Localization of reaction center and B800-850 antenna pigment proteins in membranes of Rhodobacter sphaeroides.

The localization of the N- and C-terminal regions of pigment-binding polypeptides of the bacterial photosynthetic apparatus of Rhodobacter sphaeroides was investigated by proteinase K treatment of chromatophore and spheroplast-derived vesicles and amino acid sequence determination. Under conditions of proteinase K treatment of chromatophores, which left the in vivo absorption spectrum and the membrane intact, 15 and 46 amino acyl residues from the N-terminal regions of the L and M subunits, respectively, of the reaction center polypeptides were removed. The N termini are therefore exposed on the cytoplasmic surface of the membrane. The C-terminal domain of the light-harvesting B800-850 alpha and B870 alpha polypeptides was found to be exposed on the periplasmic surface of the membrane. A total of 9 and 13 amino acyl residues were cleaved from the B800-850 alpha and B870 alpha polypeptides, respectively, when spheroplasts were treated with proteinase K. The N-terminal regions of the alpha polypeptides were not digested in either membrane preparation and were apparently protected from proteolytic attack. Seven N-terminal amino acyl residues of the B800-850 beta polypeptide were removed after the digestion of chromatophores. C-terminal residues were not removed after the digestion of chromatophores or spheroplasts. The C termini seem to be protected from protease attack by interaction with the membrane. Therefore, the N-terminal regions of the beta polypeptides are exposed on the cytoplasmic membrane surface. The C termini of the beta polypeptides are believed to point to the periplasmic space.     

Localization of the exposed N-terminal region of the B800-850 alpha and beta light-harvesting polypeptides on the cytoplasmic surface of Rhodopseudomonas capsulata chromatophores.

Proteinase K and trypsin were used to determine the orientation of the light-harvesting B800-850 alpha and beta polypeptides within the chromatophores (inside-out membrane vesicles) of the mutant strain Y5 of Rhodopseudomonas capsulata. With proteinase K 7 amino acid residues of the B800-850 alpha polypeptide were cleaved off up to position Trp-7--Thr-8 of the N terminus, and 11 residues were cleaved off up to position Leu-11-Ser-12 of the beta chain N terminus. The C termini of the B800-850 alpha and beta polypeptides, including the hydrophobic transmembrane portions, remained intact. It is proposed that the N termini of the alpha and beta subunits, each containing one transmembrane alpha-helical span, are exposed on the cytoplasmic membrane surface and the C termini are exposed to or directed toward the periplasm.     

The localization of pigment-binding polypeptides in membranes of Rhodopseudomonas viridis

Abstract Inside-out and right-side out vesicles were isolated from the intracytoplasmic membrane system of the photosynthetic bacterium Rhodopseudomonas viridis and treated with proteinase K. Afterwards the pigment-binding proteins of the photosynthetic apparatus were extracted from the membrane, purified and the N- and C-terminal amino acyl sequences determined.
Forty-eight amino acids were found to be removed from the N-terminal domain of the M-subunit and twenty-eight amino acids split off the L-subunit of reaction center when inside-out vesicles were digested with proteinase K.
Six amino acids of the N-terminal region of the beta polypeptide of the light-harvesting complex B1020 were removed when inside-out vesicles were treated with proteinase K. The N-terminal domains of alpha and gamma polypeptides of the antenna complex B1020 were not cleaved by proteinase K either in right-side out or in inside-out vesicles. It is concluded that the N-terminal domains of M-, L- and β-subunits are exposed and accessible to proteinase K on the cytoplasmic surface of the membrane. This is in agreement with results obtained with other photosynthetic bacteria. The orientation of the other light-harvesting polypeptides is discussed.     

Molecular organization of photochemical reaction complex in chromatophore membrane from Rhodospirillum rubrum as detected by immunochemical and proteolytic analyses

