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Ultrafast excited-state dynamics and fluorescence deactivation of near-infrared fluorescent proteins engineered from bacteriophytochromes.

Zhu J, Shcherbakova DM, Hontani Y, Verkhusha VV, Kennis JT - Sci Rep (2015)

Bottom Line: Their functions depend on the corresponding fluorescence efficiencies and electronic excited state properties.Significant kinetic isotope effects (KIE) were observed with a factor of ~1.8 in D2O, and are interpreted in terms of an excited-state proton transfer (ESPT) process that deactivates the excited state in competition with fluorescence and chromophore mobility.On this basis, new approaches for rational molecular engineering may be applied to iRFPs to improve their fluorescence.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Section, Department of Physics and Astronomy, Faculty of Sciences, VU University, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.

ABSTRACT
Near-infrared fluorescent proteins, iRFPs, are recently developed genetically encoded fluorescent probes for deep-tissue in vivo imaging. Their functions depend on the corresponding fluorescence efficiencies and electronic excited state properties. Here we report the electronic excited state deactivation dynamics of the most red-shifted iRFPs: iRFP702, iRFP713 and iRFP720. Complementary measurements by ultrafast broadband fluorescence and absorption spectroscopy show that single exponential decays of the excited state with 600~700 ps dominate in all three iRFPs, while photoinduced isomerization was completely inhibited. Significant kinetic isotope effects (KIE) were observed with a factor of ~1.8 in D2O, and are interpreted in terms of an excited-state proton transfer (ESPT) process that deactivates the excited state in competition with fluorescence and chromophore mobility. On this basis, new approaches for rational molecular engineering may be applied to iRFPs to improve their fluorescence.

No MeSH data available.


Related in: MedlinePlus

Transient absorption spectra and global analysis for iRFP702.(a) Time resolved spectra at different delay time; (b) EADS extracted by global fitting with a sequential decay kinetic model; (c) experimental data of time decay traces and global fitted ones. Data for iRFP713 and iRFP720 are in Figs S4 and S5, respectively.
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f3: Transient absorption spectra and global analysis for iRFP702.(a) Time resolved spectra at different delay time; (b) EADS extracted by global fitting with a sequential decay kinetic model; (c) experimental data of time decay traces and global fitted ones. Data for iRFP713 and iRFP720 are in Figs S4 and S5, respectively.

Mentions: Figure 3 depicts the result of ultrafast transient absorption spectroscopy on iRFP702. In Fig. 3a, time resolved spectra were shown at different delay time after excitation with 660 nm laser pulse. We observe a simultaneous decay of the spectra throughout the whole band. Global analysis indicated that two time constants are required to adequately describe the spectral evolution, with time constants of 19 ps and 692 ps. The global fitting extracted spectra, which termed as evolution-associated difference spectra (EADS) are shown in Fig. 3b, together with a sequential decay kinetic model. Figure 3c shows kinetics at selected wavelengths along with the result from the global fit. The first EADS (black line) shows ground state bleach from 600–680 nm, excited-state absorption at 730 nm and stimulated emission at 780 nm. It evolves in 19 ps to the 2-nd EADS (red line), which only shows a small amplitude decrease, except in the stimulated emission region around 780 nm where a slight signal increase is observed. The latter observation implies that the 19 ps component is not associated with excited-state decay. We therefore assign the 19 ps component to a structural relaxation process in the excited state, in line with ps timescale processes observed earlier on other phytochromes262728. The second EADS decays in 692 ps to baseline, which indicates that this component represents BV excited state decay. The time constant of 692 ps is similar to that observed in time-resolved fluorescence (749 ps, Fig. 2). Both EADS show an absorption band around 530 nm, which can be assigned to absorption from the (fluorescent) excited state to a higher excited state, as its decay lifetime is the same as the stimulated emission at 780 nm. No long-lived species are indicated in the global fit, in contrast to wild-type phytochromes where the 15Ea primary photoproduct Lumi-R is formed, indicating that the photocycle has been successfully abolished in the engineered iRFP proteins. The signal amplitude of any residual long-living species was too low for inclusion in the global analysis procedure; to further investigate this point, inspection of the raw ΔA spectra at 3 ns revealed a structure with an amplitude of about 2% of the initial signal, that might be interpreted as a photoproduct but is most likely a residual excited state (SI5-Fig. S6). Hence, we can put an upper limit of about 1% to photoproduct formation in iRFP702. H2O/D2O buffer exchange lead to longer excited-state lifetimes, in agreement with the streak camera results of Fig. 2.


