Herbert Zuber’s research while affiliated with Hochschule für Technik Zürich and other places

The antenna complex B880 of Rp. marina has been isolated by applying ion-exchange chromatography on Whatman DE-52 resin and sucrose density centrifugation of LDAO-solubilized photosynthetic membranes. The antenna polypeptides B880-alpha and B880-beta were prepared by organic solvent extraction of extensively dialyzed and freeze-dried B880 antenna complex material or photosynthetic membranes. Gel filtration on Sephadex LH-60 and ion-exchange chromatography on Whatman DE-32 resin in the presence of organic solvents and an additional step on a C-8 reversed phase column yielded pure alpha- and beta-apoproteins. Their complete primary structures have been elucidated using automated Edman degradation and carboxypeptidase digestion. According to quantitative Edman degradation the ratio of B880-alpha and B880-beta has been determined as 1:1 in the isolated antenna complex as well as in the photosynthetic membrane. B880-alpha of Rp. marina, presumably N-formylated, consists of 52 amino acid residues and is 75, 56, 52 and 44% homologous to the corresponding core antenna polypeptides of Rs. rubrum, Rp. viridis, Rb. capsulatus and Rb. sphaeroides. In contrast, B880-beta (56 amino acid residues) is less homologous to the corresponding core beta-antenna polypeptides of the same strains (57, 51, 41 and 42%). It shows an extended N-terminal domain as compared to the B880-alpha polypeptide. Apart from the typical structural features of bacterial membrane-bound antenna polypeptides (three domain structure. His-residue in the hydrophobic stretch) the antenna polypeptides of Rp. marina are structurally related to polypeptides of core antenna complexes with strong near infrared circular dichroism signals.

Spectrally pure B 800-850 light harvesting complexes of Rhodopseudomonas acidophila 7750 were prepared by chromatography of LDAO-solubilised photosynthetic membranes on Whatmann DE-52 ion exchange resin. Two low molecular mass polypeptides (α, β) have been isolated by organic solvent extraction of the lyophilised B 800-850 light harvesting complexes. Their primary structures were determined by liquid phase sequencer runs, by the sequence analyses of C-terminal o-iodosobenzoic acid fragments, by hydrazinolysis and by carboxypeptidase degradation. B 800-850-a consists of 53 amino acids and is 45.3% and 50.9% homologous to the B 800-850- a antenna polypeptides of Rhodobacter sphaeroides and Rhodobacter capsulatus, respectively. The second very short polypeptide (B800-850-β, 41 amino acids) is 61.0% and 56.1% homologous to the corresponding polypeptides of Rb. sphaeroides and Rb. capsulatus. The molar ratio of the two polypeptides is about 1:1. Both polypeptides show a hydrophilic N-terminal domain, a very hydrophobic central domain and a short C-terminal domain. In both polypeptides the typical His residues, identified in all antenna polypeptides of purple nonsulphur bacteria as possible bacteriochlorophyll binding sites, were found

In addition to the previous isolated and sequenced polypeptides from green photosynthetic sulfur bacteria, which are presumably involved in binding BChl c and e, an analogous polypeptide has been purified from the BChl d-containing bacterium Chlorobium vibrioforme f. thiosulfatophilum. The primary structure of this 6.15 kDa polypeptide was determined. It shows an extremely high homology (98.3%) to the corresponding polypeptide from Pelodictyon luteolum, indicative of an important functional role.

A rod-core complex, (αβ)6PC. LRC29.5,(αβ)3APC.LC8.9, composed of hexameric phycocyanin (PC) and trimeric allophycocyanin (APC) subcomplexes associated to the CpcG2 gene product, has been isolated from the phycobilisome of Mastigocladus laminosus. Exactly the same complex was obtained by reconstitution using the subcomplexes(αβ)3PC. LRC 29.5 and (aβ)3APC.LC8.9 as educts. Spectroscopic analysis of the isolated PC-LR and PC-LRC complexes from M. laminosus shows that the LRC cause the largest red-shift in the absorbance and fluorescence emission maxima of PC. These results indicate that LRC mediate PC-APC interactions in vitro. Only the CpcG2 polypeptide was able to promote this specific interaction, as neither CpcG3-PC nor, as a negative control, CpcC-PC complexes showed any reconstitution products with the core subcomplex (αβ)3APC.Lc8.9. This is an indication that each of the four LRC in the phycobilisomes of M. laminosus and Anabaena sp. PCC 7120 attaches two peripheral rods specifically to one of four different core binding sites.

