins can be applied to the construction of functionalized membranes upon which stoichiometrically conjugated proteins are fixed. Acknowledgment. We would like to thank Prof. J. Lanyi (University of California, Irvine) for kindly providing the H. salinarum host L33, Dr. K. Ihara (Nagoya University, Japan) for generously offering the expression vector pUCNov∆bop, Prof. Y. Sugiyama (Nagoya University, Japan) for the generous gift of an antibody, Mr. M. Kimura (Hitachi Science Systems Co. Ltd., Japan) for the preparation of the freeze-fracture replicas, and Prof. T. Gotow (Osaka University, Japan) for technical training in ultrathin sectioning of specimens and their immunostaining. We are grateful to Dr. Y. Kimura (Biomolecular Engineering Research Institute) for helpful discussions. M.M. wishes to thank the Israel Science Foundation for a grant on a subject related to this manuscript. LA980742U