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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

Properties of iRPF702, iRFP713 and iRFP720.(a) Biliverdin attachment to Cys in PAS domain of BphPs and these iRFPs; (b) Steady state absorption and emission spectra. (c) Close-up of the biliverdin chromophore binding pocket of RpBphP2 (PDB code 4E04, ref. 19). Note that the model structure is only approximate.
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f1: Properties of iRPF702, iRFP713 and iRFP720.(a) Biliverdin attachment to Cys in PAS domain of BphPs and these iRFPs; (b) Steady state absorption and emission spectra. (c) Close-up of the biliverdin chromophore binding pocket of RpBphP2 (PDB code 4E04, ref. 19). Note that the model structure is only approximate.

Mentions: Recently, the new class of NIR FPs was developed from the subclass of phytochromes, called bacteriophytochromes (BphPs)91011121314 Phytochromes constitute a family of light sensors in plants, fungi and bacteria15 ; their light-sensing module comprises, so-called PAS, GAF and PHY domains. The PAS-GAF fragment, which has a molecular weight of 35 kDa16, is sufficient for chromophore binding. Weak fluorescence of mutated phytochromes were first reported by Fischer and Lagarias17 and Vierstra and co-workers18. Phytochromes do not possess an autocatalytically formed chromophore as in the GFP-like proteins, but instead bind a bilin tetrapyrrole to a conserved cysteine residue in the PAS domain. In particular, BphPs bind biliverdin (BV) tetrapyrrole compound (Fig. 1a), which being a heme degradation product is a ubiquitous naturally occurring cofactor in mammalian tissues. iRFPs engineered from BphPs have their absorption and emission between 670 and 720 nm (Fig. 1b), both within the NIR optical transparency window. Fig. 1c shows a close up of the BV binding pocket in the RpBphP2 X-ray structure19, the template protein from which iRFP713 and iRFP720 were derived, revealing the ZZZssa configuration of BV. Note that this structure provides only an approximate structural model for the iRFPs as various residues in the vicinity of BV were altered914.


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)

Properties of iRPF702, iRFP713 and iRFP720.(a) Biliverdin attachment to Cys in PAS domain of BphPs and these iRFPs; (b) Steady state absorption and emission spectra. (c) Close-up of the biliverdin chromophore binding pocket of RpBphP2 (PDB code 4E04, ref. 19). Note that the model structure is only approximate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Properties of iRPF702, iRFP713 and iRFP720.(a) Biliverdin attachment to Cys in PAS domain of BphPs and these iRFPs; (b) Steady state absorption and emission spectra. (c) Close-up of the biliverdin chromophore binding pocket of RpBphP2 (PDB code 4E04, ref. 19). Note that the model structure is only approximate.
Mentions: Recently, the new class of NIR FPs was developed from the subclass of phytochromes, called bacteriophytochromes (BphPs)91011121314 Phytochromes constitute a family of light sensors in plants, fungi and bacteria15 ; their light-sensing module comprises, so-called PAS, GAF and PHY domains. The PAS-GAF fragment, which has a molecular weight of 35 kDa16, is sufficient for chromophore binding. Weak fluorescence of mutated phytochromes were first reported by Fischer and Lagarias17 and Vierstra and co-workers18. Phytochromes do not possess an autocatalytically formed chromophore as in the GFP-like proteins, but instead bind a bilin tetrapyrrole to a conserved cysteine residue in the PAS domain. In particular, BphPs bind biliverdin (BV) tetrapyrrole compound (Fig. 1a), which being a heme degradation product is a ubiquitous naturally occurring cofactor in mammalian tissues. iRFPs engineered from BphPs have their absorption and emission between 670 and 720 nm (Fig. 1b), both within the NIR optical transparency window. Fig. 1c shows a close up of the BV binding pocket in the RpBphP2 X-ray structure19, the template protein from which iRFP713 and iRFP720 were derived, revealing the ZZZssa configuration of BV. Note that this structure provides only an approximate structural model for the iRFPs as various residues in the vicinity of BV were altered914.

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