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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

Time resolved fluorescence of iRFP702.(a) Decay of fluorescence at 702 nm in H2O and D2O with global fitted curves. (b) The same decay curves as in Fig. 2a but in logarithmic scale intensity. (c) DAS extracted by global fitting with a single exponential function. Data for iRFP713 and iRFP720 are shown in Fig. S3 and Fig. S2, respectively.
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f2: Time resolved fluorescence of iRFP702.(a) Decay of fluorescence at 702 nm in H2O and D2O with global fitted curves. (b) The same decay curves as in Fig. 2a but in logarithmic scale intensity. (c) DAS extracted by global fitting with a single exponential function. Data for iRFP713 and iRFP720 are shown in Fig. S3 and Fig. S2, respectively.

Mentions: Figure 2 shows the results of a spectrally- and time-resolved fluorescence experiment on iRFP702 with excitation wavelength at 660 nm. Figure 2a (grey symbols) shows the kinetics at 702 nm in aqueous buffer. Global analysis indicates that the fluorescence decay can be described by a single exponential time constant of 749 ps (Fig. 2a, red line). Figure 2b are the same decay curves as in Fig. 2a but replotted on a logarithmic intensity scale. Figure 2c shows the decay-associated spectrum (DAS), which peaks at 702 nm and exhibits a vibronic shoulder near 760 nm. Experiments on iRFP713 and iRFP720 gave similar results, with single exponential decay time constants of 676 and 648 ps, respectively. Complete raw data and global analysis results are shown in Supplementary Information (SI3-Fig. S1). Time-resolved fluorescence experiments with iRFPs dissolved in D2O buffer showed a significant H/D effect on the fluorescence lifetime. In iRFP702, the fluorescence lifetime increased to 1.35 ns (Fig. 2a, open circles and blue line), corresponding to a kinetic isotope effect (KIE) of 1.8. Almost the same KIEs were obtained for iRFP713 and iRFP720 (SI3-Fig. S2 and Fig. S3). These observations are in line with those reported for wild type and point mutants of RpBphP2 and RpBphP326.


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)

Time resolved fluorescence of iRFP702.(a) Decay of fluorescence at 702 nm in H2O and D2O with global fitted curves. (b) The same decay curves as in Fig. 2a but in logarithmic scale intensity. (c) DAS extracted by global fitting with a single exponential function. Data for iRFP713 and iRFP720 are shown in Fig. S3 and Fig. S2, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Time resolved fluorescence of iRFP702.(a) Decay of fluorescence at 702 nm in H2O and D2O with global fitted curves. (b) The same decay curves as in Fig. 2a but in logarithmic scale intensity. (c) DAS extracted by global fitting with a single exponential function. Data for iRFP713 and iRFP720 are shown in Fig. S3 and Fig. S2, respectively.
Mentions: Figure 2 shows the results of a spectrally- and time-resolved fluorescence experiment on iRFP702 with excitation wavelength at 660 nm. Figure 2a (grey symbols) shows the kinetics at 702 nm in aqueous buffer. Global analysis indicates that the fluorescence decay can be described by a single exponential time constant of 749 ps (Fig. 2a, red line). Figure 2b are the same decay curves as in Fig. 2a but replotted on a logarithmic intensity scale. Figure 2c shows the decay-associated spectrum (DAS), which peaks at 702 nm and exhibits a vibronic shoulder near 760 nm. Experiments on iRFP713 and iRFP720 gave similar results, with single exponential decay time constants of 676 and 648 ps, respectively. Complete raw data and global analysis results are shown in Supplementary Information (SI3-Fig. S1). Time-resolved fluorescence experiments with iRFPs dissolved in D2O buffer showed a significant H/D effect on the fluorescence lifetime. In iRFP702, the fluorescence lifetime increased to 1.35 ns (Fig. 2a, open circles and blue line), corresponding to a kinetic isotope effect (KIE) of 1.8. Almost the same KIEs were obtained for iRFP713 and iRFP720 (SI3-Fig. S2 and Fig. S3). These observations are in line with those reported for wild type and point mutants of RpBphP2 and RpBphP326.

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