Topography of reaction center subunits in the membrane of the photosynthetic bacterium, rhodopseudomonas sphaeroides.

Valkirs, G E; Fehér, G.

doi:10.1083/jcb.95.1.179

Cited by 51 publications

(24 citation statements)

References 32 publications

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“…Both the cofactors and the L-and M-subunits exhibit an approximate twofold symmetry about a line joining the donor and the Fe. It was gratifying to us that the main features of the structure corroborated our previous findings, i.e., the L-and M-subunits have each five transmembrane helices, and the H-subunit has one (157) with most of it exposed on the cytoplasmic side (152). The cofactors are all associated with the Land M-subunits (117), the primary donor is a bacteriochlorophyll dimer (51), Q A is associated with the M-subunit (102), and the Fe is approximately equidistant from Q A and Q B (18) [for additional details, see (55)].…”

Section: Fehersupporting

confidence: 72%

“…Most anti-H preparations labeled the cytoplasmic side, and only one out of six labeled the periplasmic side. From this we concluded that H is asymmetrically oriented with respect to the membrane, most of it being on the cytoplasmic side (152).…”

Section: Further Characterization Of the Rcmentioning

confidence: 96%

“…The topography of the RC in the bacterial membrane was investigated by Gunars Valkirs, a graduate student, by using indirect immunoferritin labeling (152). Ferritin is an electron-dense molecule, which permits the localization of the binding site by direct electron-microscopic visualization.…”

Section: Further Characterization Of the Rcmentioning

confidence: 99%

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My Road to Biophysics: Picking Flowers on the Way to Photosynthesis

Fehér

2002

Annu. Rev. Biophys. Biomol. Struct.

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Section: Fehersupporting

confidence: 72%

Section: Further Characterization Of the Rcmentioning

confidence: 96%

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My Road to Biophysics: Picking Flowers on the Way to Photosynthesis

Fehér

2002

Annu. Rev. Biophys. Biomol. Struct.

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“…The M subunit has been shown to span the cytoplasmic membrane by several labeling techniques (31,32). The sequence obtained from the gene was examined for potential membrane-spanning regions by the method of Kyte and Doolittle (33).…”

Section: Discussionmentioning

confidence: 99%

Primary structure of the M subunit of the reaction center from Rhodopseudomonas sphaeroides

Williams

Steiner

Ogden

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

176

126

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The reaction center is a membrane-bound bacteriochlorophyll-protein complex that mediates the primary photochemical events in the photosynthetic bacterium Rhodopseudomoas sphaeroides. The previously determined amino-terminal sequences of the three subunits of the reaction center protein were used to design synthetic mixed oligonucleotide probes for the structural genes encoding the subunits. One of these probes was used to isolate and clone a fragment of DNA from R. aphaeroides that contained the gene encoding the M subunit. The nucleotide sequence of this gene was determined by the dideoxy method. In addition, a number of tryptic and chymotryptic peptides from the M protein were isolated and subjected to sequence analysis, and the sequence of the carboxyl terminus was determined. Together with the amino-terminal sequence, the data establish the primary structure of the M protein. The distribution of hydrophobic residues in the amino acid sequence suggests the presence of five membrane-spanning segments. A significant homology was found between the amino acid sequence of the M subunit and a thylakoid membrane protein (Mr 32,000) from spinach that has been implicated in herbicide and quinone binding.The reaction center (RC) is the site of the initial photochemical charge separation in the electron transfer process of photosynthesis (for reviews, see refs. 1 and 2). In the purple bacterium Rhodopseudomnas sphaeroides, the RC is part of the photosynthetic apparatus found in the highly invaginated cytoplasmic membrane that is synthesized by the bacterium under anaerobic conditions (3). The RC protein (Mr 100,000) contains three highly hydrophobic subunits, designated L, M, and H, in a 1:1:1 stoichiometry (4, 5). The cofactors found in the RC are four bacteriochlorophylls, two bacteriopheophytins, two ubiquinones, and one iron.The determination of the amino acid sequence of the RC polypeptides is essential to the detailed elucidation of the RC structure. The sequences of 25-28 amino-terminal residues of each subunit have been determined previously by automated Edman degradation (6). These were used to design mixed-sequence oligonucleotide hybridization probes for the structural genes of each of the subunits. The probe for the gene encoding the M subunit was used to identify a restriction fragment of R. sphaeroides DNA that contained the gene. The nucleotide sequence of this gene was determined by the dideoxy method and is presented together with the amino acid sequence derived from it. The sequences of the carboxyl terminus and of several peptides were determined for corroboration of the sequence obtained from the DNA. Preliminary accounts of this work have been given (7,8).EXPERIMENTAL PROCEDURES Materials. Large fragment Escherichia coli DNA polymerase I, M13mp7 replicative form (RF) DNA, M13 24-base-pair primer fragment, dideoxynucleotides, and the restriction enzymes Acc I, BamHI, EcoRI, HindIII, Hpa II, Kpn I, Sal I, Sma I, and Taq I were obtained from Bethesda Research Laboratories. T4 polynucleotide k...

