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Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human γD-Crystallin Studied by Time-Resolved Fluorescence
[Image: see text] Human γD-crystallin (HγD-Crys) is a two-domain, β-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HγD-Crys has four highly conserved buried tryptophans (Trps), with two in each of...
Autores principales: | , , , |
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Formato: | Texto |
Lenguaje: | English |
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American Chemical Society
2008
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2758765/ https://www.ncbi.nlm.nih.gov/pubmed/18795792 http://dx.doi.org/10.1021/bi800499k |
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author | Chen, Jiejin Toptygin, Dmitri Brand, Ludwig King, Jonathan |
author_facet | Chen, Jiejin Toptygin, Dmitri Brand, Ludwig King, Jonathan |
author_sort | Chen, Jiejin |
collection | PubMed |
description | [Image: see text] Human γD-crystallin (HγD-Crys) is a two-domain, β-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HγD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous β-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HγD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552−11563]. Hybrid quantum mechanical−molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, τ ∼0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, τ ∼3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from ∼3 to ∼1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HγD-Crys from excited-state reactions causing permanent covalent damage. |
format | Text |
id | pubmed-2758765 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-27587652009-10-08 Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human γD-Crystallin Studied by Time-Resolved Fluorescence Chen, Jiejin Toptygin, Dmitri Brand, Ludwig King, Jonathan Biochemistry [Image: see text] Human γD-crystallin (HγD-Crys) is a two-domain, β-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HγD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous β-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HγD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552−11563]. Hybrid quantum mechanical−molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, τ ∼0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, τ ∼3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from ∼3 to ∼1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HγD-Crys from excited-state reactions causing permanent covalent damage. American Chemical Society 2008-09-17 2008-10-07 /pmc/articles/PMC2758765/ /pubmed/18795792 http://dx.doi.org/10.1021/bi800499k Text en Copyright © 2008 American Chemical Society http://pubs.acs.org This is an open-access article distributed under the ACS AuthorChoice Terms & Conditions. Any use of this article, must conform to the terms of that license which are available at http://pubs.acs.org. |
spellingShingle | Chen, Jiejin Toptygin, Dmitri Brand, Ludwig King, Jonathan Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human γD-Crystallin Studied by Time-Resolved Fluorescence |
title | Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human γD-Crystallin Studied by Time-Resolved Fluorescence |
title_full | Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human γD-Crystallin Studied by Time-Resolved Fluorescence |
title_fullStr | Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human γD-Crystallin Studied by Time-Resolved Fluorescence |
title_full_unstemmed | Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human γD-Crystallin Studied by Time-Resolved Fluorescence |
title_short | Mechanism of the Efficient Tryptophan Fluorescence Quenching in Human γD-Crystallin Studied by Time-Resolved Fluorescence |
title_sort | mechanism of the efficient tryptophan fluorescence quenching in human γd-crystallin studied by time-resolved fluorescence |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2758765/ https://www.ncbi.nlm.nih.gov/pubmed/18795792 http://dx.doi.org/10.1021/bi800499k |
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