title: Photodynamics of red fluorescent proteins studied by fluorescence correlation spectroscopy.
abstract: Red fluorescent proteins are important tools in fluorescence-based life science research. Recently, we have introduced eqFP611, a red fluorescent protein with advantageous properties from the sea anemone Entacmaea quadricolor. Here, we have studied the submillisecond light-driven intramolecular dynamics between bright and dark states of eqFP611 and, for comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on protein solutions. A three-state model with one dark and two fluorescent states describes the power-dependence of the flickering dynamics of both proteins at different excitation wavelengths. It involves two light-driven conformational transitions. We have also studied the photodynamics of individual (monomeric) eqFP611 molecules immobilized on surfaces. The flickering rates and dark state fractions of eqFP611 bound to polyethylene glycol-covered glass surfaces were identical to those measured in solution, showing that the bound FPs behaved identically. A second, much slower flickering process was observed on the 10-ms timescale. Deposition of eqFP611 molecules on bare glass surfaces yielded bright fluorescence without any detectable flickering and a >10-fold decreased photobleaching yield. These observations underscore the intimate connection between protein motions and photophysical processes in fluorescent proteins.
authors.affiliation: Department of Biophysics, University of Ulm, Ulm, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schenk
authors.forename: Sergey
authors.initials: S
authors.lastname: Ivanchenko
authors.forename: Carlheinz
authors.initials: C
authors.lastname: Röcker
authors.forename: Jörg
authors.initials: J
authors.lastname: Wiedenmann
authors.forename: G Ulrich
authors.initials: GU
authors.lastname: Nienhaus
mesh_terms: D007700:Kinetics; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry* / Q000528:radiation effects*; D008956:Models, Chemical; D010777:Photochemistry / Q000379:methods*; D011487:Protein Conformation / Q000528:radiation effects; D013050:Spectrometry, Fluorescence / Q000379:methods*; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C413663:fluorescent protein 583