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.

EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion

“…(26). By contrast, eqFP611 was monomeric under the conditions of single-molecule experiments on immobilized preparations (9,17). Whereas only very few red spots are visible in Fig.…”

“…and a piezoelectric sample scanning stage (9,17). For EosFP, we used an excitation dichroic mirror Q495LP (AHF, Tübingen, Germany), green-red beam-splitting dichroic 560 DCXR (AHF), and two bandpass filters, HQ 535͞70 and HQ 610͞75 (AHF).…”

Targeted Green-Red Photoconversion of EosFP, a Fluorescent Marker Protein

“…Images were taken with a confocal laser scanning microscope of our own design, based on a Zeiss Axiovert 35 inverted microscope with two-channel fluorescence detection and a piezoelectric sample scanning stage (Tritor 102 Cap, Piezosysteme Jena, Jena, Germany) [11,12]. The 488-nm line of an Ar + ion laser (Innova Sabre, Coherent Inc., Santa Clara, CA) was fed into the microscope objective (UPLAPO 60×/1,2w, Olympus, Hamburg, Germany) via a single-mode quartz fiber (QSMJ 320, OZ Optics, Carp, Canada) and a Q495LP dichroic mirror (AHF, Tübingen, Germany).…”

“…Red FPs are highly attractive as marker proteins due to the lower cellular autofluorescence background in the red spectral region. Moreover, longer-wavelength excitation light is less cytotoxic, and the red emission can be properly separated from green emitting fluorophores in double labeling experiments [11,12]. The β-barrel fold of GFP is almost perfectly conserved in the novel FPs.…”

Global analysis of fluorescence fluctuation data

“…Comprehensive reviews (Hess et al 2002;Thompson et al 2002;Bacia and Schwille 2003) and a book (Rigler and Elson 2001) dedicated to this technique clearly demonstrate its versatility. FCS measurements of molecules in solution include the observation of translational diffusion , rotational diffusion (Ehrenberg and Rigler 1974;Pecora 1975, 1976), intersystem crossing (Widengren et al 1995), chemical reactions (LaClair 1997, conformational dynamics of DNA hairpins (Bonnet et al 1998), photobleaching (Eggeling et al 2001), flavins and flavoproteins ; Van den Berg et al 2001), viscosity and photodynamics of red fluorescent proteins (Schenk et al 2004). FCS has also proved to be a suitable technique to detect nanoparticles and flowing particles (Akcakir et al 2000;Kunst et al 2002) and to study membrane-mimetic systems (Bastiaens et al 1994;Hink and Visser 1998;Hink et al 1999;Korlach et al 1999;Schwille et al 1999a;Fradin et al 2003).…”