The molecular organization of photochemical reaction (PR) complex in chromatophores from Rhodospirillum rubrum was studied by a combination of proteolytic analysis with proteinase K followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical analysis with rabbit polyclonal antibodies against its five subunits (H, M, L, alpha, and beta). The preparations used for comparison were reaction center complex (RC) (composed of H, M, and L), PR complex, and chromatophores (closed membranous vesicles of polar lipid bilayer having PR complex buried in the membrane). 1. RC was bound with anti-H, anti-M, and anti-L antibodies, whereas PR complex and chromatophores were bound with anti-H and anti-beta antibodies, but not with the other antibodies. 2. With PR complex, H (Mr 31,000 (31K)) was rapidly degraded into two peptides with Mr of 16K and 14.5K (abbreviated as 16K and 14.5K, respectively), M (27K) into 25.5K, and beta (11K) into 10K. Significantly later, the 25.5K of M was degraded into 24K, L (23K) into 19K, and alpha (12K) into 11K. With chromatophores, H and beta were degraded in a manner similar to that with PR complex, whereas M, L, and alpha were not degraded at all. With RC, H, M, and L were rapidly degraded. 3. With RC, the activity for photooxidation of P870 (photochemical activity) was hardly affected till H, M, and L had been degraded into less than 10K, 24K, and 19K, respectively. With PR complex, the absorbance spectrum due to the bacteriochlorophylls of light-harvesting complex-1 composed of alpha and beta (LH1-Bchl) changed in parallel to the degradation of alpha or 10K (a part of beta). 4. Together with the previous results (Ueda et al. (1985) J. Biochem. 98, 1487-1498), the present findings suggest that: 1) RC is directly surrounded by 12 alpha and further by 12 beta; 2) H and beta are mostly and partially exposed, respectively, on the outer surface of the membranous vesicle; 3) a small part of M is exposed on the inner surface of the membranous vesicle.     

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.     

Comparison, by freeze-fracture electron microscopy, of chromatophores, spheroplast-derived membrane vesicles, and whole cells of Rhodopseudomonas sphaeroides.

By using freeze-fracture electron microscopy, chromatophores and spheroplast-derived membrane vesicles from photosynthetically grown Rhodopseudomonas sphaeroides were compared with cytoplasmic membrane and intracellular vesicles of whole cells. In whole cells, the extracellular fracture faces of both cytoplasmic membrane and vesicles contained particles of 11-nm diameter at a density of about 5 particles per 10(4) nm2. The protoplasmic fracture faces contained particles of 11 to 12-nm diameter at a density of 14.6 particles per 10(4) nm2 on the cytoplasmic membrane and a density of 31.3 particles per 10(4) nm2 on the vesicle membranes. The spheroplast-derived membrane fraction consisted of large vesicles of irregular shape and varied size, often enclosing other vesicles. Sixty-six percent of the spheroplast-derived vesicles were oriented in the opposite way from the intracellular vesicle membranes of whole cells. Eighty percent of the total vesicle surface area that was exposed to the external medium (unenclosed vesicles) showed this opposite orientation. The chromatophore fractions contained spherical vesicles of uniform size approximately equal to the size of the vesicles in whole cells. The majority (79%) of the chromatophores purified on sucrose gradients were oriented in the same way as vesicles in whole cells, whereas after agarose filtration almost all (97%) were oriented in this way. Thus, on the basis of morphological criteria, most spheroplast-derived vesicles were oriented oppositely from most chromatophores.     

Probing the topology of proteins in the chromatophore membrane of Rhodospirillum rubrum G-9 with proteinase K

All the major membrane proteins of isolated chromatophore vesicles are eventually degraded upon incubation with the unspecific proteinase K. These proteins must therefore be exposed at least partially or temporarily on the cytosolic surface of the membrane which is exclusively accessible to the proteinase in intact chromatophore vesicles. That the vesicles are intact during the incubation with proteinase is demonstrated by the finding that cytochrome c₂, which is located in the interior of the vesicles, is protected from proteolytic attack. The degree of degradation of the various chromatophore proteins and the time taken for degradation differ characteristically. From the changes in intensity of the gel bands during the course of digestion it appears that reaction center subunit H is digested first, much faster than are subunits M and L. The near-infrared absorption spectrum of the chromatophores changes only after proteolytic degradation of these two pigment-carrying subunits. Fading of the band of the light-harvesting polypeptide is evident only after prolonged incubation. It seems that this is the most stable component of the chromatophore membrane. The light-harvesting polypeptide appears to be somewhat shortened eventually, leaving the protein conformation necessary for holding the pigments unchanged, as shown by the absorption spectrum. The possible topology of these major membrane components is discussed in the light of these findings.     

Orientation of chromatophores and spheroplast-derived membrane vesicles of Rhodopseudomonas sphaeroides: analysis by localization of enzyme activities.