The excited state dynamics of trimeric phycoerythrocyanin has been studied by two-color femtosecond transient absorption spectroscopy with a time-resolution better than 200 fs. Upon selective excitation of the short-wavelength phycobiliviolin chromophore at 575 nm absorption bleachings are observed. An isotropic ultrafast decay of the initial bleaching of 585 ± 40 fs has been resolved at short detection wavelength (578 nm). Upon stepwise increase of the detection wavelength up to 617 nm, the bleaching showed a delayed rise above 593 nm with rrice=380–580 fs. All other isotropic kinetic components in this wavelength range were longer than 100 ps. The ultrafast component is discussed in terms of an energy transfer process from the α-84 phycobiliviolin chromophore to the β-84 chromophore in adjacent monomer subunits of the trimer. It is concluded that the β-155 chromophore is the longest-wavelength chromophore. Exciton relaxation between closely spaced chromophore pairs is discussed as an alternative interpretation for the ultrafast component.

The spectral effects of a number of endo- and exoproteases to the detergent-solubilized antenna complexes B802–858(sphaeroides like) of strain Rhodopseudomonas acidophila 10050 and B802–824of strain 7050 were analyzed. Upon protease digestion the time course of spectral modification between 250 and 900 nm was recorded. In addition, circular dichroism (CD) signals were measured between 450 and 950 nm. The endoprotease elastase as well as the exoprotease carboxypeptidase A (CP A) altered the B802–858antenna complex, by changing the 858 nm band hypochromically (30-50%) with an additional small hypsochromic shift (3–6nm). A combined carboxypeptidase digestion, e.g. CP A and CP B, yielded a further modification to a complex with absorption bands at 800 nm and ˜840 nm (ε˜ 50% of the 800 nm band). By exposing the isolated B803–824antenna complex to the different proteases no significant spectral change was observed. The B802–858antenna complex, modified by CP A, exhibits a60–70% decrease of the biphasic CD signal in the near infrared. The limited proteolysis experiments provide conclusive experimental evidence that the C-terminal domains of the antenna polypeptides and β contribute to the formation of (dimeric) bacteriochlorophyll (BChl) a molecules absorbing at around 850 nm. This agrees with the hypothesis, based on comparative amino acid sequence analysis, that in purple bacterial antenna complexes the structural requirements (specific amino acids in the vicinity of the BChl molecules, e.g. aromatic amino acids) for batho- and hyperchromicity of BChl molecules apparently reside to a considerable extent in the C-terminal portions of their antenna apoproteins.

Phycobilisomes of the cyanobacteria Mastigocladus laminosus and Anabaena sp. PCC7120 differ from typical tricylindrical, hemidiscoidal phycobilisomes in three respects. Firstly, size comparisons of the core-membrane linker phycobiliproteins (LCM)in different cyanobactertia by SDS/PAGE reveal an apparent molecular mass of 120 kDa for the LCM of M. laminusus and Anabaena sp. PCC7120. This observation suggests that the polypeptides of these species have four linker-repeat domains. Secondly, phycobilisomes of M. laminosus are shown to contain at least three, but most probably four, different rod-core linker polypeptides (LRC). These LRC, which attach the peripheral rods to the core and thereby make phycocyanin/allophycocyanin contacts, have been identified and characterized by N-terminal amino acid sequence analysis. Additionally, electron microscopy of phycobilisomes isolated from M. laminosus and Anabaena sp. PCC7120 reveals similar structures which differ from those of Calorthrix sp. PCC7601 with their typical six, peripheral rods. Based upon protein-analytical results and a reinterpretation of the data of [Isono, T. & Katoh, T. (1987) Arch. Biochem. Biophys. 256, 317–324], we discuss structural implications of recent findings on the established hemidiscoidal model for the phycobilisomes of M. laminosus and Anabaena sp. PCC7120. Up to eight peripheral rods are suggested to radiate from a modified core substructure which contains two additional peripheral allophycocyanin hexamer equivalents that serve as the core-proximal discs for two peripheral rods.

This chapter presents a comprehensive overview of what is currently known about the structure of purple bacterial antenna complexes, with special emphasis upon modeling their three-dimensional structure and intramembrane organization. Purple bacterial antenna complexes can be grouped into two major types, the ‘core’ B875-type which forms a close association with the reaction centers and the ‘variable’ B800-850- or B800-820-type. Both these types of complex are constructed on a similar modular principle and are oligomers of α and β-apoproteins. These antenna apoproteins have been sequenced form a range of species and form a rather homologous group of proteins. The light-absorbing pigments, BChl and carotenoids, are non-covalently bound to these apoproteins. As is described below, in a few cases these antenna complexes have been crystallized and structure determinations are under way. However, in the absence of three-dimensional structural information a great deal can be deduced by a comparison of their primary structures. The main part of this chapter illustrates the type of models for antenna structure that can be constructed by a detailed analysis of the conserved structural elements which can be seen by such an examination of the database of antenna apoprotein primary structures.