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Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 3. Short- and long-time dynamics of the excited-state proton transfer.
Biochemistry. 2007 Oct 30; 46(43):12026-36.B

Abstract

Steady-state emission, femtosecond pump-probe spectroscopy, and time-correlated single-photon counting (TCSPC) measurements were used to study the photophysics and the excited-state proton transfer (ESPT) reactions in the green fluorescent protein (GFP) variant S65T/H148D at three pH values: 6.0, 7.9, and 9.5. Selective mutation of GFP caused a dramatic change in the steady-state and excited-state behavior as compared to the wild-type GFP (wt-GFP) studied earlier. An excitation wavelength dependence of the quantum yield of the strong emission band at 510 nm (I* band) indicates the competition between adiabatic and non-adiabatic excited-state proton-transfer reactions. Pump-probe measurements show that the signal buildup probed at 510 nm is biphasic, where 0.8 of the signal amplitude is ultrashort, <150 fs, and 0.2 of the signal decreases with a approximately 10 ps time constant. This effect is a summary result of adiabatic ESPT to the carboxylate group of Asp148 and nonradiative processes. When compared with the luminescence of wt-GFP, the steady-state intensity at 450 nm is lower by a factor of about 10. This very weak emission at 450 nm has a nonexponential decay. It is relatively pH insensitive and, at about 25 ps, is almost twice as long as in wt-GFP. The former exhibits a surprisingly small kinetic deuterium isotope effect (KDIE), compared with the KDIE of about 5 for wt-GFP. Such weak proton dependence may indicate that this emission comes from the species not directly involved in the ESPT. In contrast, pH and H/D isotope dependence of the intense nonexponential luminescence decay of the S65T/H148D deprotonated form measured at 510 nm may result from an isomerization-induced deactivation that is accompanied by the proton recombination quenching. The data are complementary to the femtosecond time-resolved emission data obtained by ultrafast fluorescence up-conversion spectroscopy, found in the preceding paper (Shi et al.). The spectroscopic results are discussed on the basis of the detailed X-ray structure of the mutant published in the preceding paper (Shu et al.).

Authors+Show Affiliations

Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

17918961

Citation

Leiderman, Pavel, et al. "Ultrafast Excited-state Dynamics in the Green Fluorescent Protein Variant S65T/H148D. 3. Short- and Long-time Dynamics of the Excited-state Proton Transfer." Biochemistry, vol. 46, no. 43, 2007, pp. 12026-36.
Leiderman P, Genosar L, Huppert D, et al. Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 3. Short- and long-time dynamics of the excited-state proton transfer. Biochemistry. 2007;46(43):12026-36.
Leiderman, P., Genosar, L., Huppert, D., Shu, X., Remington, S. J., Solntsev, K. M., & Tolbert, L. M. (2007). Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 3. Short- and long-time dynamics of the excited-state proton transfer. Biochemistry, 46(43), 12026-36.
Leiderman P, et al. Ultrafast Excited-state Dynamics in the Green Fluorescent Protein Variant S65T/H148D. 3. Short- and Long-time Dynamics of the Excited-state Proton Transfer. Biochemistry. 2007 Oct 30;46(43):12026-36. PubMed PMID: 17918961.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Ultrafast excited-state dynamics in the green fluorescent protein variant S65T/H148D. 3. Short- and long-time dynamics of the excited-state proton transfer. AU - Leiderman,Pavel, AU - Genosar,Liat, AU - Huppert,Dan, AU - Shu,Xiaokun, AU - Remington,S James, AU - Solntsev,Kyril M, AU - Tolbert,Laren M, Y1 - 2007/10/06/ PY - 2007/10/9/pubmed PY - 2008/1/11/medline PY - 2007/10/9/entrez SP - 12026 EP - 36 JF - Biochemistry JO - Biochemistry VL - 46 IS - 43 N2 - Steady-state emission, femtosecond pump-probe spectroscopy, and time-correlated single-photon counting (TCSPC) measurements were used to study the photophysics and the excited-state proton transfer (ESPT) reactions in the green fluorescent protein (GFP) variant S65T/H148D at three pH values: 6.0, 7.9, and 9.5. Selective mutation of GFP caused a dramatic change in the steady-state and excited-state behavior as compared to the wild-type GFP (wt-GFP) studied earlier. An excitation wavelength dependence of the quantum yield of the strong emission band at 510 nm (I* band) indicates the competition between adiabatic and non-adiabatic excited-state proton-transfer reactions. Pump-probe measurements show that the signal buildup probed at 510 nm is biphasic, where 0.8 of the signal amplitude is ultrashort, <150 fs, and 0.2 of the signal decreases with a approximately 10 ps time constant. This effect is a summary result of adiabatic ESPT to the carboxylate group of Asp148 and nonradiative processes. When compared with the luminescence of wt-GFP, the steady-state intensity at 450 nm is lower by a factor of about 10. This very weak emission at 450 nm has a nonexponential decay. It is relatively pH insensitive and, at about 25 ps, is almost twice as long as in wt-GFP. The former exhibits a surprisingly small kinetic deuterium isotope effect (KDIE), compared with the KDIE of about 5 for wt-GFP. Such weak proton dependence may indicate that this emission comes from the species not directly involved in the ESPT. In contrast, pH and H/D isotope dependence of the intense nonexponential luminescence decay of the S65T/H148D deprotonated form measured at 510 nm may result from an isomerization-induced deactivation that is accompanied by the proton recombination quenching. The data are complementary to the femtosecond time-resolved emission data obtained by ultrafast fluorescence up-conversion spectroscopy, found in the preceding paper (Shi et al.). The spectroscopic results are discussed on the basis of the detailed X-ray structure of the mutant published in the preceding paper (Shu et al.). SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/17918961/Ultrafast_excited_state_dynamics_in_the_green_fluorescent_protein_variant_S65T/H148D__3__Short__and_long_time_dynamics_of_the_excited_state_proton_transfer_ L2 - https://dx.doi.org/10.1021/bi7009053 DB - PRIME DP - Unbound Medicine ER -