Dramatic reduction in fluorescence quantum yield in mutants of Green Fluorescent Protein due to fast internal conversion

Kummer, Andreas D.; Kompa, Christian; Lossau, H.; Pöllinger-Dammer, Florian; Michel‐Beyerle, M. E.; Silva, Christopher M.; Bylina, Edward J.; Coleman, William J.; Yang, Mary M.; Youvan, Douglas C.

doi:10.1016/s0301-0104(98)00245-6

Cited by 91 publications

(121 citation statements)

References 28 publications

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“…Proton transfer becomes less efficient at acidic pH due to structural rearrangements that result in the loss of an adequate proton-transfer relay. The rate of ESPT can no longer effectively compete with other processes such as internal conversion back to the ground state (30), and emission is primarily from the neutral chromophore. At all pHs, A* f I* is slowed compared to wild-type GFP and results in the visible 460 nm steady-state fluorescence that makes deGFP4 a useful ratiometric pH sensor.…”

Section: Discussionmentioning

confidence: 99%

Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 2. Excited-State Dynamics

et al. 2002

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], novel mutants of the green fluorescent protein (GFP) that exhibit dual steady-state emission properties were characterized structurally and discussed as potential intracellular pH probes. In this work, the excited-state dynamics of one of these new dual emission GFP variants, deGFP4 (C48S/ S65T/H148C/T203C), is studied by ultrafast fluorescence upconversion spectroscopy. Following excitation of the high-energy absorption band centered at 398 nm and assigned to the neutral form of the chromophore, time-resolved emission was monitored from the excited state of both the neutral and intermediate anionic chromophores at both high and low pH and upon deuteration of exchangeable protons. The time-resolved emission dynamics and isotope effect appear to be very different from those of wild-type GFP [Chattoraj, M., King, B. A., Bublitz, G. U., and Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 8362-8367]; however, due to overlapping emission bands, the apparent difference can be analyzed quantitatively within the same framework used to describe GFP excited-state dynamics. The results indicate that the pH-sensitive steady-state emission characteristics of deGFP4 are a result of a pH-dependent modulation of the rate of excited-state proton transfer. At high pH, a rapid interconversion from the excited state of the higher energy neutral chromophore to the lower energy intermediate anionic chromophore is achieved by proton transfer. At low pH, excited-state proton transfer is slowed to the point where it is no longer rate limiting.The green fluorescent protein (GFP) 1 from the jellyfish Aequorea Victoria has become a ubiquitous tool in cell and molecular biology (1). The chromophore of wild-type GFP is formed spontaneously from the internal cyclization and oxidation of the Ser65-Tyr66-Gly67 tripeptide unit (2, 3) and is protectively housed along a coaxial helix threaded through the center of an 11-stranded -barrel (4). The surrounding protein environment is responsible for many of the emission properties of GFP such as the emission wavelength, relative contributions from neutral and anionic forms of the chromophores, and constraints that are responsible for the high fluorescence quantum yield. There has been a significant effort to change the surrounding environment by both random and semirational mutagenesis to produce GFP variants with desirable properties such as new emission wavelengths and improved quantum yields (5, 6), more rapid chromophore maturation (7), greater structural stability (8, 9), and altered sensitivity to environmental properties such as pH (10-12), redox potential (13), and ion concentration (14-16). In the preceding companion paper, a new class of pH-sensitive dual emission GFP (deGFP) mutants is reported (17). These mutants exhibit emission that changes from green (λ max ) 514 nm) to blue (λ max ) 465 nm) as the pH is lowered (Figure 1) and should be useful ratiometric pH sensors in vitro and in vivo. Structural studies of a related dual emission GFP, deGFP1 (S65T/H148G/T203C), show that wh...

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Section: Discussionmentioning

confidence: 99%

Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 2. Excited-State Dynamics

et al. 2002

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“…Correspondingly, dramatic reductions in fluorescence also have been observed in solution-phase studies of related GFP mutants (18,19). The molecular origins of these phenomena are largely inexplicable on basis of the canonical three-state model.…”

Section: Introductionmentioning

confidence: 89%

“…The chromophore, phydroxybenzylideneimidazolidinone, forms autocatalytically on the nascent apoprotein backbone by cyclization and subsequent oxidation of the Ser65-Tyr66-Gly67 sequence (7). Because of its great practical and commercial interest as a cloneable reporter of gene expression (8), GFP has been studied intensively over the past few years (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)39). Crystallographic studies of wild-type (11,12) and mutant GFPs (13,14) have revealed the cylindrical shape protecting its buried chromophore from solvent.…”

