Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120.

The phycobilisome (PBS) of Anabaena sp. PCC 7120 was allowed to dissociate into its constituents and the resulting allophycocyanin (AP) fraction was purified. Its reconstitution yielded a complex which according to negative stain electron microscopy and spectral analysis was identical to the native pentacylindrical PBS core domain. Each cylinder of the central tricylindric unit was comprised of four AP (alphabeta)3 disks. Mass analysis using the scanning transmission electron microscope (STEM) showed the presence of 16 AP trimers in the intact reconstitute, which had a total mass of 1966(+/-66) kDa. Composition analysis indicated an AP trimer distribution of (AP-II):(AP-LCM):(AP-B):(AP-I)=6:2:2:6, i.e. an addition of two AP-I and two AP-II complexes compared to a tricylindrical PBS core domain. Therefore, we suggest that each supplementary half-core cylinder found in pentacylindrical AP core domains is comprised of one AP-I and one AP-II trimer, in agreement with the current model. The structural significance of the 127 kDa core membrane linker polypeptide was further investigated by subjecting the AP core reconstitute to mild chymotryptic degradation. After isolation, the digested complex exhibited a tricylindrical appearance while STEM mass analysis confirmed the presence of only 12 AP complexes. Polypeptide analysis by SDS-PAGE and Edman degradation related the half-cylinder loss to cleavage of the Rep4 domain of the core membrane linker polypeptide. On the basis of these data, a general model for the assembly of the three hemidiscoidal PBS types known to date is discussed.

The antenna complexes of the purple non-sulfur photosynthetic bacterium Rhodocyclus tenuis. Structural and spectral characterization.

The photoreceptor complex (B885-RC) and the peripheral antenna complex (B800-860) were isolated from photosynthetic membranes of the purple non-sulfur bacterium Rhodocyclus tenuis DSM 109 using a detergent combination of Deriphate-160 and octyl glucoside and subsequent linear sucrose gradient centrifugation. The two complexes were characterized by room-temperature absorption, circular dichroism and fluorescence spectroscopy. The B800-860 complex has a more red-shifted B860 absorbance band. The alpha,beta-polypeptides were purified with a reverse-phase HPLC system and resolved at a ratio of 1:1 in the B800-860 complex and at an overall ratio of 1:1 for the B885-RC complex. The complete amino acid sequences of the alpha and beta polypeptides of the B800-860 and B885-RC complexes were determined by micro-sequencing analysis and mass spectrometry. The B800-860-alpha polypeptide possesses an identical N-terminal domain (the first 15 residues) to Rhodobacter sphaeroides B800-850-alpha polypeptide. The central hydrophobic and C-terminal domains of the B800-860-alpha,beta polypeptides show a number of B870/880-like structural elements in which, of special interest, is the WWSEF cluster in the C-terminal domain of the B800-860-alpha polypeptide which is very similar to the WWEF cluster in the same region of Rhodopseudomonas viridis B1015-alpha polypeptide. The more red-shifted absorption characteristic of the 860-nm bacteriochlorophylls could most probably be related to the B870/880-like polypeptide features in the central hydrophobic domains and the C-terminal domains of the B800-860-alpha,beta polypeptides. The hydropathy plot of the B800-860-alpha polypeptide exhibits an extended C-terminal hydrophobic segment indicative of a second membrane-contacting domain, which has not been found in the antenna polypeptides of the purple bacteria with intracytoplasmic membranes. Further sequence analysis revealed the existence of multiple forms of the B885-alpha,beta polypeptides, the B885-alpha 1,alpha 2 polypeptides, and the B885-beta 1,beta 2 polypeptides. The B885-Alpha 2 polypeptide shows an identical sequence to the B885-alpha 1 polypeptide, but it is 12 amino acid residues shorter than the B885-alpha 1 polypeptide at the C-terminal. The two species of the B885-beta polypeptides were identified as an identical sequence with only one amino acid residue variation at sequence position 34, where the B885-beta 1 has a valine residue and the B885-beta 2 polypeptide an isoleucine residue. The possible correlation between the intensity of the near-infrared circular dichroic signal and the specific structural features of the alpha and beta core antenna polypeptides is also discussed.

