Alexander N. Glazer

Characterization and structural properties of the major biliproteins of Anabaena sp.

AbstractStudies are presented of the biliproteins of Anabaena sp. This filamentous cyanobacterium contains three major biliproteins. Whereas two of these, C-phycocyanin and allophycocyanin, are common to all cyanobacteria, the third, phycoerythrocyanin (λmax∼568nm) has hitherto not been described and its distribution among cyanobacteria appears to be limited. Anabaena variabilis and Anabaena sp. 6411 allophycocyanin, C-phycocyanin, and phycoerythrocyanin were purified to homogeneity and characterized with respect to molecular weight, isoelectric point, absorption spectrum and amino acid composition. The α and β subunits of each of these proteins were also purified to homogeneity and characterized in the same manner. The tetrapyrrole chromophore content was determined for each of the proteins and subunits. The α subunit of phycoerythrocyanin carries a novel phycobiliviolin-like chromophore. This chromophore has not previously been detected in cyanobacterial biliproteins, but has been noted as a prosthetic group of a cryptophytan phycocyanin.Sedimentation equilibrium studies show that at pH 7.0, at protein concentrations of 0.2–0.6 mg/ml, allophycocyanin, C-phycocyanin and phycoerythrocyanin, each exists as a trimeric aggregate, (αβ)3, of molecular weight of approximately 105000. Structural studies of microcrystals of these three biliproteins by electron microscopy and X-ray diffraction reveal a common plan for the construction of higher assembly forms. The major building block appears to be the trimer (αβ)3. It is proposed that this is a dise-like structure about 3.0×12.0 nm. The individual α or β subunits are roughly spherical, 3 nm in diameter. Allophycocyanin trimers stack to form bundles of rods which form long needles. Both phycocyanin and phycoerythrocyanin form double dises (αβ)6 which are visible as ring-shaped structures by electron microscopy. The mode of assembly of the biliproteinstructures in the phycobilisome is, as yet, unknown.

Allophycocyanin B (λmax 671, 618 nm)

AbstractA hitherto undescribed red fluorescent phycobiliprotein (maximum emission at ∼ 680 nm), characterized by long wavelength absorption maxima in the visible region at 671 nm (ε=172000 M−1·cm−1 per monomer of mol. wt. 30600) and 618 nm, has been purified to homogeneity from a unicellular cyanobacterium, Synechococcus sp., and from a filamentous cyanobacterium, Anabaena variabilis. The name allophycocyanin B has been proposed for the new protein. A. variabilis allophycocyanin B is characterized by a native molecular weight of 89000 ± 5000 (in 0.05 M phosphate at pH 7.2), an isoelectric point of 5.09, and a subunit molecular weight, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of 15300. The protein contains one phycocyanobilin chromophore per subunit. In common with allophycocyanin from the same organism, allophycocyanin B does not contain either histidine or tryptophan. In other respects, the amino acid compositions of the two proteins are significantly different. Synechococcus sp. (Anacystis nidulans) allophycocyanin B gives two components of 16000 and 17000 mol. wt., of equal staining intensity, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Allophycocyanins B from both organisms cross-react with rabbit antisera directed against either Synechococcus sp. or Anabaena sp. allophycocyanin, but not with antisera against the phycocyanins of the same organisms.It is suggested that allophycocyanin B occupies a position between allophycocyanin and chlorophyll a in the energy transfer path from the accessory pigments to species of chlorophyll a with absorption maxima at λ>670 nm.

