Article pubs.acs.org/JPCB
Excited State Dynamics of Photoswitchable Fluorescent Protein Padron Eduard Fron,* Mark Van der Auweraer, Johan Hofkens, and Peter Dedecker* Molecular Imaging and Photonics, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium S Supporting Information *
ABSTRACT: The key events in the light-induced switching mechanism of the photochromic green fluorescent protein Padron have been investigated by employing femtosecond fluorescence up-conversion, femtosecond transient absorption, and time-correlated single photon counting techniques. In contrast to Dronpa, excitation of protein’s neutral state at 395 nm triggers an efficient and complex photoswitching to a dark state whereas irradiation with 495 nm light reverses the protein to its initial state restoring the bright fluorescence. On the basis of the kinetics observed upon irradiation of the chromophore in the protonated state, we suggest that the switching mechanism consists of a lightinitiated excited state process (presumably ESPT) with a time constant of 1 ps producing an unstable intermediate state, tentatively assigned to the excited state of the cis-anionic form, that is followed by a cis- to trans- isomerization (14.5 ps) forming the trans-anionic state in which the dark chromophore resides. In the trans-state, the protonation equilibrium strongly favors the anionic form. Consequently, upon excitation of the formed anionic species a trans− cis isomerization of the chromophore was found to occur with a time constant as fast as 5.2 ps switching the chromophore quantitatively to the bright (anionic) state.
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INTRODUCTION Fluorophores with controllable fluorescence properties are rapidly becoming essential tools in fluorescence imaging, as they allow the circumvention of traditional barriers such as the diffraction limit.1 While a range of different fluorophores has emerged, however, a particularly notable class of “dynamic fluorophores” are those that allow the fluorescence emission to be switched on and off in an entirely light-induced fashion.2 One of the first and still most promising examples of such a fluorophore is the fluorescent protein Dronpa.3 Irradiation of the deprotonated form of Dronpa with blue light induces bright fluorescence emission but also causes the emission to disappear in time due to light-induced conversion of the individual molecules to a protonated form. However, the original fluorescence can be nearly completely recovered by irradiating the protonated form with UV light. The effective and reversible photoswitching of Dronpa has caused the development of several mutants with modified switching properties. Recently, however, a novel mutant called “Padron” was reported by Jakobs et al.4 Intriguingly, Padron displays opposite switching to Dronpa: irradiation with blue light causes the appearance of more emission (Φf = 0.64, bright form), while irradiation with UV light causes the disappearance of the fluorescence emission due to conversion to a dark form with Φf = 0.007. The mechanism behind this conversion has been studied in more detail using a related mutant and revealed that the underlying reason behind its opposite switching is due © 2013 American Chemical Society
to changes in the pKa of the trans and cis chromophores induced by the V167G and M159Y mutations.5 However, little is known about the fast light-induced dynamics responsible for this switching. Therefore, we decided to explore the photophysics involved in this process using ultrafast spectroscopy. Similar to Dronpa and other fluorescent proteins, the Padron chromophore can exists in neutral and anionic states, reflecting different protonation states of the phenolic oxygen of the chromophore.5 The data obtained from the crystal structure has established the cis conformation as the bright state of the chromophore.5 The absorption spectra of Padron in the fluorescent and nonfluorescent form and the emission spectrum are shown in Figure 1. To study the photoconversion mechanism in Padron, we investigated the ground- and excited-state dynamics using time correlated single photon counting (TCSPC), femtosecond fluorescence up-conversion, and transient absorption techniques, selectively exciting the neutral and anionic forms.