Isolation, characterization and electron microscopy analysis of a hemidiscoidal phycobilisome type from the cyanobacterium Anabaena sp. PCC 7120.

In this work we present the characterization of a hemidiscoidal phycobilisome type of the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. The phycobilisome of this organism contains allophycocyanin, phycocyanin and phycoerythrocyanin, similar to the closely related thermophilic cyanobacterium Mastigocladus laminosus. Intact phycobilisomes exhibit an absorption maximum at 619 nm and two fluorescence maxima at 664 nm and 680 nm, corroborating the presence of a complete energy pathyway along the antenna. Upon dissociation, the phycobiliproteins were released from the phycobilisome. One phycoerythrocyanin, one phycocyanin and three allophycocyanin complexes were isolated by ion-exchange chromatography and characterized by absorption and fluorescence spectroscopy and by SDS/PAGE. The amino-terminal sequences of the polypeptides belonging to the phycoerythrocyanin and phycocyanin families were identical with the derived sequences of their corresponding genes. Partial amino-terminal sequences of the polypeptides belonging to the allophycocyanin family are presented here. Our results show that the phycobiliproteins and linker polypeptides from Anabaena sp. PCC 7120 are similar to the phycobilisome components characterized in other cyanobacteria. The phycobilisome of Anabaena sp. PCC 7120 was extensively analyzed by electron microscopy. It differs from the common hemidiscoidal tricylindrical, six-rod phycobilisome type by a core domain consisting of five core cylinders surrounded by up to eight rods radiating in a hemidiscoidal manner. One rod is linked to each basal core cylinder, whereas the remaining core cylinders bind two rods each. On the basis of the data presented in this work, a revised model for the hemidiscoidal pentacylindrical phycobilisome of Anabaena sp. PCC 7120, M. laminosus and Anabaena variabilis is proposed. This model accounts more accurately for the 'grape' pattern typically exhibited by these phycobilisomes in electron micrographs.

Proteolytic modifications of the B800-860 complex of the photosynthetic bacterium Rhodocyclus tenuis: Structural and spectral effects.

The modification effects on the absorption and cirular dichroic (CD) spectra of the isolated B800-860 antenna complex of Rhodocyclus tenuis by a number of proteolytic enzymes were investigated. The chymotrypsin modifications of the B800-860 complex led to an about 40% decrease of the 860-nm band and a blue-shift to 841 nm. The biphasic CD signal related to the B860 BChl disappeared and a new double CD signal with a zero-crossing point at 842 nm appeared. These absorption and CD spectral changes suggested that a B800-841 complex resulted after chymotrypsin digestion. The polypeptide components of the chymotrypsin-modified B800-860 complex were separated by reverse-phase chromatography, and their amino acid sequences determined by protein sequencing and mass spectrometry. Sequence analyses showed that the C-terminal 25 residues of the B800-860-α polypeptide and the C-terminal 8 residues of the B800-860-ß polypeptide were cleaved by chymotrypsin, and the remaining α, ß polypeptide fragments apparently form the structural basis for the newly-formed B800-841 complex. No significant spectral change was observed from exposing the isolated B800-860 complex to trypsin, carboxypeptidase A and the combination of carboxypeptidase A and carboxypeptidase B. Short-term proteinase K incubation of the B800-860 complex of Rc. tenuis led to a preferential decrease of the 860-nm absorbance band and its related CD signals, as compared to the 800-nm absorbance and CD bands, suggesting that the C-terminal portions of the antenna polypeptides are possibly exposed to the exterior of the B800-860 complex micelles. Whereas, long-term proteinase K digestion resulted in the spectral collapse of the B800-860 complex and the release of free BChls. Our proteolysis experiments support the hypothesis that the C-terminal portions of the antenna polypeptides play a key role in the redshift and strong molar extinction of the Qy band of the B850 BChls.

The six fold symmetry of the B880 light-harvesting complex and the structure of the photosynthetic membranes of Rhodopseudomonas marina.

