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Site-Specific Tryptophan Labels Reveal Local Microsecond–Millisecond Motions of Dihydrofolate Reductase
Many enzymes are known to change conformations during their catalytic cycle, but the role of these protein motions is not well understood. Escherichia coli dihydrofolate reductase (DHFR) is a small, flexible enzyme that is often used as a model system for understanding enzyme dynamics. Recently, nat...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503464/ https://www.ncbi.nlm.nih.gov/pubmed/32842574 http://dx.doi.org/10.3390/molecules25173819 |
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author | Vaughn, Morgan B. Biren, Chloe Li, Qun Ragupathi, Ashwin Dyer, R. Brian |
author_facet | Vaughn, Morgan B. Biren, Chloe Li, Qun Ragupathi, Ashwin Dyer, R. Brian |
author_sort | Vaughn, Morgan B. |
collection | PubMed |
description | Many enzymes are known to change conformations during their catalytic cycle, but the role of these protein motions is not well understood. Escherichia coli dihydrofolate reductase (DHFR) is a small, flexible enzyme that is often used as a model system for understanding enzyme dynamics. Recently, native tryptophan fluorescence was used as a probe to study micro- to millisecond dynamics of DHFR. Yet, because DHFR has five native tryptophans, the origin of the observed conformational changes could not be assigned to a specific region within the enzyme. Here, we use DHFR mutants, each with a single tryptophan as a probe for temperature jump fluorescence spectroscopy, to further inform our understanding of DHFR dynamics. The equilibrium tryptophan fluorescence of the mutants shows that each tryptophan is in a different environment and that wild-type DHFR fluorescence is not a simple summation of all the individual tryptophan fluorescence signatures due to tryptophan–tryptophan interactions. Additionally, each mutant exhibits a two-phase relaxation profile corresponding to ligand association/dissociation convolved with associated conformational changes and a slow conformational change that is independent of ligand association and dissociation, similar to the wild-type enzyme. However, the relaxation rate of the slow phase depends on the location of the tryptophan within the enzyme, supporting the conclusion that the individual tryptophan fluorescence dynamics do not originate from a single collective motion, but instead report on local motions throughout the enzyme. |
format | Online Article Text |
id | pubmed-7503464 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-75034642020-09-23 Site-Specific Tryptophan Labels Reveal Local Microsecond–Millisecond Motions of Dihydrofolate Reductase Vaughn, Morgan B. Biren, Chloe Li, Qun Ragupathi, Ashwin Dyer, R. Brian Molecules Article Many enzymes are known to change conformations during their catalytic cycle, but the role of these protein motions is not well understood. Escherichia coli dihydrofolate reductase (DHFR) is a small, flexible enzyme that is often used as a model system for understanding enzyme dynamics. Recently, native tryptophan fluorescence was used as a probe to study micro- to millisecond dynamics of DHFR. Yet, because DHFR has five native tryptophans, the origin of the observed conformational changes could not be assigned to a specific region within the enzyme. Here, we use DHFR mutants, each with a single tryptophan as a probe for temperature jump fluorescence spectroscopy, to further inform our understanding of DHFR dynamics. The equilibrium tryptophan fluorescence of the mutants shows that each tryptophan is in a different environment and that wild-type DHFR fluorescence is not a simple summation of all the individual tryptophan fluorescence signatures due to tryptophan–tryptophan interactions. Additionally, each mutant exhibits a two-phase relaxation profile corresponding to ligand association/dissociation convolved with associated conformational changes and a slow conformational change that is independent of ligand association and dissociation, similar to the wild-type enzyme. However, the relaxation rate of the slow phase depends on the location of the tryptophan within the enzyme, supporting the conclusion that the individual tryptophan fluorescence dynamics do not originate from a single collective motion, but instead report on local motions throughout the enzyme. MDPI 2020-08-22 /pmc/articles/PMC7503464/ /pubmed/32842574 http://dx.doi.org/10.3390/molecules25173819 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Vaughn, Morgan B. Biren, Chloe Li, Qun Ragupathi, Ashwin Dyer, R. Brian Site-Specific Tryptophan Labels Reveal Local Microsecond–Millisecond Motions of Dihydrofolate Reductase |
title | Site-Specific Tryptophan Labels Reveal Local Microsecond–Millisecond Motions of Dihydrofolate Reductase |
title_full | Site-Specific Tryptophan Labels Reveal Local Microsecond–Millisecond Motions of Dihydrofolate Reductase |
title_fullStr | Site-Specific Tryptophan Labels Reveal Local Microsecond–Millisecond Motions of Dihydrofolate Reductase |
title_full_unstemmed | Site-Specific Tryptophan Labels Reveal Local Microsecond–Millisecond Motions of Dihydrofolate Reductase |
title_short | Site-Specific Tryptophan Labels Reveal Local Microsecond–Millisecond Motions of Dihydrofolate Reductase |
title_sort | site-specific tryptophan labels reveal local microsecond–millisecond motions of dihydrofolate reductase |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503464/ https://www.ncbi.nlm.nih.gov/pubmed/32842574 http://dx.doi.org/10.3390/molecules25173819 |
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