N5-methylasparagine and energy-transfer efficiency in C-phycocyanin

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.