Molecular architecture of a light-harvesting antenna. Isolation and characterization of phycobilisome subassembly particles.
Synechococcus 6301 mutant, strain AN112, produces phycobilisomes containing two major biliproteins, phycocyanin and allophycocyanin, and two major linker polypeptides of 27 and 75 kilodaltons (27K and 75K). These phycobilisomes have a molecular weight of approximately 2.5 X 10(6) and are the smallest of these particles known to date. Sucrose density gradient centrifugation of AN112 phycobilisomes partially dissociated in 50 mM N-[tris(hydroxymethyl)methyl]glycine, 5 mM CaCl2, 10% (w/v) glycerol, pH 7.8, separated three distinct fractions: (1) free trimeric biliproteins, (2) hexameric complexes of phycocyanin with 27K (11 S particles), and (3) phycobilisome subassemblies equivalent in mass to approximately 25% of the intact phycobilisome (18 S particles). The 18 S particles contained equimolar amounts of phycocyanin and allophycocyanin, which represented approximately 30 and 50%, respectively, of the content of these biliproteins in the AN112 phycobilisome. The 18 S particles also contained 75% and 100%, respectively, of 27K and 75K polypeptides; i.e. 75K was present in a 2-fold higher amount than in the intact phycobilisome. The absorption spectrum (lambda max 648 nm) of the 18 S particles was similar to that of allophycocyanin. Upon excitation at 580 nm, these particles exhibited a fluorescence emission spectrum consisting of 680 and 660 nm components, identical with that of intact phycobilisomes. The circular dichroism spectra of AN112 phycobilisomes and of the 18 S particles, in the region between 650 and 700 nm, were also very similar. Allophycocyanin B, which fluoresces at 680 nm, was found in fraction 1, and was totally absent from the 18 S particle. Thus, the long wavelength emission of the 18 S particle must have arisen from another terminal energy acceptor. The most probable candidate is the 75K polypeptide, which has been shown to carry a bilin chromophore and emit near 680 nm (Lundell, D. J., Yamanaka, G., and Glazer, A. N. (1980) J. Cell Biol. 91, 315-319). The 27K polypeptide, present in both fractions 2 and 3, was a component of different complexes in the two fractions. Fraction 2 displayed the physical and spectroscopic properties characteristic of the phycocyanin-linker complex, (alpha beta)6.27K. However, in the 18 S particle, 27K functioned in the assembly and attachment of phycocyanin trimers to a core domain. Based on the analysis of the components in fractions 1-3, a model is proposed which describes the structure of the AN112 phycobilisome, with emphasis on the roles of the linker polypeptides in the assembly of the core.