Intact spheroplasts, vesicles obtained from French-press lysates (chromatophores), and spheroplast-derived vesicles were isolated from photosynthetically grown cells of Rhodopseudomonas sphaeroides. Lysed spheroplasts showed specific activities of succinate, NADH, and l-lactate dehydrogenase which were eight-, six-, and seven-fold higher, respectively, than those of intact spheroplasts when ferricyanide was used as electron acceptor. Mg²⁺-ATPase activity of lysed spheroplasts, measured using an assay system coupled to the oxidation of NADH, was seven-fold higher than the activity of intact sheroplasts. Toluene-treated spheroplast-derived vesicles displayed higher succinate dehydrogenase (ferricyanide reduction) and Mg²⁺-ATPase activities than untreated vesicles whereas no differences were measured between untreated and toluene-treated chromatophores. However, NADH dehydrogenase (ferricyanide reduction) activities of both toluene-treated vesicles and chromatophores were higher than the activities of untreated vesicles and chromatophores. When chromatophores and spheroplast-derived vesicles were preincubated with trypsin, the l-lactate and succinate dehydrogenase activities of chromatophores were preferentially inactivated when phenazine methosulfate was used as electron acceptor. The data indicate that chromatophores are oriented in an opposite direction to the spheroplast-derived vesicles. At least 80% of the latter are oriented in a direction equivalent to the cytoplasmic membrane of intact cells and spheroplasts. Spheroplast-derived vesicles from cells grown with higher light intensities seem to be more uniformly oriented than those obtained from cells grown with lower light intensities.     

Molecular architecture of a light-harvesting antenna. Structure of the 18 S core-rod subassembly of the Synechococcus 6301 phycobilisome

The 18 S subassembly particles obtained by partial dissociation of phycobilisomes from Synechococcus 6301 (Anacystis nidulans) strain AN 112 contain approximately one-half of the mass of the phycobilisome and include core-rod junctions (Yamanaka, G., Lundell, D. J., and Glazer, A. N. (1982) J. Biol. Chem. 257, 4077-4086). The polypeptide composition of 18 S complexes, determined by analysis of uniformly 14C-labeled phycobilisomes, gave the following stoichiometry: 75K:27K:18.3K:alpha beta allophycocyanin monomer: alpha beta phycocyanin monomer of 1:2:1:5:6; where 75K, 27K, etc. represent polypeptides of 75, 27 kilodaltons, etc. The 18.3K polypeptide is a hitherto underscribed biliprotein bearing a single phycocyanobilin. The NH2-terminal sequence of this subunit was determined to be homologous to that of the beta subunit of allophycocyanin. Chromatography of products resulting from limited trypsin treatment of the 18 S complex led to the isolation of three subcomplexes: a mixture of (alpha beta)3 . 22K and (alpha beta)3 . 24K phycocyanin complexes, an (alpha beta)3 allophycocyanin trimer, and an (alpha beta)2 . 18.3K.40K.11K allophycocyanin-containing complex. The 22K and 24K components were products of the degradation of the 27K polypeptides, whereas the 40K and 11K components were derived from the 75K polypeptide. The subcomplexes accounted for the composition of the 18 S complex. Determination of the composition, stoichiometry, and spectroscopic properties of the subcomplexes has led to a model of the polypeptide arrangement within the 18 S complex and of the pathway of energy transfer among these polypeptides.     

The light-harvesting polypeptides of Rhodospirillum rubrum. II. Localisation of the amino-terminal regions of the light-harvesting polypeptides B 870-alpha and B 870-beta and the reaction-centre subunit L at the cytoplasmic side of the photosynthetic membrane of Rhodospirillum rubrum G-9+