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EXPERIMENTAL PROCEDURES Stationary Measurements. The stationary measurements were recorded using a spectrophotometer (Lambda 40, PerkinElmer) and a fluorimeter (Fluorolog FL3-11, Perkin-Elmer) Received: September 27, 2013 Revised: November 26, 2013 Published: December 5, 2013 16422
dx.doi.org/10.1021/jp409654f | J. Phys. Chem. B 2013, 117, 16422−16427
The Journal of Physical Chemistry B
Article
Picosecond Fluorescence Time-Correlated Single Photon Counting (TCSPC) Experiments. A time-correlated single photon timing PC module (SPC 830, Becker & Hickl) was used to obtain the fluorescence decay histogram in 4096 channels. The decays were recorded with 10 000 counts in the peak channel in time windows of 15 ns corresponding to 3.7 ps/channel and analyzed globally with time-resolved fluorescence analysis (TRFA) software. The full width at halfmaximum (fwhm) of the IRF was typically in the order of 40 ps. The quality of the fits was judged by the fit parameters χ2 (
Excited State Dynamics of Photoswitchable Fluorescent Protein Padron Eduard Fron,* Mark Van der Auweraer, Johan Hofkens, and Peter Dedecker* Molecular Imaging and Photonics, Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium S Supporting Information *
ABSTRACT: The key events in the light-induced switching mechanism of the photochromic green fluorescent protein Padron have been investigated by employing femtosecond fluorescence up-conversion, femtosecond transient absorption, and time-correlated single photon counting techniques. In contrast to Dronpa, excitation of protein’s neutral state at 395 nm triggers an efficient and complex photoswitching to a dark state whereas irradiation with 495 nm light reverses the protein to its initial state restoring the bright fluorescence. On the basis of the kinetics observed upon irradiation of the chromophore in the protonated state, we suggest that the switching mechanism consists of a lightinitiated excited state process (presumably ESPT) with a time constant of 1 ps producing an unstable intermediate state, tentatively assigned to the excited state of the cis-anionic form, that is followed by a cis- to trans- isomerization (14.5 ps) forming the trans-anionic state in which the dark chromophore resides. In the trans-state, the protonation equilibrium strongly favors the anionic form. Consequently, upon excitation of the formed anionic species a trans− cis isomerization of the chromophore was found to occur with a time constant as fast as 5.2 ps switching the chromophore quantitatively to the bright (anionic) state.
■
INTRODUCTION Fluorophores with controllable fluorescence properties are rapidly becoming essential tools in fluorescence imaging, as they allow the circumvention of traditional barriers such as the diffraction limit.1 While a range of different fluorophores has emerged, however, a particularly notable class of “dynamic fluorophores” are those that allow the fluorescence emission to be switched on and off in an entirely light-induced fashion.2 One of the first and still most promising examples of such a fluorophore is the fluorescent protein Dronpa.3 Irradiation of the deprotonated form of Dronpa with blue light induces bright fluorescence emission but also causes the emission to disappear in time due to light-induced conversion of the individual molecules to a protonated form. However, the original fluorescence can be nearly completely recovered by irradiating the protonated form with UV light. The effective and reversible photoswitching of Dronpa has caused the development of several mutants with modified switching properties. Recently, however, a novel mutant called “Padron” was reported by Jakobs et al.4 Intriguingly, Padron displays opposite switching to Dronpa: irradiation with blue light causes the appearance of more emission (Φf = 0.64, bright form), while irradiation with UV light causes the disappearance of the fluorescence emission due to conversion to a dark form with Φf = 0.007. The mechanism behind this conversion has been studied in more detail using a related mutant and revealed that the underlying reason behind its opposite switching is due © 2013 American Chemical Society
to changes in the pKa of the trans and cis chromophores induced by the V167G and M159Y mutations.5 However, little is known about the fast light-induced dynamics responsible for this switching. Therefore, we decided to explore the photophysics involved in this process using ultrafast spectroscopy. Similar to Dronpa and other fluorescent proteins, the Padron chromophore can exists in neutral and anionic states, reflecting different protonation states of the phenolic oxygen of the chromophore.5 The data obtained from the crystal structure has established the cis conformation as the bright state of the chromophore.5 The absorption spectra of Padron in the fluorescent and nonfluorescent form and the emission spectrum are shown in Figure 1. To study the photoconversion mechanism in Padron, we investigated the ground- and excited-state dynamics using time correlated single photon counting (TCSPC), femtosecond fluorescence up-conversion, and transient absorption techniques, selectively exciting the neutral and anionic forms.
■
EXPERIMENTAL PROCEDURES Stationary Measurements. The stationary measurements were recorded using a spectrophotometer (Lambda 40, PerkinElmer) and a fluorimeter (Fluorolog FL3-11, Perkin-Elmer) Received: September 27, 2013 Revised: November 26, 2013 Published: December 5, 2013 16422
dx.doi.org/10.1021/jp409654f | J. Phys. Chem. B 2013, 117, 16422−16427
The Journal of Physical Chemistry B
Article
Picosecond Fluorescence Time-Correlated Single Photon Counting (TCSPC) Experiments. A time-correlated single photon timing PC module (SPC 830, Becker & Hickl) was used to obtain the fluorescence decay histogram in 4096 channels. The decays were recorded with 10 000 counts in the peak channel in time windows of 15 ns corresponding to 3.7 ps/channel and analyzed globally with time-resolved fluorescence analysis (TRFA) software. The full width at halfmaximum (fwhm) of the IRF was typically in the order of 40 ps. The quality of the fits was judged by the fit parameters χ2 (
