René A. Brunisholz

Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N‐ and C‐terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 Å resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox‐active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways.

The complete amino acid sequence of the single light harvesting protein from chromatophores of Rhodospirillium rubrum G-9+

Pigment-free preparations of the single light-harvesting protein from the purple photosynthetic bacterium Rhodospirillum rubrum have been reported both from the antenna bacteriochlorophyll-protein complex and directly from chromatophores by chloroform/methanol extraction [ 1,2]. Molecular mass determinations yielded values of 9000-l 9 000 [ l-4 1. Preliminary sequence information was obtained from a few very small fragments of limited acid-treated LHP from wildtype R. rubrum [5]. N-Terminal amino acid sequence data have been published for LHP from the carotenoidless mutant R. r&urn G-9+, which was found to be blocked, presumably by a formyl group [6]. Aside from the elucidation of the initial N-termin’al sequences of the 10 000 Mr and the 8000 J4, polypeptides from the light-harvesting B800-850 complex from Rhodopseudomonas capsulata strain Y5 [7], this was the first report on extensive N-terminal sequence data of a LHP from a purple photosynthetic bacterium. Here we present the complete amino acid sequence of LHP from R. rubrum G-9+, thus opening the way for progress in localizing it within the chromatophore membrane and for studying its in vivo aggregational state in the BChl-protein complex. On the basis of the amino acid sequence, LHP consists of 52 amino acid residues yielding 6106 Mr. Of particular interest

Structure, function and organization of antenna polypeptides and antenna complexes from the three families of Rhodospirillaneae.

Comparative primary structural analysis of polypeptides from antenna complexes from species of the three families of Rhodospirillaneae indicates the structural principles responsible for the formation of spectrally distinct light-harvesting complexes. In many of the characterized antenna systems the basic structural minimal unit is an alpha/beta polypeptide pair. Specific clusters of amino acid residues, in particular aromatic residues in the C-terminal domain, identify the antenna polypeptides to specific types of antenna systems, such as B880 (strong circular dichroism (CD)), B870 (weak CD), B800-850 (high), B800-850 (low) or B800-820. The core complex B880 (B1020) of species from Ectothiorhodospiraceae and Chromatiaceae apparently consists of four (alpha 1 alpha 2 beta 1 beta 2) or three (2 alpha beta 1 beta 2) chemically dissimilar antenna polypeptides respectively. There is good evidence that the so-called variable antenna complexes, such as the B800-850 (high), B800-850 (low) or B800-820 of Rp. acidophila, Rp. palustris and Cr. vinosum, are comprised of multiple forms of peripheral light-harvesting polypeptides. Structural similarities between prokaryotic and eukaryotic antenna polypeptides are discussed in terms of similar pigment organization. The structural basis for the strict organization of pigment molecules (bacteriochlorophyll (BChl) cluster) in the antenna system of purple bacteria is the hierarchical organization of the alpha- and beta-antenna polypeptides within and between the antenna complexes. On the basis of the three-domain structure of the antenna polypeptides with the central hydrophobic domain, forming a transmembrane alpha helix, possible arrangements of the antenna polypeptides in the three-dimensional structure of core and peripheral antenna complexes are discussed. Important structural and functional features of these polypeptides and therefore of the BChl cluster are the alpha/beta heterodimers, the alpha 2 beta 2 basic units and cyclic arrangements of these basic units. Equally important for the formation of the antenna complexes or the entire antenna are polypeptide-polypeptide, pigment-pigment and pigment-polypeptide interactions.

The complete amino acid sequence of a bacteriochlorophyll a binding polypeptide isolated from the cytoplasmic membrane of the green photosynthetic bacterium Chloroflexus aurantiacus

A polypeptide soluble in organic solvents was isolated from whole membrane fractions of the green thermophilic bacterium Chloroflexus aurantiacus by chromatography on Sephadex LH‐60, Whatman DE‐32 and Bio Gel P‐10. The complete amino acid sequence of this 4.9 kDa polypeptide (44 amino acid residues) was determined. The polypeptide shows a 3‐domain structure, similar to the domain structure of the antenna BChI polypeptides of purple photosynthetic bacteria, and sequence homologies (27–39%) to the light‐harvesting α‐polypeptides of the B870 (890) antenna complexes from purple bacteria. Therefore, the 4.9 kDa polypeptide is designated B(808‐866)‐α. The typical His residue (conserved His residue identified in all antenna polypeptides of purple bacteria as possible BChI binding site) is found within the hydrophobic domain, which extends from Asn 10 to Leu 30.

The light-harvesting core-complex and the B820-subunit from Rhodopseudomonas marina. Part I. Purification and characterisation

The BChla‐containing B880‐complex (core‐complex) of Rhodopseudomonas marina (Rhodospirillaceae) was isolated with a new purification method. The isolation of the B880‐complex was performed by solubilisation of the photosynthetic membranes with the detergent LDAO and subsequent fractionated ammonium‐sulfate precipitation with about 50% recovery. The B880‐complex retained its original spectral properties as revealed with absorption, fluorescence and circular dichroism spectroscopy. Furthermore, we dissociated the B880‐complex with the detergent n‐octyl‐β‐glucoside (OG) and purified the developed subcomplex by the method of Miller et al. [1], which showed an absorption maximum at 820 nm (B820). The α‐ to β‐polypeptide ratio and the α‐ or β‐polypeptide to BChla ratio, respectively, were estimated to be 1:1 in both complexes. The molecular weights of the B880 and the B820‐complexes, determined by gel filtration chromatography, were 181 and 32 kDa, respectively. Thus, it appears that the B880‐complex of RP. marina consists of 24 polypeptides and the B820‐complex of four polypeptides. Six B820‐complexes or possible subunits could form the B880‐complex. On the basis of these data we propose a model for the structure or BChla containing core‐complexes.

