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

Abstract

Recent studies on the newly cloned red fluorescence protein DsRed from the Discosoma genus have shown its tremendous advantages: bright red fluorescence and high resistance against photobleaching. However, it has also become clear that the protein forms closely packed tetramers, and there is indication for incomplete protein maturation with unknown proportion of immature green species. We have applied single-molecule methodology to elucidate the nature of the fluorescence emission in the DsRed. Real-time fluorescence trajectories have been acquired with polarization sensitive detection. Our results indicate that energy transfer between identical monomers occurs efficiently with red emission arising equally likely from any of the chromophoric units. Photodissociation of one of the chromophores weakly quenches the emission of adjacent ones. Dual color excitation (at 488 and 568 nm) single-molecule microscopy has been performed to reveal the number and distribution of red vs. green species within each tetramer. We find that 86% of the DsRed contain at least one green species with a red-to-green ratio of 1.2-1.5. On the basis of our findings, oligomer suppression would not only be advantageous for protein fusion but will also increase the fluorescence emission of individual monomers.

Figure 1

Near-field fluorescence images of individual DsRed molecules embedded in a poly(acrylamide) gel. Both images correspond to the same area in the sample, 3.2 × 3.2 μm², 256 × 256 pixels, and λ_det > 590 nm. (a) Circularly polarized excitation at 568 nm, 0.7 kW/cm², and integration time of 5 ms/pixel. Circles highlight molecules that exhibit intermittent emission and photodissociation. (b) Circularly polarized excitation at 488 nm, 0.3 kW/cm², and 10 ms/pixel. Molecules labeled B are comparatively brighter in b than in a, whereas molecules labeled D are comparatively dimmer.

Figure 2

(a) Real-time fluorescence trajectory in both polarization channels of a DsRed molecule. Excitation intensity was 0.5 kW/cm². Acquisition time was 1 ms per point, although the signal has been binned to 100 ms per point to reduce noise. The total emitted signal s is obtained by adding the counts of both x and y detectors. The real-time degree of polarization P is shown in upper section. (b) Correlation plot between P and the total number of counts emitted by the molecule in a. The clusters of points corresponding to the emission of the four different levels are marked on the plot. (c) Histogram of the total number of counts of another DsRed molecule exhibiting four intensity levels. The peak values of the intensity (after fitting to gaussians) are: 1,329 (w = 192), 816 (w = 392), 484 (w = 107), and 153 (w = 210), for the fourth, third, second, and first levels, respectively, where w is the width of the Gaussian. (d) P vs. total number of counts for molecule in c.

Figure 3

Real-time fluorescence trajectories of two different DsRed proteins exhibiting blinking of a time scale of milliseconds to few seconds. Excitation intensity was 0.5 kW/cm² at 568 nm by using circularly polarized light. Acquisition time was 1 ms per point, with 100-ms binning time.

Figure 4

(a) Fluorescence intensity distribution for 122 DsRed molecules excited with circularly polarized light at 568 nm. To compare the intensity levels between different trajectories, each recorded signal has been normalized to the excitation intensity with background subtraction. The histogram has been constructed after analyzing, one by one, all of the trajectories and building independent distributions for each intensity level. In the case of molecules showing only one or two levels, the assignment to a particular distribution was done by looking at its normalized intensity level after background subtraction and including it in the best-fitting distribution. The peak intensities of the four count distributions are: 966 (w = 338), 633 (w = 267), 357 (w = 220), and 163 (w = 170) for fourth, third, second, and first levels, respectively. a Inset shows the percentage of molecules displaying four, three, two, and only one intensity level. (b) P distribution of all four different levels for the same number of molecules as in a. For comparison, b Inset shows the P distribution for 232 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine molecules embedded in a thin polymer film. The width of the distribution reflects the random orientation of individual transition dipoles, as expected for single molecules embedded in an amorphous matrix. The P distributions for the first and second levels display similar width, whereas for the third and fourth levels, a dip develops around P = 0.

Figure 5

Absorption spectra of DsRed taken before (gray dotted line) and after (black line) 1 h of continuous irradiation at 568 nm. The dashed line results from subtraction of both spectra showing photobleaching in the absorption peak at 568 nm and the generation of new species red-shifted with respect to the unbleached sample.

Figure 6

(a) Distribution of red and green species within the DsRed, as derived from single-molecule data. (b) The cartoon illustrates the mechanism of fluorescence in DsRed. For simplicity, only red species are considered. (Left) Energy transfer occurs between all chromophores, and emission results with equal probability in time from any of the four chromophores. (Right) A damaged chromophore absorbs efficiently but partially quenches the fluorescence of the remaining undamaged chromophores. η₁, η₂, and η₃ are the quenching fractions for the nearest, intermediate, and most distant pairs, respectively.