Ultrafast excited-state dynamics and fluorescence deactivation of near-infrared fluorescent proteins engineered from bacteriophytochromes.

Zhu J, Shcherbakova DM, Hontani Y, Verkhusha VV, Kennis JT - Sci Rep (2015)

Transient absorption spectra and global analysis for iRFP702.(a) Time resolved spectra at different delay time; (b) EADS extracted by global fitting with a sequential decay kinetic model; (c) experimental data of time decay traces and global fitted ones. Data for iRFP713 and iRFP720 are in Figs S4 and S5, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526943&req=5

f3: Transient absorption spectra and global analysis for iRFP702.(a) Time resolved spectra at different delay time; (b) EADS extracted by global fitting with a sequential decay kinetic model; (c) experimental data of time decay traces and global fitted ones. Data for iRFP713 and iRFP720 are in Figs S4 and S5, respectively.
Mentions: Figure 3 depicts the result of ultrafast transient absorption spectroscopy on iRFP702. In Fig. 3a, time resolved spectra were shown at different delay time after excitation with 660 nm laser pulse. We observe a simultaneous decay of the spectra throughout the whole band. Global analysis indicated that two time constants are required to adequately describe the spectral evolution, with time constants of 19 ps and 692 ps. The global fitting extracted spectra, which termed as evolution-associated difference spectra (EADS) are shown in Fig. 3b, together with a sequential decay kinetic model. Figure 3c shows kinetics at selected wavelengths along with the result from the global fit. The first EADS (black line) shows ground state bleach from 600–680 nm, excited-state absorption at 730 nm and stimulated emission at 780 nm. It evolves in 19 ps to the 2-nd EADS (red line), which only shows a small amplitude decrease, except in the stimulated emission region around 780 nm where a slight signal increase is observed. The latter observation implies that the 19 ps component is not associated with excited-state decay. We therefore assign the 19 ps component to a structural relaxation process in the excited state, in line with ps timescale processes observed earlier on other phytochromes262728. The second EADS decays in 692 ps to baseline, which indicates that this component represents BV excited state decay. The time constant of 692 ps is similar to that observed in time-resolved fluorescence (749 ps, Fig. 2). Both EADS show an absorption band around 530 nm, which can be assigned to absorption from the (fluorescent) excited state to a higher excited state, as its decay lifetime is the same as the stimulated emission at 780 nm. No long-lived species are indicated in the global fit, in contrast to wild-type phytochromes where the 15Ea primary photoproduct Lumi-R is formed, indicating that the photocycle has been successfully abolished in the engineered iRFP proteins. The signal amplitude of any residual long-living species was too low for inclusion in the global analysis procedure; to further investigate this point, inspection of the raw ΔA spectra at 3 ns revealed a structure with an amplitude of about 2% of the initial signal, that might be interpreted as a photoproduct but is most likely a residual excited state (SI5-Fig. S6). Hence, we can put an upper limit of about 1% to photoproduct formation in iRFP702. H2O/D2O buffer exchange lead to longer excited-state lifetimes, in agreement with the streak camera results of Fig. 2.

Bottom Line: Their functions depend on the corresponding fluorescence efficiencies and electronic excited state properties.Significant kinetic isotope effects (KIE) were observed with a factor of ~1.8 in D2O, and are interpreted in terms of an excited-state proton transfer (ESPT) process that deactivates the excited state in competition with fluorescence and chromophore mobility.On this basis, new approaches for rational molecular engineering may be applied to iRFPs to improve their fluorescence.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Section, Department of Physics and Astronomy, Faculty of Sciences, VU University, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands.

ABSTRACT
Near-infrared fluorescent proteins, iRFPs, are recently developed genetically encoded fluorescent probes for deep-tissue in vivo imaging. Their functions depend on the corresponding fluorescence efficiencies and electronic excited state properties. Here we report the electronic excited state deactivation dynamics of the most red-shifted iRFPs: iRFP702, iRFP713 and iRFP720. Complementary measurements by ultrafast broadband fluorescence and absorption spectroscopy show that single exponential decays of the excited state with 600~700 ps dominate in all three iRFPs, while photoinduced isomerization was completely inhibited. Significant kinetic isotope effects (KIE) were observed with a factor of ~1.8 in D2O, and are interpreted in terms of an excited-state proton transfer (ESPT) process that deactivates the excited state in competition with fluorescence and chromophore mobility. On this basis, new approaches for rational molecular engineering may be applied to iRFPs to improve their fluorescence.

No MeSH data available.


Related in: MedlinePlus