The reaction center free light-harvesting core complex of Rp. marina was purified by DEAE 52 ion exchange chromatography in the presence of the detergent OG. The protein complex was crystallised by microdialysis yielding two-dimensional crystals with a diameter of up to 10 microns. The crystals were negatively stained with uranyl acetate or prepared in vitrified ice and electron micrographs were taken. They exhibited a hexagonal lattice with a lattice constant of 102 +/- 3 A. The optical diffraction pattern of the best ordered areas of electron micrographs showed spots up to a resolution of 29 A. Image processing revealed a six fold symmetry of the ring like B880-complex. The protein ring is hexagonal with one subunit in each corner of the hexagon and two subunits forming the connection site to the neighbouring B880-complex in the crystal. In freeze fracture preparations of whole cells the intra-cytoplasmic photosynthetic membranes are seen to be organised into large stacks that affect the organisation of the photosynthetic complexes. Most notably, the stacked membrane regions exhibit hexagonally packed photosynthetic complexes with a repeat of approximately 100 A, which is very similar to the lattice of the artificial B880-complex crystals. The same quasi-crystalline structure appeared in the cytoplasmic membrane of the contact sites with the intra-cytoplasmic membrane stack, but was absent from the end membrane of the stack. Thus, membrane stacking appears to induce the formation of the crystalline arrays, presumably through interactions between the cytoplasmic surface domains of the photosynthetic complexes. Tight packing of the photosynthetic particles is not sufficient to induce the crystalline order. The intra cytoplasmic membranes form a continuum with the cytoplasmic membrane via their origins at the round invagination sites.

Structural and spectral characterisation of the antenna complexes of Rhodocyclus gelatinosus. Indications of a hairpin-like-arranged antenna apoprotein with an unusually high alanine content.

The core antenna/reaction-centre complex RC-B875 and the peripheral antenna complex B800-850 of the two strains DSM 149 and DSM 151 of the purple non-sulphur bacterium Rhodocyclus gelatinosus have been isolated from photosynthetic membranes by means of lauryl-N,N-dimethyl-amineoxide as a detergent and subsequent sucrose-gradient centrifugation. The two complexes were characterised spectroscopically by absorption and circular dichroism (CD) spectroscopy at room temperature. CD measurements revealed very weak signals for the core antenna B875 whereas for the peripheral antenna B800-850, a strong biphasic CD signal was observed, attributable to the B850 pigments. There is apparently no CD signal present for the B800 pigments. The core and the peripheral antenna complex are built up by a distinct alpha/beta-polypeptide pair. The pigment/protein ratio in the peripheral antenna complex is 3 bacteriochlorophyll/(alpha/beta)-polypeptide pair. The amino acid sequences of the alpha and beta polypeptides of both complexes from the two strains of Rc. gelatinosus were established by automated Edman degradation, chemical and enzymic digestion, amino acid composition analyses and carboxypeptidase digestion. In the case of the beta polypeptides, the amino acid sequence determination was confirmed by ion-spray MS of the isolated antenna apoproteins. The inter-strain (DSM 149 and 151) positional identity between the equivalent apoproteins is extremely large and varies in the range 90-100%. The B875-beta polypeptide from Rc. gelatinosus exhibits shortened C-termini, as detected for the analogous antenna apoproteins of Rhodobacter sphaeroides and Rhodobacter capsulatus, which can be correlated with weak core antenna near-infrared CD signals. However, the B800-850-alpha polypeptide of Rc. gelatinosus, with 71 amino acids, exhibits an extended C-terminal portion indicative of the formation of a second transmembrane domain, which so far has not been observed for bacterial antenna apoproteins. This part of the molecule is extremely rich in alanine and proline residues. All the sequenced antenna apoproteins of Rc. gelatinosus exhibit a characteristic membrane-buried histidine which is thought to ligate the B875 or the B850 pigments. In the B800-850-beta apoprotein, a second, so far beta-antenna-apoprotein-specific histidine, is replaced by a glutamine residue. A careful inspection of the determined antenna structures of Rc. gelatinosus revealed some remarkable structural similarities within presumed cofactor-binding sites of Fe-S-type-reaction-centre apoproteins, indicating possible basic structural motifs for complexing bacteriochlorophyll molecules.