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“…The RC is an oligomeric integral membrane protein complex in which each subunit may span the membrane (1,18,39), and it has been proposed that L and M each contain five membrane-spanning regions (42,43). Therefore, the arrangment of the subunits in the membrane is, undoubtedly, quite complex.…”

Section: Discussionmentioning

confidence: 99%

In vitro biosynthesis and membrane association of photosynthetic reaction center subunits from Rhodopseudomonas sphaeroides

Hoger¹,

Chory²,

Kaplan³

1986

J Bacteriol

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The reaction center of Rhodopseudomonas sphaeroides is an integral membrane protein complex responsible for primary photochemical charge separation in photosynthesis. We report the synthesis of two of the three subunits of the photosynthetic reaction center using a DNA-directed in vitro transcription-translation system prepared from R. sphaeroides. The in vitro-synthesized polypeptides, as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had apparent Mrs of 24,000 and 21,000 and were shown to be synthesized in equimolar amounts. This corresponds precisely to the in vivo reaction center subunits M and L, respectively. The in vitro-synthesized polypeptides were immunoprecipitated with antibody prepared against whole native reaction centers. In addition, the identity of the in vitro-synthesized polypeptides as L and M was verified by comparing the protease digestion products of in vivo-with in vitro-synthesized reaction center subunits. Both of the in vitro-synthesized polypeptides were also found to partition with the particulate material in the transcription-translation system and to associate with added membranes.When Rhodopseudomonas sphaeroides is grown chemoheterotrophically in the presence of oxygen, it is a typical gram-negative bacterial cell; when the oxygen tension is reduced below threshold levels, the cell responds by synthesizing an extensive intracytoplasmic membrane system (13). This new membrane houses the numerous components of the photosynthetic apparatus. In addition to regulation by oxygen tension, the synthesis of the components necessary for photophosphorylation is also modulated by light intensity (19). Given these attributes, the photosynthetic bacteria, and R. sphaeroides in particular, have been employed as a model system for the study of membrane biosynthesis and photosynthesis at the molecular level (20).One of the key complexes within the intracytoplasmic membrane is the photosynthetic reaction center (RC). The RC used light energy in the formation of high-energy electrons, and it is these electrons which are the driving force of photosynthetic electron transport. Studies have compared R. sphaeroides RC with the photosystem II RC of chloroplasts (17). Three integral membrane protein subunits, designated H, M, and L, make up the R. sphaeroides RC. In addition to the three polypeptides, there are four bacteriochlorophylls, two bacteriopheophytins, two quinones, and one iron in a functional RC (27). Polypeptides H, M, and L are extremely hydrophobic and have apparent Mrs of 28,000, 24,000, and 21,000, respectively, based on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, although these are not the true molecular weights (27). The actual role of each subunit is unclear, but dissociation of H from L and M leaves the L-M complex with spectral and functional properties similar to those of the whole RC (10).Despite extensive biochemical analysis of the photosynthetic membranes and RCs, very little is known about the assembly of these proteins or the regul...

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Topography of reaction center subunits in the membrane of the photosynthetic bacterium, rhodopseudomonas sphaeroides.

Cited by 51 publications

References 32 publications

My Road to Biophysics: Picking Flowers on the Way to Photosynthesis

My Road to Biophysics: Picking Flowers on the Way to Photosynthesis

Primary structure of the M subunit of the reaction center from Rhodopseudomonas sphaeroides

In vitro biosynthesis and membrane association of photosynthetic reaction center subunits from Rhodopseudomonas sphaeroides

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