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confidence: 99%

“…The molecular origins of these phenomena are largely inexplicable on basis of the canonical three-state model. Although small alterations of the protein environment apparently influence the quantum yield of fluorescence significantly, the wild type and all of these mutants contain chemically identical chromophores (17)(18)(19)(20)(21). This observation prompted us to search for an underlying mechanism largely intrinsic to the photophysics of the chromophore itself.…”

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Shedding light on the dark and weakly fluorescent states of green fluorescent proteins

Weber

Helms

McCammon

et al. 1999

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

341

423

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Recent experiments on various similar green f luorescent protein (GFP) mutants at the single-molecule level and in solution provide evidence of previously unknown short-and long-lived ''dark'' states and of related excited-state decay channels. Here, we present quantum chemical calculations on cis-trans photoisomerization paths of neutral, anionic, and zwitterionic GFP chromophores in their ground and first singlet excited states that explain the observed behaviors from a common perspective. The results suggest that favorable radiationless decay channels can exist for the different protonation states along these isomerizations, which apparently proceed via conical intersections. These channels are suggested to rationalize the observed dramatic reduction of f luorescence in solution. The observed single-molecule fast blinking is attributed to conversions between the f luorescent anionic and the dark zwitterionic forms whereas slow switching is attributed to conversions between the anionic and the neutral forms. The predicted nonadiabatic crossings are seen to rationalize the origins of a variety of experimental observations on a common basis and may have broad implications for photobiophysical mechanisms in GFP.Green fluorescent protein (GFP) (1) is a uniquely important member of the exciting class of photoactive proteins that includes, among others, bacteriorhodopsin (2) and the photoactive yellow protein (3-5). Wild-type GFP is a highly efficient fluorescent emitter that performs wavelength shifting in vivo by resonance energy transfer (6). The chromophore, phydroxybenzylideneimidazolidinone, forms autocatalytically on the nascent apoprotein backbone by cyclization and subsequent oxidation of the Ser65-Tyr66-Gly67 sequence (7). Because of its great practical and commercial interest as a cloneable reporter of gene expression (8), GFP has been studied intensively over the past few years (6-25, 39). Crystallographic studies of wild-type (11, 12) and mutant GFPs (13,14) have revealed the cylindrical shape protecting its buried chromophore from solvent. The remarkable rigidity of the protein inferred from recent molecular dynamics simulations (39) further explains its high fluorescence efficiency. Indeed, the bare chromophore in solution is not significantly fluorescent at room temperature (10). Based on optical absorbance spectra and ultrafast time-resolved fluorescence excitation and emission measurements (15, 16), a three-state model has been suggested for wild-type GFP (15,16). In this, the chromophore exists in either a neutral (A) or an anionic form; the latter occurs in two different protein environments, in a kinetically accessible but thermodynamically unstable (I) form and in a thermodynamically stable (B) form.Recently, single-molecule spectroscopic measurements of fluorescence in GFP triple mutants under constant illumination have revealed on-off blinking and switching (17), a behavior that is completely obscured when a large number of molecules is observed at once. Correspondingly, dramatic redu...

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“…In the native fold, the inhibition of molecular vibration or rotation along the chromophore double exo-metilene bond, prevents emission quenching by a rapid internal conversion process [13,15,16]. Inside the β-barrel, chromophore's fluorescence extinction by O 2 [17] and by hydron ions [18] is also avoid.…”

Section: Introductionmentioning

confidence: 99%

Thermal Effect on Aequorea Green Fluorescent Protein Anionic and Neutral Chromophore Forms Fluorescence

Santos

2011

J Fluoresc

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The emission behaviour of Aequorea green fluorescent protein (A-GFP) chromophore, in both neutral (N) and anionic (A) form, was studied in the temperature range from 20°C to 75°C and at pH=7. Excitation wavelengths of 399 nm and 476 nm were applied to probe the N and A forms environment, respectively. Both forms exhibit distinct fluorescence patterns at high temperature values. The emission quenching rate, following a temperature increase, is higher for the chromophore N form as a result of the hydrogen bond network weakening. The chromophore anionic form emission maximum is red shifted, upon temperature increase, due to a charge transfer process occurring after A form excitation.

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Dramatic reduction in fluorescence quantum yield in mutants of Green Fluorescent Protein due to fast internal conversion

Cited by 91 publications

References 28 publications

Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 2. Excited-State Dynamics

Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 2. Excited-State Dynamics

Shedding light on the dark and weakly fluorescent states of green fluorescent proteins

Thermal Effect on Aequorea Green Fluorescent Protein Anionic and Neutral Chromophore Forms Fluorescence

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