Photophysics of DsRed, a Red Fluorescent Protein, from the Ensemble to the Single-Molecule Level

Abstract

DsRed, a tetrameric fluorescent protein cloned from the Discosoma genus of coral, has shown promise as a longer-wavelength substitute for green fluorescent protein (GFP) mutants for in vivo protein labeling. Bulk and single-molecule studies of the recombinant protein revealed that the DsRed chromophore shows high stability against photobleaching as compared to GFP mutants. Stark modulation spectra confirm that the electronic structure of the DsRed chromophore is similar to that of GFP. However, the tetrameric nature of DsRed leads to intersubunit energy transfer, as evidenced by the molecule's unusually low fluorescence anisotropy when immobilized (0.23 ( 0.02). This value is approximately consistent with an estimate of the energy transfer rates based on preliminary crystallographic information. The fluorescence emission bleaches at a rate linear in the applied excitation intensity, implying that the cessation of emission during pumping at 532 nm is light-driven and, consistent with the tetrameric structure, several photobleaching "steps" were observed for individual complexes. Because more photons are emitted before bleaching, this study suggests that DsRed may be superior to some GFP-based labeling technologies as long as tetramerization is not an issue in physiological studies. IntroductionSingle-molecule spectroscopy (SMS) continues to play a unique role in the elucidation of the dynamics of an increasingly broad range of complex systems. 14,25 By removing the averaging inherent in ensemble measurements, SMS yields a measure of the distribution of molecular properties which is of importance in systems that display static or time-dependent heterogeneity. Concurrently, developments in fluorescence microscopy have made the observation of single molecules of biological interest possible under a wide range of experimental conditions. 26 This versatility can be exploited to examine the molecular complexity of many biological systems.One critical challenge to the application of SMS techniques to biomolecules, especially in vivo, is the introduction of a fluorophore that acts as a reporter of activity, local environment, or spatial location. While certain important proteins display useful amounts of visible fluorescence arising from native cofactors, 12 most require the attachment of an extrinsic fluorophore to serve as the probe. The genetic fusion of naturally fluorescent proteins to a protein of interest ensures a consistent 1:1 stoichiometry in the cell, without the need for external chemical reactions. While the green fluorescent protein (GFP) and its mutants have proved suitable for many applications, 22,27 the relatively high quantum yield of photobleaching (∼10× that for a rhodamine dye 16 ) leaves room for improvement in applications that require a large number of emitted photons, such as in single-molecule studies. Also, no mutant of GFP has been demonstrated to be a strong emitter at long (>550 nm) wavelengths, where the interference from endogenous fluorophores is substantially less severe.Considering th...

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

“…Upon expression in E. coli, the wild-type form of EosFP appears as a tetramer in size-exclusion chromatography (Fig. 1), and its apparent molecular mass (M W ), of Ϸ66 kDa, is identical to that of DsRed, which is known to be tetrameric (18,19). Homology modeling of the interfaces of DsRed; its monomerized version, mRFP1; and EosFP guided our mutation strategy.…”

The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection

“…Of the molecules investigated, 50% exhibited such blinking dynamics. To explain the occurrence of one-step blinking in DsRed, one would have to postulate that the four chromophores simultaneously jump to an ''off'' state, or that the chromophores are coupled in some fashion, as suggested by Lounis et al (8), so that the resultant emission and on-off blinking proceed from one emitter in the complex. Intensity jumps to the background have also been observed in strongly coupled multichromophoric systems and are understood in terms of photogenerated traps, which quench the fluorescence of all of the other subunits (20)(21)(22)(23).…”

“…The assumption is based on the crystallographic data (11,12), the single-molecule experiments at concentrations as low as 10 Ϫ9 and 10 Ϫ10 M (8,18), which also show multistep emission, and the oligomer dissociation constant of DsRed of Ͻ10 Ϫ9 M (6), which is one order of magnitude lower than the protein concentration used in our near-field scanning optical microscope experiments. We point out that premature photobleaching of species before recording the fluorescence emission is negligible.…”

“…An extinction coefficient of 75,000 mol Ϫ1 ͞cm Ϫ1 and a fluorescence quantum yield of 0.7 at 558-nm excitation wavelength have been reported (7), much higher than initially published by Matz et al (1). The protein proves to be stable under harsh pH conditions and is extremely resistant to photobleaching (7,8). However, two major deficiencies have been discovered: the slow and possibly incomplete maturation for the red fluorescence, which apparently passes through an obligatory greenemitting intermediate similar to avGFP, and its tendency to oligomerize even at dilute concentrations (7,9,10).…”

Novel Fluorophores for Single-Molecule Imaging

“…Blinking is observed for ~15% of the molecules on the time scale of the measurement (100 ms), and this degree of blinking is less severe than for many other single-molecule emitters. The total number of detected photons (N tot ) was determined by integrating individual single-molecule time traces such as those shown in Figures 6B and 6C to construct histograms of the total number of detected photons per molecule 6,34 . The values in the table are the means (equivalent to the exponential decay parameter) of these (exponential) distributions from >100 single molecules each as shown in Figure 7.…”