Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules

Abstract

Real-time single-molecule fluorescence detection using confocal and near-field scanning optical microscopy has been applied to elucidate the nature of the ''on-off'' blinking observed in the Ser-65 3 Thr (S65T) mutant of the green fluorescent protein (GFP). Fluorescence time traces as a function of the excitation intensity, with a time resolution of 100 s and observation times up to 65 s, reveal the existence of a nonemissive state responsible for the long dark intervals in the GFP. We find that excitation intensity has a dramatic effect on the blinking. Whereas the time during which the fluorescence is on becomes shorter as the intensity is increased, the off-times are independent of excitation intensity. Statistical analysis of the on-and off-times renders a characteristic off-time of 1.6 ؎ 0.2 s and allows us to calculate a transition yield of Ϸ0.5 ؋ 10 ؊5 from the emissive to the nonemissive state. The saturation excitation intensity at which on-and off-times are equal is Ϸ1.5 kW͞cm 2 . On the basis of the single-molecule data we calculate an absorption cross section of 6.5 ؋ 10 ؊17 cm 2 for the S65T mutant. These results have important implications for the use of the GFP to follow dynamic processes in time at the single-molecular level. Since the cloning and subsequent expression of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria (1, 2) the research interest for this protein has increased dramatically. The protein has been used successfully in a large and ever-growing number of applications, including gene expression and cell dynamics (3, 4). It is the only cloned protein that exhibits strong intrinsic fluorescence without the need of external chromophores. In the native protein [wild-type (wt)-GFP] the chromophore is formed in an autocatalytic, posttranslational cyclization and oxidation of the tripeptide unit at residues 65-67 (2, 5, 6). The wt-GFP absorbs blue light at Ϸ395 nm, with a weak peak at Ϸ475 nm, and emits green light at Ϸ508 nm (7). Substitution of one or more amino acids at or in close proximity to the chromophore results in mutants with different absorption and emission properties, and in some cases, improved emission and photostability (5,8,9). A widely used mutant is the S65T, in which Ser-65 is replaced by Thr (10). The mutant shows only an absorption peak at Ϸ475 nm, has larger absorption cross section than wt-GFP, and shows no photo-isomerization (8, 9).Because of the rapidly increasing number of applications, great attention has been focused on the photophysical properties of the wt-GFP and a number of its mutants. Investigation of the photophysical properties has been carried out in ensemble measurements, at room and low temperatures (7,(11)(12)(13)(14), and at the single-molecular level (15)(16)(17)(18)(19). When molecules are observed individually, the fluorescence emission of GFP shows intensity fluctuations, on-off blinking, and fluorescence switching, a behavior that is hidden in ensemble experiments. These intriguing phenomena also manifest in many other...

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

“…This technique is extremely powerful in revealing photodynamic properties in molecular systems by avoiding population averaging (15). Features hidden in the ensemble, such as rare fluctuations and on-off blinking, have been directly observed, including the case of GFP (16,17). Our single-molecule DsRed data show the multistep emission characteristic of closely packed multichromophoric systems, consistent with recent DsRed experiments (8, Abbreviations: GFP, green fluorescent protein; FRET, fluorescence resonance energy transfer.…”

“…Fig. 3 shows time traces of two different DsRed molecules with multiple intensity steps as well as ''on-off'' blinking on time scales comparable to those observed in the S65T mutant of the GFP (17). It is interesting to note the character of both polarization emission and blinking in the time traces.…”

“…Samples for single-molecule experiments were prepared by immobilizing the proteins [10 Ϫ8 M in water-filled pores of poly(acrylamide)], following a procedure similar to that described in ref. 17.…”

Nanotechnologies in proteomics

“…Attempts of investigators to descend to subfemtomolar concentration range at the cost of large amounts of biological material have thus far been unsuccessful [5]. Indeed, it is very difficult to secure high concentration sensitivity of low-abundance proteins in the presence of other proteins with much higher concentrations in the assayed sample, if the dynamic concentration range is about 10 15 .…”

“…These are scanning atomic force microscopes [14], optical microscopes [15], cryomassdetectors [16] and others.…”