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An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET
With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynam...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8500717/ https://www.ncbi.nlm.nih.gov/pubmed/34623258 http://dx.doi.org/10.7554/eLife.70236 |
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author | Zagotta, William N Sim, Brandon S Nhim, Anthony K Raza, Marium M Evans, Eric GB Venkatesh, Yarra Jones, Chloe M Mehl, Ryan A Petersson, E James Gordon, Sharona E |
author_facet | Zagotta, William N Sim, Brandon S Nhim, Anthony K Raza, Marium M Evans, Eric GB Venkatesh, Yarra Jones, Chloe M Mehl, Ryan A Petersson, E James Gordon, Sharona E |
author_sort | Zagotta, William N |
collection | PubMed |
description | With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose-binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions. |
format | Online Article Text |
id | pubmed-8500717 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-85007172021-10-13 An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET Zagotta, William N Sim, Brandon S Nhim, Anthony K Raza, Marium M Evans, Eric GB Venkatesh, Yarra Jones, Chloe M Mehl, Ryan A Petersson, E James Gordon, Sharona E eLife Biochemistry and Chemical Biology With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose-binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions. eLife Sciences Publications, Ltd 2021-10-08 /pmc/articles/PMC8500717/ /pubmed/34623258 http://dx.doi.org/10.7554/eLife.70236 Text en © 2021, Zagotta et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Biochemistry and Chemical Biology Zagotta, William N Sim, Brandon S Nhim, Anthony K Raza, Marium M Evans, Eric GB Venkatesh, Yarra Jones, Chloe M Mehl, Ryan A Petersson, E James Gordon, Sharona E An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET |
title | An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET |
title_full | An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET |
title_fullStr | An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET |
title_full_unstemmed | An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET |
title_short | An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET |
title_sort | improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion fret |
topic | Biochemistry and Chemical Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8500717/ https://www.ncbi.nlm.nih.gov/pubmed/34623258 http://dx.doi.org/10.7554/eLife.70236 |
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