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Green fluorescent protein variants as ratiometric dual emission pH sensors. 3. Temperature dependence of proton transfer.
Biochemistry. 2005 Jun 21; 44(24):8701-11.B

Abstract

In parts 1 and 2 of this series [Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E. P., Yarbrough, D. K., Chu, S. Y., Xi, L. X., Boxer, S. G., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488; McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], we described the structure, excited-state dynamics, and applications of pH-sensitive, ratiometric dual emission green fluorescent protein (deGFP) variants with fluorescence emission that is modulated between blue (lambda(max) approximately equal 465 nm) and green (lambda(max) approximately equal 515 nm) depending on the pH of the bulk solvent. In this paper, we consider the energetic origin of the dual emission properties of these GFP variants by examining the temperature dependence of the steady-state absorption and fluorescence emission. In most cases, the quantum yield of the green emission decreased as the temperature was lowered, indicating that the excited-state proton transfer (ESPT) which produces the green emitting form is an activated process. The activation energies of ESPT, determined by modeling the quantum yields of both blue and green emissions between 260 and 298 K in the context of a simple photocycle, were found to be larger at low pH than at high pH. These results indicate that the ratiometric dual emission properties of deGFP mutants are due to this pH-sensitive ESPT rate, combined with a modulation of the ground-state neutral and anionic chromophore populations with pH. The time-resolved fluorescence of one of the deGFP mutants was studied in detail. The time-resolved emission spectra of this mutant are the first ultrafast spectra obtained for a GFP. These spectra demonstrate that the rising kinetics for green emission, considered a hallmark of ESPT, is the sum of the contribution from both the neutral and intermediate anionic forms of the chromophore at the probe wavelength and may not be observed in all mutants that undergo ESPT, depending on the relative contributions of the two forms.

Authors+Show Affiliations

Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15952777

Citation

McAnaney, Tim B., et al. "Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 3. Temperature Dependence of Proton Transfer." Biochemistry, vol. 44, no. 24, 2005, pp. 8701-11.
McAnaney TB, Shi X, Abbyad P, et al. Green fluorescent protein variants as ratiometric dual emission pH sensors. 3. Temperature dependence of proton transfer. Biochemistry. 2005;44(24):8701-11.
McAnaney, T. B., Shi, X., Abbyad, P., Jung, H., Remington, S. J., & Boxer, S. G. (2005). Green fluorescent protein variants as ratiometric dual emission pH sensors. 3. Temperature dependence of proton transfer. Biochemistry, 44(24), 8701-11.
McAnaney TB, et al. Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 3. Temperature Dependence of Proton Transfer. Biochemistry. 2005 Jun 21;44(24):8701-11. PubMed PMID: 15952777.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Green fluorescent protein variants as ratiometric dual emission pH sensors. 3. Temperature dependence of proton transfer. AU - McAnaney,Tim B, AU - Shi,Xinghua, AU - Abbyad,Paul, AU - Jung,Henry, AU - Remington,S James, AU - Boxer,Steven G, PY - 2005/6/15/pubmed PY - 2005/9/27/medline PY - 2005/6/15/entrez SP - 8701 EP - 11 JF - Biochemistry JO - Biochemistry VL - 44 IS - 24 N2 - In parts 1 and 2 of this series [Hanson, G. T., McAnaney, T. B., Park, E. S., Rendell, M. E. P., Yarbrough, D. K., Chu, S. Y., Xi, L. X., Boxer, S. G., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488; McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489-15494], we described the structure, excited-state dynamics, and applications of pH-sensitive, ratiometric dual emission green fluorescent protein (deGFP) variants with fluorescence emission that is modulated between blue (lambda(max) approximately equal 465 nm) and green (lambda(max) approximately equal 515 nm) depending on the pH of the bulk solvent. In this paper, we consider the energetic origin of the dual emission properties of these GFP variants by examining the temperature dependence of the steady-state absorption and fluorescence emission. In most cases, the quantum yield of the green emission decreased as the temperature was lowered, indicating that the excited-state proton transfer (ESPT) which produces the green emitting form is an activated process. The activation energies of ESPT, determined by modeling the quantum yields of both blue and green emissions between 260 and 298 K in the context of a simple photocycle, were found to be larger at low pH than at high pH. These results indicate that the ratiometric dual emission properties of deGFP mutants are due to this pH-sensitive ESPT rate, combined with a modulation of the ground-state neutral and anionic chromophore populations with pH. The time-resolved fluorescence of one of the deGFP mutants was studied in detail. The time-resolved emission spectra of this mutant are the first ultrafast spectra obtained for a GFP. These spectra demonstrate that the rising kinetics for green emission, considered a hallmark of ESPT, is the sum of the contribution from both the neutral and intermediate anionic forms of the chromophore at the probe wavelength and may not be observed in all mutants that undergo ESPT, depending on the relative contributions of the two forms. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/15952777/Green_fluorescent_protein_variants_as_ratiometric_dual_emission_pH_sensors__3__Temperature_dependence_of_proton_transfer_ L2 - https://dx.doi.org/10.1021/bi050132a DB - PRIME DP - Unbound Medicine ER -