title: N5-methylasparagine and energy-transfer efficiency in C-phycocyanin.
abstract: A posttranslationally methylated asparagine residue, N5-methylasparagine, is found at the beta-72 site in many phycobiliproteins. Two mutations (Asp and Gln) in the beta-72 position of Agmenellum quadruplicatum C-phycocyanin were investigated to clarify the role of the wild-type N5-methylasparagine near the beta-84 "fluorescing" bilin tetrapyrrole chromophore. Chemical analysis for amide modification revealed that the beta-72Q protein was partially methylated with a stoichiometry of 0.27, suggesting that either the asparagine methyltransferase is nonspecific or a glutamine methyltransferase exists. Urea denaturation studies could detect no difference in protein stability for any of the C-phycocyanin species. Steady-state spectroscopic measurements demonstrate that Asp and Gln substitution for the C-phycocyanin beta-72 NMA affects both the ground to excited state transition and the excited-state characteristics of the beta-84 chromophore, while the rate of radiative energy transfer is unaffected. Energy-transfer efficiency within phycobilisomes (represented by steady-state fluorescence quantum yields) was also negatively impacted by the beta-72 substitutions. Time-resolved fluorescence emission spectroscopic studies with C-phycocyanin reveal three distinguishable fluorescence lifetimes. The longest fluorescence lifetime is diminished 7-10% by the Asp and Gln mutations in comparison to a control sample where beta-72 is NMA. Molecular dynamics calculations implicate a change in the bilin tetrapyrrole chromophore ring geometry as a likely source of the altered photophysics induced by the mutations. We conclude that N5-methylasparagine plays a special role in establishing the environment surrounding the beta-84 chromophore which minimizes the rates of nonradiative energy losses that would otherwise defeat the high quantum yield for energy transfer within the phycobilisomes.
authors.affiliation: Department of Biochemistry, Louisiana State University, Baton Rouge 70803.
authors.forename: B A
authors.initials: BA
authors.lastname: Thomas
authors.forename: L P
authors.initials: LP
authors.lastname: McMahon
authors.forename: A V
authors.initials: AV
authors.lastname: Klotz
mesh_terms: D001216:Asparagine / Q000031:analogs & derivatives* / Q000737:chemistry; D002942:Circular Dichroism; D000458:Cyanobacteria / Q000737:chemistry*; D004735:Energy Transfer; D016297:Mutagenesis, Site-Directed; D045524:Phycobilisomes; D010798:Phycocyanin / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D045524:Phycobilisomes; C054431:gamma-N-methylasparagine; D010798:Phycocyanin; D001216:Asparagine