Bilin deletions and subunit stability in cyanobacterial light‐harvesting proteins

Abstract

SummaryLight-har vesting in cyanobacteria and red algae is a function of the biliproteins, which have covalently bound bilin chromophores. The biliproteins are assembled with linker proteins into the phycobilisome, a large complex that resides on the surface of the photosynthetic membranes. Early steps in the phycobilisome assembly pathway include the folding of biliprotein ␣-and ␤-subunits, covalent modification of subunits by bilin attachment and formation of the primary assembly unit, the ␣␤ heterodimer. The potential role of bilins in subunit structure and assembly is examined in this study by site mutagenesis of biliprotein genes. Phycocyanin subunits from Synechocystis sp. 6701 that were unable to bind chromophores at specific sites were generated by changing the codons for bilin-binding cysteines to alanine residues. The altered genes were then expressed in a phycocyaninminus mutant of the transformable Synechocystis sp. strain 6803. Single and multiple chromophore deletions cause specific and reproducible variations in phycobilisome-associated phycocyanin that do not correlate with transcript levels. Sedimentation equilibrium studies with purified proteins showed that bilin absence reduces the strength of ␣␤ interaction in the heterodimer. These results suggest that phycocyanin instability in bilin-deletion mutants is a consequence of diversion of unassembled ␣-and ␤-subunits to a degradation pathway. Attachment of the central bilin, which is common to all biliprotein subunits, may facilitate ␣␤ interaction by completing the final stage of subunit folding and stabilizing the contact domains of binding partners in the heterodimer.

Identification and Characterization of a New Class of Bilin Lyase

“…It is unclear why we did not observe ␤*-PC paired with holo-␣-PC in the upper regions of the sucrose gradients. One explanation may be that the absence of a bilin at Cys-␤153 reduces the binding interaction between the ␣-and ␤-subunits, as has been shown with sedimentation equilibrium studies (62), and that dimers of ␤*-PC may be more stable and less susceptible to degradation than dimers of holo-␣-PC. Absence of the ␤153 chromophore does not appear to impair PC assembly as much as absence of the ␤82 chromophore (59,62,66,67), however.…”

“…Second, the gradients loaded with extracts from the cpcT mutant had an intense, blue-colored band near the top of the gradient that was not observed for gradients loaded with extracts from wild type. Because this fraction typically contains PBPs from dissociated PBS and becomes enriched when there is a defective or missing PBP or linker polypeptide (18,21,(57)(58)(59)(60)(61)(62), the enrichment of PBPs in this fraction in the cpcT mutant indicates that there is a defect in the assembly or stability of a PBP or PBS component.…”

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

“…Bilin strongly affects the conformation of the subunits, and contributes to the stability of the higher-order structure (Fischer and Scheer 1992;Toole et al 1998). Specific lyases are responsible for bilin attachment (Fairchild and Glazer 1994;Jung et al 1995;Scheer and Zhao 2008).…”

Lyase Activities of CpcS- and CpcT-like Proteins from Nostoc PCC7120 and Sequential Reconstitution of Binding Sites of Phycoerythrocyanin and Phycocyanin β-Subunits

“…Assembly of ␣ and ␤ subunits to holo-CPC is affected in mutants lacking either chromophore, which may relate to pleiotropic effects in lyase mutants (9,21,23,(55)(56)(57)(58). The results obtained here by using a combination of CpcS1 and CpcT1 indicate for the first time that a specific attachment sequence may be required for multichromophoric biliprotein subunits.…”