Spectroscopic studies of cyanobacterial phycobilisomes lacking core polypeptides

Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum PR6) genes encoding two highly conserved phycobilisome core polypeptides, a small linker polypeptide (LC8, apcC) and the allophycocyanin-B alpha-subunit (alpha APB, apcD), respectively, were interrupted by insertion of restriction fragments carrying the neomycin phosphotransferase gene of Tn5. The interrupted genes were used to transform Synechococcus sp. PCC 7002 to kanamycin resistance. The apcC- mutant assembled phycobilisomes lacking the LC8 polypeptide and the apcD- mutant assembled phycobilisomes lacking alpha APB. No other differences between the compositions of the mutant and wild-type phycobilisomes were detected. The apcC- strain grew about 25% more slowly than the wild-type, and its phycobilisomes dissociated more rapidly in 0.33 M Na/K-PO4 (pH 8.0) or in 0.75 M Na/K-PO4 at pH 8.0, at 40 degrees C, than did those of the wild-type. The phycobilisomes of this mutant were indistinguishable from those of the wild-type with respect to absorption and circular dichroism spectra, as well as time-resolved fluorescence emission. Steady-state emission spectra indicate a small decrease in long wavelength (680 nm) emission from the apcC- phycobilisomes and a complementary increase in shorter wavelength (665 nm) emission, relative to wild-type phycobilisomes. Strain apcD- phycobilisomes appear to be functionally indistinguishable from those of the wild-type, in spite of the absence of the two alpha APB subunits which bear terminal acceptor bilins. The only spectroscopic difference was seen in the steady-state fluorescence emission, for which the emission of the mutant was about 15% higher than that of the wild-type and was slightly blue-shifted. A phenotype has yet to be found for the apcD- mutation.

The formation of bacterial flagella: III. Characterization of the subunits of the flagella of Bacillus subtilis and Spirillum serpens*

The flagellins of Bacillus subtilis strain 19 and Spirillum serpens have been characterized by physical, chemical and immunochemical methods. The purified flagellar structures of both organisms consist of a single type of protein subunit of molecular weight approximately 40,000.

A comparison of cryptophytan phycocyanins

The spectroscopically different phycocyanins present in the type strain of Hemiselmis virescens, Millport 64, and in a second strain of this cryptophytan species, Plymouth 157, have been purified and compared. They are similar in native molecular weight and in subunit structure, both containing α and β subunits with molecular weights of approximately 10 000 and 19 000 respectively. However, they do not have the same chromophore composition. Both subunits of the phycocyanin of Plymouth 157 contain two bilins, with absorption maxima at 600 and 660 nm, respectively. Only the β subunit of Millport 64 carries these chromophores; its α subunit bears another chromophore, with absorption maxima at 368 and 694 nm. The spectroscopic differences between the two native phycocyanins can be entirely accounted for by their differing chromophore compositions. The phycocyanin of Millport 64 is the only biliprotein so far described which contains three chemically different chromophores.

Blue-green algal proteins: assembly forms of C-phycocyanin from Synechococcus sp.

Aggregation states of C-phycocyanin isolated from Synechococcus sp. strain 6301 (Anacystis nidulans) were examined both by spectrophotometric and electron microscopic methods. The electron microscope observations support previous results which showed that the separated α and β chains of the protomer can be recombined in vitro to form a reversible aggregate (hexamer) composed of 6 copies of each chain. This aggregate has the same disc shape as native phycocyanin, and the assembly process can be followed in solution by distinctive changes in circular dichroism properties. A new observation was the occurrence of higher assembly forms (short rods and ordered bundles) probably related to the structure of the intact phycobilisome. However, conditions were not found which allow assembly of these structural forms in vitro from highly purified phycocyanin alone.

Homology of amino-terminal regions of C-phycocyanins from a prokaryote and a eukaryote.

Summary Comparison of the amino-terminal sequences of the α and β subunits of C-phycocyanin of a prokaryotic blue-green alga of Synechococcus sp. ( Anacystis nidulans ), determined in this laboratory, with the corresponding sequences for the subunits of C-phycocyanin of the eukaryotic red alga Cyanidium caldarium , recently presented by Troxler et al . (1974), Federation Proc. 33 , 1258 Abs., shows extensive homology between these proteins. This confirms earlier conclusions on the evolutionary relationship of Cyanophyta and Rhodophyta based on immunological studies (Glazer et al . (1971) Proc. US Nat Acad. Sci. 68 , 3005–3008).