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 BChl 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 (delta A/A740) 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.

The complete amino acid sequence of R-phycocyanin-I alpha and beta subunits from the red alga Porphyridium cruentum. Structural and phylogenetic relationships of the phycocyanins within the phycobiliprotein families.

We present here the complete primary structure of R-phycocyanin-I alpha and beta subunits from the red alga Porphyridium cruentum. The alpha chain is composed of 162 amino acid residues (18049 Da, calculated from sequence, including chromophore) and carries a phycocyanobilin pigment covalently linked to Cys84. The beta chain contains 172 amino acids (19344Da, calculated from sequence, including chromophores) and carries a phycocyanobilin pigment covalently linked at Cys82 and a phycoerythrobilin pigment at Cys153. A gamma-N-methyl asparagine residue was also characterised at position beta 72 similar to other phycobiliprotein beta subunits. R-phycocyanin-I from Porphyridium cruentum shares high sequence identity with C-phycocyanins (69-83%), R-phycocyanins (66-70%) and in a less extent with phycoerythrocyanins (57-65%) from various sources. The presented phylogenetic trees are based on a comparison of all phycobiliprotein amino acid sequences known so far and confirm the clear affiliation of the R-phycocyanins in the phycocyanin family. In spite of their particular phycobilin pattern, they do not represent intermediate forms between the phycocyanin and the phycoerythrin family. Phycoerythrocyanin, a phycocyanin-related phycobiliprotein adapted to green light harvesting, is also shown to belong to the phycocyanin family. However, the phycoerythrocyanins diverge from phycocyanins in their different function and it is suggested that they should be assigned to a separate group within the phycocyanin family.

Polypeptide sequence of the chlorophyll a/b/c-binding protein of the prasinophycean alga Mantoniella squamata.

The primary structure of the Chla/b/c-binding protein from Mantoniella squamata is determined. This is the first report that protein sequencing reveals one modified amino acid resulting in a LHCP-specific TFA-cleavage site. The comparison of the sequence of Mantoniella with other Chla/b-and Chla/c-binding proteins shows that the modified amino acid is located in a region which is highly conserved in all these proteins. The alignment also reveals that the LHCP of Mantoniella is related to the Chla/b-binding proteins. Finally, possible Chl-binding regions are discussed.

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.

Genes encoding two chlorosome components from the green sulfur bacteriaChlorobium vibrioforme strain 8327D andChlorobium tepidum.

Chlorosomes of the thermophilic green sulfur bacteriumChlorobium tepidum have been isolated and their polypeptides analyzed by polyacrylamide gel electrophoresis and amino acid sequencing. These chlorosomes were shown to contain nine different polypeptides ranging in mass from approximately 6 to 27 kDa. ThecsmA gene, encoding a highly abundant chlorosome protein with a mass of 6.2 kDa, were cloned and sequenced from bothChlorobium vibrioforme strain 8327D andChlorobium tepidum. The gene from both species predicts identical proteins of 79 amino acid residues, and a comparison of the deduced sequence with that determined for the protein indicates that 20 amino acid residues are post-translationally removed from the carboxyl-terminus of the CsmA precursor. Transcript analyses showed that inChlorobium tepidum thecsmA gene is encoded on two transcripts of approximately 350 and 940 nucleotides; the smaller transcript probably results from processing of the larger RNA molecule. Transcription of the longer mRNA initiates 68 basepairs upstream from the start codon of a second open reading frame that is located 154 nucleotides 5' tocsmA and that predicts a protein of 139 amino acid residues. The amino-terminal sequence determined for a 14.5 kDa polypeptide in the chlorosomes ofChlorobium tepidum matched the sequence deduced from this open reading frame except for the absence of the initiator methionine residue; accordingly, this gene has been namedcsmC. A comparison of the genomic organization of thecsmA loci inChlorobium vibrioforme, Chlorobium tepidum, andChloroflexus aurantiacus were found to be surprisingly similar.

Reconstitution of the core complex (alpha beta)3APCLC8.9 of the phycobilisome from Mastigocladus laminosus using the Lc8.9 linker polypeptide overexpressed in Escherichia coli.

