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Uncovering the hidden ground state of green fluorescent protein.
Proc Natl Acad Sci U S A. 2004 Dec 28; 101(52):17988-93.PN

Abstract

The fluorescence properties of GFP are strongly influenced by the protonation states of its chromophore and nearby amino acid side chains. In the ground state, the GFP chromophore is neutral and absorbs in the near UV. Upon excitation, the chromophore is deprotonated, and the resulting anionic chromophore emits its green fluorescence. So far, only excited-state intermediates have been observed in the GFP photocycle. We have used ultrafast multipulse control spectroscopy to prepare and directly observe GFP's hidden anionic ground-state intermediates as an integral part of the photocycle. Combined with dispersed multichannel detection and advanced global analysis techniques, the existence of two distinct anionic ground-state intermediates, I(1) and I(2), has been unveiled. I(1) and I(2) absorb at 500 and 497 nm, respectively, and interconvert on a picosecond timescale. The I(2) intermediate has a lifetime of 400 ps, corresponding to a proton back-transfer process that regenerates the neutral ground state. Hydrogen/deuterium exchange of the protein leads to a significant increase of the I(1) and I(2) lifetimes, indicating that proton motion underlies their dynamics. We thus have assessed the complete chain of reaction intermediates and associated timescales that constitute the photocycle of GFP. Many elementary processes in biology rely on proton transfers that are limited by slow diffusional events, which seriously precludes their characterization. We have resolved the true reaction rate of a proton transfer in the molecular ground state of GFP, and our results may thus aid in the development of a generic understanding of proton transfer in biology.

Authors+Show Affiliations

Department of Biophysics, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1081, Amsterdam 1018HV, The Netherlands. j.kennis@few.vu.nlNo 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

Language

eng

PubMed ID

15608070

Citation

Kennis, John T M., et al. "Uncovering the Hidden Ground State of Green Fluorescent Protein." Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 52, 2004, pp. 17988-93.
Kennis JT, Larsen DS, van Stokkum IH, et al. Uncovering the hidden ground state of green fluorescent protein. Proc Natl Acad Sci USA. 2004;101(52):17988-93.
Kennis, J. T., Larsen, D. S., van Stokkum, I. H., Vengris, M., van Thor, J. J., & van Grondelle, R. (2004). Uncovering the hidden ground state of green fluorescent protein. Proceedings of the National Academy of Sciences of the United States of America, 101(52), 17988-93.
Kennis JT, et al. Uncovering the Hidden Ground State of Green Fluorescent Protein. Proc Natl Acad Sci USA. 2004 Dec 28;101(52):17988-93. PubMed PMID: 15608070.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Uncovering the hidden ground state of green fluorescent protein. AU - Kennis,John T M, AU - Larsen,Delmar S, AU - van Stokkum,Ivo H M, AU - Vengris,Mikas, AU - van Thor,Jasper J, AU - van Grondelle,Rienk, Y1 - 2004/12/17/ PY - 2004/12/21/pubmed PY - 2005/3/1/medline PY - 2004/12/21/entrez SP - 17988 EP - 93 JF - Proceedings of the National Academy of Sciences of the United States of America JO - Proc. Natl. Acad. Sci. U.S.A. VL - 101 IS - 52 N2 - The fluorescence properties of GFP are strongly influenced by the protonation states of its chromophore and nearby amino acid side chains. In the ground state, the GFP chromophore is neutral and absorbs in the near UV. Upon excitation, the chromophore is deprotonated, and the resulting anionic chromophore emits its green fluorescence. So far, only excited-state intermediates have been observed in the GFP photocycle. We have used ultrafast multipulse control spectroscopy to prepare and directly observe GFP's hidden anionic ground-state intermediates as an integral part of the photocycle. Combined with dispersed multichannel detection and advanced global analysis techniques, the existence of two distinct anionic ground-state intermediates, I(1) and I(2), has been unveiled. I(1) and I(2) absorb at 500 and 497 nm, respectively, and interconvert on a picosecond timescale. The I(2) intermediate has a lifetime of 400 ps, corresponding to a proton back-transfer process that regenerates the neutral ground state. Hydrogen/deuterium exchange of the protein leads to a significant increase of the I(1) and I(2) lifetimes, indicating that proton motion underlies their dynamics. We thus have assessed the complete chain of reaction intermediates and associated timescales that constitute the photocycle of GFP. Many elementary processes in biology rely on proton transfers that are limited by slow diffusional events, which seriously precludes their characterization. We have resolved the true reaction rate of a proton transfer in the molecular ground state of GFP, and our results may thus aid in the development of a generic understanding of proton transfer in biology. SN - 0027-8424 UR - https://www.unboundmedicine.com/medline/citation/15608070/Uncovering_the_hidden_ground_state_of_green_fluorescent_protein_ L2 - http://www.pnas.org/cgi/pmidlookup?view=long&pmid=15608070 DB - PRIME DP - Unbound Medicine ER -