One- and Two-Photon Excited Fluorescence Lifetimes and Anisotropy Decays of Green Fluorescent Proteins

Volkmer, Andreas; Subramaniam, Vinod; Birch, David J. S.; Jovin, Thomas M.

doi:10.1016/s0006-3495(00)76711-7

Cited by 176 publications

(165 citation statements)

References 51 publications

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“…The anisotropy then approaches zero on the nanosecond time scale, presumably with the rotational lifetime of YFP. It is important to note that control experiments on S65T GFP at pH 6 produced anisotropy results in agreement with those reported previously (26) and that a solution of coumarin- 480, a freely rotating laser dye, exhibited a uniformly zero anisotropy following rotational depolarization indicating no significant instrumental bias of the parallel or perpendicular signals.…”

Section: Absorption and Fluorescence Spectrasupporting

confidence: 89%

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

et al. 2005

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Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP -anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale. Femtosecond spectroscopy revealed that the protonated excited-state (YFPH*) decayed predominantly by a radiationless mechanism, but emission at 460 nm was detected within the first picosecond. Limited excited-state proton transfer leads to 527 nm emission characteristic of the YFP -* anion. Prolonged continuous wave illumination at the peak of YFP -absorbance (514 nm) yields, irreversibly, a weakly fluorescent product that absorbs at 390 nm. This "photobleaching" process also gives a different species (YFPHrb) that absorbs at 350/430 nm and spontaneously regenerates YFP -in the dark on the time scale of hours but can be photoactivated by UV light to regenerate YFP -within seconds, via a ground-state protonated intermediate. Using a pulsed laser for photobleaching resulted in decarboxylation of YFP as indicated by the mass spectrum. These observations are accounted for in a unifying kinetic scheme.Green fluorescent protein (GFP) 1 and its color variants have found wide use in biology to probe protein dynamics in vitro and within cells (1). They have also attracted the attention of spectroscopists owing to their complex photochemistry (2-5). In particular, those in the YFP class (i.e. containing a T203Y or T203F mutation to shift the emission to 527 nm) have been shown to undergo fluctuations of emission intensity over a wide range of times scales and reversible photobleaching (6-8). Fluctuations in emission were observed by wide field microscopy at the single molecule level by immobilizing individual YFP molecules in acrylamide or agarose gels and using TIRF excitation (6, 9). Molecules were found to blink on the seconds time scale at low laser powers (<500 W cm -2 ). Increasing laser power reduced the lifetime of the emitting state, but not in direct proportion to the laser power. Initial studies discussed the possibility that the fluctuations were related to the protonation of the phenolate moiety of the fluorophore derived from Y66 (6), but later studies found little pH dependence of the onand off-times when monitored at a laser power of 5000 W cm -2 (9).Members of the YFP class also showed a photochromic switching behavior. Molecules that were bleached by 488 nm irradiation, could be induced to fluoresce by illumination with near UV or blue light (6-8). This reversible photobleaching reaction of YFP was observed in FRET measurements (10). Illumination of YFP at 514 nm resulted in bleaching of the YFP acceptor and dequenching of a donor CFP molecule. Once the YFP was bleached, illumina...

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Section: Absorption and Fluorescence Spectrasupporting

confidence: 89%

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

et al. 2005

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“…The rotational correlation time ( ) is the time constant of this slow anisotropy decay component and serves as a measure of molecular rotation (25). This value was similar to rotational correlation times measured for GFP (23,28). The two-photon limiting anisotropy of V␣(1-315) (the anisotropy at t ϭ 0) was found to be 0.48.…”

Section: Catalytic Domains Are Arranged As Pairs In the Intact Autoinsupporting

confidence: 66%

Structural rearrangement of CaMKIIα catalytic domains encodes activation

Thaler

Koushik²,

Puhl³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…In proteins there are several contributions to the loss of anisotropy [9]. In case of GFP the main sources of depolarization are an intrinsic one leading to the fundamental anisotropy (the anisotropy at time zero) and protein tumbling, since the fluorophore is rigidly embedded within the protein matrix [7,10,11]. The fundamental anisotropy contains information about the angle θ between absorption and emission transition moments:…”

Section: Time-resolved Fluorescence Anisotropymentioning

confidence: 99%

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

et al. 2005

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The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Timeresolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water-glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.

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One- and Two-Photon Excited Fluorescence Lifetimes and Anisotropy Decays of Green Fluorescent Proteins

Cited by 176 publications

References 51 publications

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

Structural rearrangement of CaMKIIα catalytic domains encodes activation

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

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