The apcC gene from Mastigocladus laminosus encodes the linker polypeptide LC8.9 located in the phycobilisome core. A T7 RNA polymerase expression system was used to express the linker polypeptide LC8.9 from M. laminosus in Escherichia coli. The apcC gene product was expressed as an inclusion body which was solubilized in a buffer containing 8M urea. Final purification was achieved by ion exchange chromatography on Fractogel TSK CM 650 (S). In addition, a method for preparative isolation of the LC8.9 linker polypeptide from M. laminosus by reverse phase chromatography is presented. Both LC8.9 isolated from M. laminosus and overexpressed in E. coli were capable of reconstituting the complex (alpha beta)3APCLC8.9. The reconstituted complex was identical to preparations isolated from M. laminosus in terms of polypeptide composition, absorption and fluorescence emission spectroscopy.

Mutations that significantly change the stability, flexibility and quaternary structure of the l-lactate dehydrogenase from Bacillus megaterium.

In order to investigate the physical basis of protein stability, two mutant L-lactate dehydrogenases (LDH) and the wild-type enzyme from Bacillus megaterium were analyzed for differences in quaternary structure, global protein conformation, thermal stability, stability against guanidine hydrochloride, and polypeptide chain flexibility. One mutant enzyme, ([T29A, S39A]LDH), differing at two positions in the alpha-B helix, exhibited a 20 degrees C increase in thermostability. Hydrogen/deuterium exchange revealed a rigid structure of this enzyme at room temperature. The substitutions Ala37 to Val and Met40 to Leu destabilize the protein. This is observable in a greater susceptibility to thermal denaturation and in an unusual monomer/dimer/tetramer equilibrium in the absence of fructose 1,6-bisphosphate Fru(1,6)P2. The stability, flexibility and protein-conformation measurements were all performed in the presence of 5 mM Fru(1,6)P2, i.e. under conditions where the three investigated LDH species are stable tetramers. Tryptophan fluorescence was used to monitor the unfolding in guanidine HCl of two local structures in or very close to the beta-sheets at the protein surface. The LDHs form folding intermediates in guanidine HCl that aggregate at elevated temperatures. Pronounced differences between the three investigated enzymes are found in their ability to aggregate. The exchange of Thr29 and Ser39 for Ala leads to significantly less aggregation in guanidine HCl than is observed for wild-type LDH. Using 8-anilinonaphthalene-1-sulfonic acid, the folding intermediates were shown to be in accordance with molten-globule-like structures. We have found, by means of molecular sieve chromatography, that the [T29A, S39A]LDH with its increased thermostability has lower susceptibility to disintegrate into monomers in guanidine HCl at 25 degrees C. Despite the differences in aggregation at low guanidine HCl concentrations and temperatures above 25 degrees C, the molten-globule-like structures of the three investigated LDH species are structurally similar, as shown by molecular-sieve chromatography. Although the thermostabilities of the three LDH species are so different in aqueous buffers, their stabilities in guanidine HCl at 20 degrees C are, surprisingly, almost identical. Some comments are made as to the origin of the observed difference between thermal and guanidine HCl stabilities of the LDH. Near-ultraviolet and far-ultraviolet circular dichroism measurements, as well as differences in the amount of activation by Fru(1,6)P2, point to small global structural rearrangements caused by the mutations. Conformational changes upon Fru(1,6)P2 binding or point mutations in the alpha-B helix show that the Fru(1,6)P2-binding site and the alpha-B helix are structurally linked together.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of chlorophyll a/b proteins of photosystem I from Chlamydomonas reinhardtii.

In this study we have isolated the chlorophyll a/b-binding proteins from a photosystem I preparation of the green alga Chlamydomonas reinhardtii and characterized them by N-terminal sequencing, fluorescence, and absorption spectroscopy and by immunochemical means. The results indicate that in this organism, the light-harvesting complex of photosystem I (LHCI) is composed of at least seven distinct polypeptides of which a minimum number of three are shown to bind chlorophyll a and b. Both sequence homology and immunological cross-reactivity with other chlorophyll-binding proteins suggest that all of the LHCI polypeptides bind pigments. Fractionation of LHCI by mildly denaturing methods showed that, in contrast to higher plants, the long wavelength fluorescence emission typical of LHCI (705 nm in C. reinhardtii) cannot be correlated with the presence of specific polypeptides, but rather with changes in the aggregation state of the LHCI components. Reconstitution of both high aggregation state and long wavelength fluorescence emission from components that do not show these characteristics confirm this hypothesis.