The light-harvesting core-complex and the B820-subunit from Rhodopseudomonas marina. Part II. Electron microscopic characterisation.

Electron micrographs of photosynthetic membranes of the BChla‐containing bacterium Rp, marina showed a quasi‐crystalline structure. The photoreceptor units are arranged in a hexagonal lattice with a reaction center to reaction center distance of 102± Å. Purified B880‐complex was concentrated up to an OD? of 60 which induced the formation of large protein vesicles. The protein complexes within these vesicles were highly ordered and showed a hexagonal lattice with the same center to center distance of 102±3 Å as was observed in the native membranes. Image processing of the micrographs revealed a ring‐like structure of the B880‐complex at 26 Å resolution and suggests that the B880‐complex consists of 5 or 6 subunits. For the first time it can be shown that an isolated core‐complex is in a stable, ring‐like structure even without the reaction center which is supposed to be located in the middle of the B880‐ring. The data indicate that the isolated B880‐complex exhibits the same structure as in the native membrane.

The complete amino acid sequence of the antenna polypeptide B806‐866‐β from the cytoplasmic membrane of the green bacterium Chloroflexus auranliacus

The bacteriochlorophyll a‐binding polypeptide B806–866‐β was extracted from membranes of the green thermophilic bacterium Chloroflexus aurantiacus with chloroform/methanol/ammonium acetate. Purification of the antenna polypeptide (6.3 kDa) was achieved by chromatography on Sephadex LH‐60, Whatman DE‐32 and by FPLC. The complete amino acid sequence (53 amino acid residues) was determined. The B806–866‐β polypeptide is sequence homologous to the antenna β‐polypeptides of purple bacteria (27–40%) and exhibits the characteristic three domain structure of the B870, B800–850 and B800–820 antenna complexes. The two typical His residues, conserved in all antenna β‐polypeptides of purple bacteria, were found: His‐24 lies within the N‐terminal hydrophilic domain and His‐42 within the central hydrophobic domain. This polypeptide together with the previously described α‐polypeptide form the basic structural unit of the B806–866 antenna complex from C. aurantiacus.

The BChlc/e-binding polypeptides from chlorosomes of green photosynthetic bacteria

A 6.3 kDa polypeptide has been isolated from chlorosomes of the green photosynthetic bacterium Pelodictyon luteolum, and its complete amino acid sequence has been determined. It exhibits an overall homology of 30% to the Bchlc‐binding protein of Chloroflexus aurantiacus. Preliminary results from the N‐terminal sequence analyses of the analogous polypeptides isolated from Chlorobium limicola, Prosthecochloris aestuarii and chlorobium phaeovibrioides revealed a highly conserved sequence. This protein is suggested to be the Bchlc/e‐binding polypeptide in the family of the Chlorobiaceae.

Giant circular dichroism of chlorosomes fromChloroflexus aurantiacus treated with 1-hexanol and proteolytic enzymes.

The circular dichroism (CD) spectrum of isolated chlorosomes fromChloroflexus aurantiacus showed a conservative, S-shaped signal with a negative maximum at 723 nm, a positive maximum at 750 nm and a zero-crossing at 740 nm. Proteolytic treatment of chlorosomes with trypsin at 37°C did not change the CD signal or the absorption spectrum in contrast to treatment with proteinase K, where a twofold increase in rotational strength and a slight decrease of the absorption band at 740 nm were observed. Treatment with saturating 1-hexanol concentrations resulted in a blue shift of the absorption band at 740 nm as well as in changes of the CD spectrum. These changes reversed when the sample was diluted to half the saturating 1-hexanol concentration. In contrast to that, we observed an irreversible formation of a giant CD signal using the combination of 1-hexanol and proteinase K treatment. Electron micrographs of chlorosomes treated with both 1-hexanol and proteinase K showed large aggregates of multiple chlorosome size. By comparison of proteinase K induced effects with trypsin effects it appeared that the 5.7 kDa polypeptide has a structural role in the organisation of BChlc in the chlorosome.

Structural differences in chlorosomes from Chloroflexus aurantiacus grown under different conditions support the BChl c-binding function of the 5.7 kDa polypeptide

Structurally different chlorosomes were isolated from the green photosynthetic bacterium Chloroflexus aurantiacus grown under different conditions. They were analysed with respect to variable pigment‐protein stoichiometries in view of the presumed BChI c‐binding function of the 5.7 kDa chlorosome polypeptide. Under high‐light conditions on substrate‐limited growth medium the pigment‐protein ratio of isolated chlorosomes was several times lower than under low‐light conditions on complex medium. Proteolytic degradation of the 5.7 kDa polypeptide in high‐light chlorosomes led to a 60% decrease of the absorbance at 740 nm. The CD spectrum of high‐light chlorosomes exhibited a sixfold lower relative intensity at 740 nm (ΔA/A 740) than low‐light chlorosomes, but it showed a fivefold increase in intensity upon degradation of the 5.7 kDa polypeptide compared to a twofold increase in low‐light chlorosomes. It seems probable that BChl c in the chlorosomes is present as oligomers bound to the 5.7 kDa polypeptide. Our data suggest further that compared to low‐light chlorosomes smaller oligomers or single BChl c molecules are bound to the 5.7 kDa polypeptide in high‐light chlorosomes resulting in lower rotational strength.