title: Watching conformational- and photo-dynamics of single fluorescent proteins in solution.
abstract: Observing the dynamics of single biomolecules over prolonged time periods is difficult to achieve without significantly altering the molecule through immobilization. It can, however, be accomplished using the Anti-Brownian ELectrokinetic (ABEL) Trap, which allows extended investigation of solution-phase biomolecules - without immobilization -through real-time electrokinetic feedback. Here we apply the ABEL trap to study an important photosynthetic antenna protein, Allophycocyanin (APC). The technique allows the observation of single molecules of solution-phase APC for more than one second. We observe a complex relationship between fluorescence intensity and lifetime that cannot be explained by simple static kinetic models. Light-induced conformational changes are shown to occur and evidence is obtained for fluctuations in the spontaneous emission lifetime, which is typically assumed to be constant. Our methods provide a new window into the dynamics of fluorescent proteins and the observations are relevant for the interpretation of in vivo single-molecule imaging experiments, bacterial photosynthetic regulation, and biomaterials for solar energy harvesting.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Randall H
authors.initials: RH
authors.lastname: Goldsmith
authors.forename: W E
authors.initials: WE
authors.lastname: Moerner
mesh_terms: D004566:Electrodes; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism*; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques; D008958:Models, Molecular; D009995:Osmosis; D038761:Photobleaching; D010798:Phycocyanin / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation / Q000528:radiation effects; D055503:Protein Multimerization; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D055767:Immobilized Proteins; D008164:Luminescent Proteins; D012996:Solutions; C002497:allophycocyanin; D010798:Phycocyanin