The unspecific proteinase K and the specific proteases alpha-chymotrypsin, trypsin and S. aureus V 8 protease were used in order to determine the orientation of the polypeptides B 870-alpha and B 870-beta from the major antenna complex B 870 of Rs. rubrum G-9+ within the chromatophore membrane (inside-out vesicle). Although B 870-alpha exhibits cleavable peptide bonds, treatment with specific proteases yielded splitting only in B 870-beta within the N-terminal region. In the case of proteinase K, which was most effective, mainly 6 (B 870-alpha) and 16 (B 870-beta) amino acid residues were removed from their N-terminal parts as proved by means of Edman degradation of cleavage products. The major peptide bonds cleaved were identified as Gln6-Leu7 in B 870-alpha and as Lys16-Glu17 in B 870-beta. The central hydrophobic stretch regions and the relatively hydrophilic C-terminal parts of both light-harvesting polypeptides were not affected by proteinase K. On the basis of these degradation experiments a transmembrane orientation of B 870-alpha and B 870-beta is postulated, with their N-terminal towards the cytoplasm and their C-termini towards periplasm with regard to the photosynthetic membrane. This hypothesis is supported by the transmembrane model proposed by Brunisholz et al. (Hoppe-Seyler's Z., Physiol. Chem., (1984) 365, 675-688) in which the hydrophobic stretch of B 870-alpha and of B 870-beta forming an alpha-helix would span the membrane once. Organic solvent extraction of chromatophores treated with proteinase K yielded a fairly pure polypeptide fragment with an apparent molecular mass of 14000 Da. Its N-terminal amino-acid sequence is identical with the sequence within the N-terminal region of the reaction centre subunit L of Rs. rubrum G-9+. Thus it is most likely that as in the case of B 870-beta, proteinase K removed 16 amino acid residues from the N-terminal part of subunit L. This subunit therefore also seems to be exposed at the surface of the cytoplasmic side of the chromatophore membrane.     

Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical characterization of Ia antigens. I. Characterization of the 31K polypeptide associated with I-A subregion Ia antigens

Procedures are presented for the preparative isolation of murine Ia antigens directly from splenocyte detergent extracts with monoclonal immunoadsorbents. Utilizing these procedures, three Ia (I-A subregion) polypeptides (alpha, 31K, beta) were isolated and their m.w. and pI values characterized. Evidence is presented that indicates that: 1) the 31K polypeptide probably does not associate with the Ia alpha and beta chain complex during the Ia isolation procedure; 2) the 31K polypeptide is not tightly bound to the alpha/beta Ia complex and can be selectively removed by freezing and thawing and by washing the Ia-immunoadsorbent with buffers containing pyrrolidinone (a polar solvent); and (3) unlike the alpha and beta chains, the 31K polypeptide is not intrinsically radiolabeled with 3H fucose and 3H glucosamine, indicating that the 31K polypeptide either contains a carbohydrate structure that is different from that of the alpha and beta chains or it is not a glycopeptide. These data suggest that although Ia antigens are probably comprised of three polypeptides in the intact cell, only two (alpha and beta) are required to maintain alloantigenic determinants.     

Orientation of the carboxyl terminus of the transposon Tn10-encoded tetracycline resistance protein in Escherichia coli

A site-directed antibody was generated against a synthetic polypeptide corresponding to the 14 amino acid residues of the carboxyl terminus of the Tn10 TetA protein. The antibody reacted preferentially with inside-out vesicles, rather than right-side-out vesicles, prepared from Escherichia coli cells harboring transposon Tn10. When inside-out vesicles were treated with trypsin, the TetA protein was completely digested in the vicinity of the carboxyl terminus, as judged on immunoblot analysis using the antibody. In contrast, when right-side-out vesicles were treated with trypsin, the TetA protein was hardly digested. These results indicate that the carboxyl terminus of TetA is exposed to the cytoplasmic side of the membrane.     

Localization of chromatophore proteins of Rhodobacter sphaeroides. I. Rapid Ca(2+)-induced fusion of chromatophores with phosphatidylglycerol liposomes for proteinase delivery to the luminal membrane surface.

A protease delivery system was developed for the exclusive and controlled digestion of proteins exposed at the morphological inside (periplasmic surface) of Rhodobacter sphaeroides chromatophores. In this procedure, proteinase K is encapsulated within large unilamellar liposomes which are fused to the chromatophores in the presence of Ca2+ ions. The liposomes were prepared by a detergent dialysis procedure from native phosphatidylglycerol and found to undergo rapid bilayer fusion with purified chromatophore preparations above a threshold concentration of 12.5 mM CaCl2. The fusion process was complete within 10 min at 35 mM Ca2+ with about 80% of the pigment located in the fusion products. Electron micrographs of freeze-fracture replicas confirmed the intermixing of the lipid bilayers and the unilamellar structure of the fused membrane vesicles. The procedure did not affect the labile B800 chromophore of the B800-850 antenna complex, but reduced slightly the absorption due to the B875 core antenna. Emission from both light-harvesting complexes was increased in the fused membranes, suggesting a partial dissociation of photosynthetic units in the expanded bilayer. The results, together with those presented in the following paper (Theiler, R., and Niederman, R. A. (1991) J. Biol. Chem. 266, 23163-23168), demonstrate that this new method fulfills the stringent requirements for a successful delivery of macromolecules to the chromatophore interior.     

