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Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation.

ABSTRACT: Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumination. An analysis of closely related red FPs reveals that DSC and irreversible photobleaching are not always connected by the same mechanistic pathway. DSC occurs out of the first-excited singlet state, and its magnitude depends predominantly on the kinetics for recovery out of the dark state. The experimental results can be replicated through kinetic simulations of a four-state model of the electronic states. The methodology presented here allows light-driven dynamics to be studied at the ensemble level over six orders of magnitude in time (microsecond to second timescales).

SUBMITTER: Dean KM

PROVIDER: S-EPMC3175071 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation.

Dean Kevin M KM Lubbeck Jennifer L JL Binder Jennifer K JK Schwall Linda R LR Jimenez Ralph R Palmer Amy E AE

Biophysical journal 20110801 4

Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness and overall photostability. However, the brightness of FPs is limited by dark-state conversion (DSC) and irreversible photobleaching, which occur on different timescales. Here, we present in vivo ensemble assays for measuring DSC and irreversible photobleaching under continuous and pulsed illumina ...[more]

PMID: 21843488

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Project description:Modulating fluorescent protein emission holds great potential for increasing readout sensitivity for applications in biological imaging and detection. Here, we identify and engineer optically modulated yellow fluorescent proteins (EYFP, originally 10C, but renamed EYFP later, and mVenus) to yield new emitters with distinct modulation profiles and unique, optically gated, delayed fluorescence. The parent YFPs are individually modulatable through secondary illumination, depopulating a long-lived dark state to dynamically increase fluorescence. A single point mutation introduced near the chromophore in each of these YFPs provides access to a second, even longer-lived modulatable dark state, while a different double mutant renders EYFP unmodulatable. The naturally occurring dark state in the parent YFPs yields strong fluorescence modulation upon long-wavelength-induced dark state depopulation, allowing selective detection at the frequency at which the long wavelength secondary laser is intensity modulated. Distinct from photoswitches, however, this near IR secondary coexcitation repumps the emissive S1 level from the long-lived triplet state, resulting in optically activated delayed fluorescence (OADF). This OADF results from secondary laser-induced, reverse intersystem crossing (RISC), producing additional nanosecond-lived, visible fluorescence that is delayed by many microseconds after the primary excitation has turned off. Mutation of the parent chromophore environment opens an additional modulation pathway that avoids the OADF-producing triplet state, resulting in a second, much longer-lived, modulatable dark state. These Optically Modulated and Optically Activated Delayed Fluorescent Proteins (OMFPs and OADFPs) are thus excellent for background- and reference-free, high sensitivity cellular imaging, but time-gated OADF offers a second modality for true background-free detection. Our combined structural and spectroscopic data not only gives additional mechanistic details for designing optically modulated fluorescent proteins but also provides the opportunity to distinguish similarly emitting OMFPs through OADF and through their unique modulation spectra.

Dataset Information

Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation.

Publications

Analysis of red-fluorescent proteins provides insight into dark-state conversion and photodegradation.

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