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Ground-State Proton Transfer Kinetics in Green Fluorescent Protein

[Image: see text] Proton transfer plays an important role in the optical properties of green fluorescent protein (GFP). While much is known about excited-state proton transfer reactions (ESPT) in GFP occurring on ultrafast time scales, comparatively little is understood about the factors governing t...

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Autores principales: Oltrogge, Luke M., Wang, Quan, Boxer, Steven G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172208/
https://www.ncbi.nlm.nih.gov/pubmed/25184668
http://dx.doi.org/10.1021/bi500147n
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author Oltrogge, Luke M.
Wang, Quan
Boxer, Steven G.
author_facet Oltrogge, Luke M.
Wang, Quan
Boxer, Steven G.
author_sort Oltrogge, Luke M.
collection PubMed
description [Image: see text] Proton transfer plays an important role in the optical properties of green fluorescent protein (GFP). While much is known about excited-state proton transfer reactions (ESPT) in GFP occurring on ultrafast time scales, comparatively little is understood about the factors governing the rates and pathways of ground-state proton transfer. We have utilized a specific isotopic labeling strategy in combination with one-dimensional (13)C nuclear magnetic resonance (NMR) spectroscopy to install and monitor a (13)C directly adjacent to the GFP chromophore ionization site. The chemical shift of this probe is highly sensitive to the protonation state of the chromophore, and the resulting spectra reflect the thermodynamics and kinetics of the proton transfer in the NMR line shapes. This information is complemented by time-resolved NMR, fluorescence correlation spectroscopy, and steady-state absorbance and fluorescence measurements to provide a picture of chromophore ionization reactions spanning a wide time domain. Our findings indicate that proton transfer in GFP is described well by a two-site model in which the chromophore is energetically coupled to a secondary site, likely the terminal proton acceptor of ESPT, Glu222. Additionally, experiments on a selection of GFP circular permutants suggest an important role played by the structural dynamics of the seventh β-strand in gating proton transfer from bulk solution to the buried chromophore.
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spelling pubmed-41722082015-09-03 Ground-State Proton Transfer Kinetics in Green Fluorescent Protein Oltrogge, Luke M. Wang, Quan Boxer, Steven G. Biochemistry [Image: see text] Proton transfer plays an important role in the optical properties of green fluorescent protein (GFP). While much is known about excited-state proton transfer reactions (ESPT) in GFP occurring on ultrafast time scales, comparatively little is understood about the factors governing the rates and pathways of ground-state proton transfer. We have utilized a specific isotopic labeling strategy in combination with one-dimensional (13)C nuclear magnetic resonance (NMR) spectroscopy to install and monitor a (13)C directly adjacent to the GFP chromophore ionization site. The chemical shift of this probe is highly sensitive to the protonation state of the chromophore, and the resulting spectra reflect the thermodynamics and kinetics of the proton transfer in the NMR line shapes. This information is complemented by time-resolved NMR, fluorescence correlation spectroscopy, and steady-state absorbance and fluorescence measurements to provide a picture of chromophore ionization reactions spanning a wide time domain. Our findings indicate that proton transfer in GFP is described well by a two-site model in which the chromophore is energetically coupled to a secondary site, likely the terminal proton acceptor of ESPT, Glu222. Additionally, experiments on a selection of GFP circular permutants suggest an important role played by the structural dynamics of the seventh β-strand in gating proton transfer from bulk solution to the buried chromophore. American Chemical Society 2014-09-03 2014-09-23 /pmc/articles/PMC4172208/ /pubmed/25184668 http://dx.doi.org/10.1021/bi500147n Text en Copyright © 2014 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html)
spellingShingle Oltrogge, Luke M.
Wang, Quan
Boxer, Steven G.
Ground-State Proton Transfer Kinetics in Green Fluorescent Protein
title Ground-State Proton Transfer Kinetics in Green Fluorescent Protein
title_full Ground-State Proton Transfer Kinetics in Green Fluorescent Protein
title_fullStr Ground-State Proton Transfer Kinetics in Green Fluorescent Protein
title_full_unstemmed Ground-State Proton Transfer Kinetics in Green Fluorescent Protein
title_short Ground-State Proton Transfer Kinetics in Green Fluorescent Protein
title_sort ground-state proton transfer kinetics in green fluorescent protein
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172208/
https://www.ncbi.nlm.nih.gov/pubmed/25184668
http://dx.doi.org/10.1021/bi500147n
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