Identification and partial characterization of a novel bipartite protein antigen associated with the outer membrane of Escherichia coli.

A study by crossed immunoelectrophoresis performed in conjunction with precipitate excision and polypeptide analysis identified a new antigen complex in the envelope of Escherichia coli ML308-225. This antigen corresponds to antigen 43 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H. R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978). Immunoprecipitation experiments conducted with specific antiserum revealed that the complex was expressed on the cell surface and that it contained, in equal stoichiometry, two chemically distinct polypeptides termed alpha and beta (Mrs of 60,000 and 53,000, respectively). The beta polypeptide was heat modifiable, displaying an apparent Mr of 37,000 when solubilized at temperatures below 70 degrees C. Analysis of fractions obtained following cell disruption, isopycnic centrifugation, and detergent extraction indicated that both alpha and beta polypeptides were components of the outer membrane. The two polypeptides were not linked by disulfide bonds, and neither was peptidoglycan associated. The complex contained no detectable lipopolysaccharide, enzyme activity, fatty acyl groups, or other cofactors. Neither correlated with E. coli proteins of similar molecular weight which had previously been shown to be associated with the outer membrane. Antibodies were raised to individual alpha and beta polypeptides. Each of these sera was shown to be subunit specific when tested against denatured membrane proteins. In contrast, each immunoglobulin preparation coprecipitated both alpha and beta polypeptides when tested against undenatured proteins derived from Triton X-100-treated membranes. The results reveal the presence of a novel bipartite protein antigen in the outer membrane of E. coli.     

Cyanobacterial phycobilisomes. Role of the linker polypeptides in the assembly of phycocyanin

The phycocyanin-containing segments of the rod substructures of Anabaena variabilis phycobilisomes consist of complexes of phycocyanin with "linker" polypeptides of 27,000 and 32,500 daltons (Yu, M.-H., Glazer, A. N., and Williams, R. C. (1981) J. Biol. Chem. 256, 13130-13136). Complexes (alpha beta)3.27,000, (alpha beta)3.32,500, (alpha beta)6.27,000, [(alpha beta)6.32,500]n, (alpha beta)6.27,000 - (alpha beta)6.32,500 were prepared, where alpha beta represents a monomer of phycocyanin, and 27,000 and 32,500 represent the 27,000- and 32,500-dalton polypeptides, respectively. Tryptic digestion of (alpha beta)3.32,500 leads to a stable (alpha beta)3.28,000 complex which does not form higher aggregates. The 32,500 polypeptide is stable to trypsin in the [(alpha beta)6.32,500]n and (alpha beta)6.27,000 - [(alpha beta)6.32,500]n=1.2 aggregates. Upon trypsin treatment of all 27,000 still assembled into higher aggregates, (alpha beta)6.21,0900 and (alpha beta)6.21,000 - (alpha beta)6.32,500. The spectroscopic properties of phycocyanin-linker polypeptide complexes were not modified by the tryptic cleavages. These results show that the 32,500 polypeptide has two distinct functional domains, a 28,000 portion necessary to the stabilization of a trimeric phycocyanin complex and a 4,500 domain which links consecutive phycocyanin hexamers in the rod substructure. The 27,000 polypeptide likewise has two distinct functional domains: a 21,000 domain stabilizes a trimeric phycocyanin complex, a 6,000 domain is exposed in all of the assembly forms examined. From these and earlier studies, it is concluded that the 6,000 domain functions in the attachment of the rod substructures to the core of the phycobilisome.     

Chromatographic and protein chemical analysis of the ubiquinol-cytochrome c2 oxidoreductase isolated from Rhodobacter sphaeroides

