title: Energy transfer and light tolerance studies in a fluorescent tandem phycobiliprotein conjugate.
abstract: Light harvesting and energy transfer between allophycocyanin (APC) and a tandem conjugate dye was investigated using single-molecule recrossings in the probe volume. By comparing saturation irradiance and recrossing events of APC and the tandem conjugate Streptavidin Allophycocyanin-AlexaFluor-680 (APC-680), it was demonstrated that the light tolerance and photostability of APC-680 were improved over APC. This improvement was due to conjugating a tandem dye to APC, which provided an alternative pathway for energy transfer. This additional pathway decreases photobleaching and triplet-state conversion. In this study, the average fluorescence intensity at different laser powers was calculated from extracted signal peaks. Saturation irradiance was determined by interpolating the average intensity as a function of excitation power. By comparing the saturation irradiance of APC and APC-680, it was demonstrated that light tolerance was not significantly increased by having a small fluorophore covalently linked to the protein. The molecular recrossing method was used to determine triplet-state conversion and photobleaching effects. The number of molecules that were detected within 2 ms of each other was defined as the number of recrossing events (N(r)). The total number of molecules detected was defined as N(t). The normalized number of recrossings events (N(r)/N(t)) showed that energy transfer between APC and AlexaFluor-680 could reduce triplet-state formation of phycobiliproteins. In this case, the saturation irradiance was not improved, but the resistance to photobleaching and triplet-state conversion was improved.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Tian
authors.forename: Dimitri
authors.initials: D
authors.lastname: Pappas
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057054:Molecular Imaging; D055668:Photochemical Processes; D010798:Phycocyanin / Q000737:chemistry*; D019809:Streptavidin / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; C002497:allophycocyanin; D010798:Phycocyanin; D019809:Streptavidin