Cloning and sequencing of the genes encoding glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase (gap operon) from mesophilic Bacillus megaterium: comparison with corresponding sequences from thermophilic Bacillus stearothermophilus.

The structural genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and the N-terminal part of triosephosphate isomerase (TIM) from mesophilic Bacillus megaterium DSM319 have been cloned as a gene cluster (gap operon) by complementation of an Escherichia coli gap amber mutant. Subsequently, the entire tpi gene, encoding TIM, was isolated by colony hybridization using a homologous probe. Nucleotide (nt) sequence analysis revealed an unidentified open reading frame (urf1) of 1029 bp located 50 nt upstream from the start codon of the gap gene. Gene expression from subclones containing different coding regions was studied by enzyme assay and SDS-PAGE. Both GAPDH and TIM are synthesized in transformed E. coli cells, whereas PGK is not. There is no unequivocal evidence for urf1 expression. Two putative promoter sites are present: one 100 nt upstream from urf1 and one 200 nt upstream from the pgk gene. An inverted repeat following the second promoter site is postulated to be involved in the transcriptional regulation of the operon. Each coding region shows a G+C content of 40% attained by the adaptation of the G+C content of the third base in the codon to compensate the G+C content of the first and second bases. The deduced amino acid (aa) sequences of B. megaterium GAPDH, PGK and TIM were compared with those from the thermophilic Bacillus stearothermophilus by antisymmetrical matrices. The detected characteristic thermophilic-mesophilic exchange pattern concerning aa substitutions between hydrophobic-polar and charged-charged residues corresponds to data obtained for thermophilic and mesophilic lactate dehydrogenases (LDH). The determination of the thermostability of these enzymes revealed two regions of stability for B. megaterium TIM at high enzyme concentrations. Heat treatment seems to be responsible for the conversion of two differently active conformations or the induction of a new quaternary structure.

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-beta-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 alpha- to beta-polypeptide ratio and the alpha- or beta-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 of 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 +/- 3 A. Purified B880-complex was concentrated up to an OD880 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 A as was observed in the native membranes. Image processing of the micrographs revealed a ring-like structure of the B880-complex at 26 A 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.

Evidence for temperature-dependent conformational changes in the L-lactate dehydrogenase from Bacillus stearothermophilus.

L-Lactate dehydrogenase from Bacillus stearothermophilus (BSLDH) has been shown to change its conformation in a temperature-dependent manner in the temperature range between 25 and 70 degrees C. To provide a more detailed understanding of this reversible structural reorganization of the tetrameric form of BSLDH, we have determined in the presence of 5 mM fructose, 1,6-bisphosphate (FBP) the effect of temperature on far-UV and near-UV circular dichroism (CD), Nile red-binding to the enzyme surface, NADH binding, fluorescence polarization of fluorescamine-labeled protein, and hydrogen-deuterium exchange. In addition, we have analyzed the temperature dependence of the dimer-tetramer equilibrium of this protein by steady-state enzyme kinetics in the absence of FBP. The results obtained from these measurements at various temperatures can be summarized as follows. No changes in the secondary-structure distribution are detectable from far-UV CD measurements. On the other hand, near-UV CD data reveal that changes in the arrangements of aromatic side chains do occur. With increasing temperature, the asymmetry of the environment around aromatic residues decreases with a small change at 45 degrees C and a more pronounced change at 65 degrees C. Nile red-binding data suggest that the BSLDH surface hydrophobicity changes with temperature. It appears that decreasing the surface hydrophobicity may be a strategy to increase the protein stability of the active enzyme. We have noted significant alterations in the thermodynamic binding parameters of NADH above 45 degrees C, indicating a conformational change in the active site at 45 degrees C. The hydrodynamic volume of BSLDH is also temperature dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

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.