The ubiquinol-cytochrome c2 oxidoreductase (cytochrome bc1 complex) purified from chromatophores of Rhodobacter sphaeroides consists of four polypeptide subunits corresponding to cytochrome b, c1, and the Rieske iron-sulfur protein, as well as a 14-kDa polypeptide of unknown function, respectively. In contrast, the complex isolated from Rhodospirillum rubrum by the same procedure lacked a polypeptide corresponding to the 14-kDa subunit. Gel-permeation chromatography of the R. sphaeroides cytochrome bc1 complex in the presence of 200 mM NaCl removed the iron-sulfur protein, while the 14-kDa polypeptide remained tightly bound to the cytochromes; this is consistent with the possibility that the latter protein is an authentic component of the complex rather than an artifact of the isolation procedure. The individual polypeptides of the R. sphaeroides complex were purified to homogeneity by gel-permeation chromatography in the presence of 50% aqueous formic acid and their amino acid compositions determined. The 14-kDa polypeptide was found to be rich in charged and polar residues. Edman degradation analysis indicated that its N terminus is blocked and not rendered accessible by de-blocking procedures. Cyanogen bromide cleavage gave rise to a blocked N-terminal fragment as well as a C-terminal peptide comprising more than one-third of the protein. Gas-phase sequence analysis of this peptide established a sequence of 48 residues and identified a putative trans-membrane segment near the C terminus. The blocked N-terminal fragment was cleaved at tryptophan with BNPS-skatole. The resulting peptides, together with tryptic fragments derived from the intact protein, yielded additional sequence information; however, none of the sequences exhibited significant homologies to any known proteins. Tryptic fragments were also used to generate sequence information for cytochrome c1.     

Purification of the energy-transducing adenosine triphosphatase complex from Rhodospirillum rubrum.

The oligomycin- and N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase complex extracted with Triton X-100 from the chromatophores of Rhodospirillum rubrum was extensively purified. The purification procedure included (diethylamino)ethylcellulose chromatography and glycerol gradient centrifugation. The specific activity of Mg2+-dependent ATP hydrolysis in the purified preparation increased about 11-fold, while that of Ca2+-dependent ATP hydrolysis increased 50-fold as compared with chromatophores. The purified adenosine triphosphatase complex dissociated into a maximum of eight different polypeptides upon electrophoresis in the presence of sodium dodecyl sulfate. The estimated subunit molecular weights were as follows: 56 000 (alpha), 50 000 (beta), 33 000 (gamma), and those ranging from 17 000 to 9400 for the remaining smaller subunits. The purified preparation was incorporated into phospholipid vesicles by using the freeze--thaw technique. The reconstituted vesicles catalyzed [32P]ATP exchange, which was almost completely inhibited by both oligomycin and N,N'-dicyclohexylcarbodiimide as well as by a protonophorous uncoupler, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone.     

Heavy riboflavin synthase of Bacillus subtilis. Primary structure of the beta subunit

Heavy riboflavin synthase is a 1,000,000-Da protein catalyzing the last two reactions of riboflavin biosynthesis. The enzyme complex consists of 60 beta subunits (Mr = 16,200) and approximately three alpha subunits (Mr = 23,000). beta subunits were isolated and cleaved with cyanogen bromide. Fragments were isolated and further digested with trypsin and staphylococcal protease. Peptides were isolated by high performance liquid chromatography. Sequences were determined by automated liquid-phase Edman degradation. The complete sequence of the beta subunit (154 amino acids) was established by direct sequencing of the NH2 terminus, sequencing of overlapping peptides, and carboxypeptidase degradation of the COOH terminus. The sequence shows no detectable homologies to other proteins. A computer prediction of secondary structure elements indicates 34% alpha helix and 30% beta sheet.     

The solution structure of Rhodobacter sphaeroides LH1beta reveals two helical domains separated by a more flexible region: structural consequences for the LH1 complex

Here, the solution structure of the Rhodobacter sphaeroides core light-harvesting complex beta polypeptide solubilised in chloroform:methanol is presented. The structure, determined by homonuclear NMR spectroscopy and distance geometry, comprises two alpha helical regions (residue -34 to -15 and -11 to +6, using the numbering system in which the conserved histidine residue is numbered zero) joined by a more flexible four amino acid residue linker. The C-terminal helix forms the membrane spanning region in the intact LH1 complex, whilst the N-terminal helix must lie in the lipid head groups or in the cytoplasm, and form the basis of interaction with the alpha polypeptide. The structure of a mutant beta polypeptide W(+9)F was also determined. This mutant, which is deficient in a hydrogen bond donor to the bacteriochlorophyll, showed an identical structure to the wild-type, implying that observed differences in interaction with other LH1 polypeptides must arise from cofactor binding. Using these structures we propose a modification to existing models of the intact LH1 complex by replacing the continuous helix of the beta polypeptide with two helices, one of which lies at an acute angle to the membrane plane. We suggest that a key difference between LH1 and LH2 is that the beta subunit is more bent in LH1. This modification puts the N terminus of LH1beta close to the reaction centre H subunit, and provides a rationale for the different ring sizes of LH1 and LH2 complexes.