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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

KIEs of iRFPs, RpBphP2 and RpBphP3 versus their fluorescence lifetimes.Dots represent current experimental results and from refs 26 and 28. The red curve is calculated according to Marcus BEBO expression model. The constant n before pKa* is a random positive scale factor. Number 1 to 11 represent life time from protein (1) WT PAS-GAF-PHY (short phase), (2) WT PAS-GAF (short phase), (3) WT PAS-GAF-PHY, (4) WT PAS-GAF, (5) WT PAS-GAF-PHY, (6) WT PAS-GAF-PHY/D202A, (7) WT PAS-GAF, (8) WT PAS-GAF-PHY/D216A, (9) iRFP720, (10) iRFP713 and (11) iRFP702.
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f4: KIEs of iRFPs, RpBphP2 and RpBphP3 versus their fluorescence lifetimes.Dots represent current experimental results and from refs 26 and 28. The red curve is calculated according to Marcus BEBO expression model. The constant n before pKa* is a random positive scale factor. Number 1 to 11 represent life time from protein (1) WT PAS-GAF-PHY (short phase), (2) WT PAS-GAF (short phase), (3) WT PAS-GAF-PHY, (4) WT PAS-GAF, (5) WT PAS-GAF-PHY, (6) WT PAS-GAF-PHY/D202A, (7) WT PAS-GAF, (8) WT PAS-GAF-PHY/D216A, (9) iRFP720, (10) iRFP713 and (11) iRFP702.

Mentions: Interestingly, there appears to be a correlation between fluorescence lifetime and KIE in natural BphPs and BphP-engineered iRFPs, as shown in Fig. 4 where current and published2628 data are summarized. Note that the lifetimes of wild type RpBphP2 appear twice because of their bi-exponential decays. We observe that for short lifetimes (<100 ps), the KIE is close to 1, while for increasing lifetimes the KIE goes up to 1.8. This observation indicates that the proposed ESPT process becomes more important when the fluorescence lifetime becomes longer.


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)

KIEs of iRFPs, RpBphP2 and RpBphP3 versus their fluorescence lifetimes.Dots represent current experimental results and from refs 26 and 28. The red curve is calculated according to Marcus BEBO expression model. The constant n before pKa* is a random positive scale factor. Number 1 to 11 represent life time from protein (1) WT PAS-GAF-PHY (short phase), (2) WT PAS-GAF (short phase), (3) WT PAS-GAF-PHY, (4) WT PAS-GAF, (5) WT PAS-GAF-PHY, (6) WT PAS-GAF-PHY/D202A, (7) WT PAS-GAF, (8) WT PAS-GAF-PHY/D216A, (9) iRFP720, (10) iRFP713 and (11) iRFP702.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: KIEs of iRFPs, RpBphP2 and RpBphP3 versus their fluorescence lifetimes.Dots represent current experimental results and from refs 26 and 28. The red curve is calculated according to Marcus BEBO expression model. The constant n before pKa* is a random positive scale factor. Number 1 to 11 represent life time from protein (1) WT PAS-GAF-PHY (short phase), (2) WT PAS-GAF (short phase), (3) WT PAS-GAF-PHY, (4) WT PAS-GAF, (5) WT PAS-GAF-PHY, (6) WT PAS-GAF-PHY/D202A, (7) WT PAS-GAF, (8) WT PAS-GAF-PHY/D216A, (9) iRFP720, (10) iRFP713 and (11) iRFP702.
Mentions: Interestingly, there appears to be a correlation between fluorescence lifetime and KIE in natural BphPs and BphP-engineered iRFPs, as shown in Fig. 4 where current and published2628 data are summarized. Note that the lifetimes of wild type RpBphP2 appear twice because of their bi-exponential decays. We observe that for short lifetimes (<100 ps), the KIE is close to 1, while for increasing lifetimes the KIE goes up to 1.8. This observation indicates that the proposed ESPT process becomes more important when the fluorescence lifetime becomes longer.

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