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...

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Autores principales:	William N Zagotta, Brandon S Sim, Anthony K Nhim, Marium M Raza, Eric GB Evans, Yarra Venkatesh, Chloe M Jones, Ryan A Mehl, E James Petersson, Sharona E Gordon
Formato:	article
Lenguaje:	EN
Publicado:	eLife Sciences Publications Ltd 2021
Materias:	Fluorescence FRET protein dynamics noncanonical amino acid maltose binding protein fluorescence lifetime Medicine R Science Q Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/93b61d38288c44e393a7774bb956124a
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spelling	oai:doaj.org-article:93b61d38288c44e393a7774bb956124a2021-11-30T11:42:54ZAn improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET10.7554/eLife.702362050-084Xe70236https://doaj.org/article/93b61d38288c44e393a7774bb956124a2021-10-01T00:00:00Zhttps://elifesciences.org/articles/70236https://doaj.org/toc/2050-084XWith 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.William N ZagottaBrandon S SimAnthony K NhimMarium M RazaEric GB EvansYarra VenkateshChloe M JonesRyan A MehlE James PeterssonSharona E GordoneLife Sciences Publications LtdarticleFluorescenceFRETprotein dynamicsnoncanonical amino acidmaltose binding proteinfluorescence lifetimeMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Fluorescence FRET protein dynamics noncanonical amino acid maltose binding protein fluorescence lifetime Medicine R Science Q Biology (General) QH301-705.5
spellingShingle	Fluorescence FRET protein dynamics noncanonical amino acid maltose binding protein fluorescence lifetime Medicine R Science Q Biology (General) QH301-705.5 William N Zagotta Brandon S Sim Anthony K Nhim Marium M Raza Eric GB Evans Yarra Venkatesh Chloe M Jones Ryan A Mehl E James Petersson Sharona E Gordon An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET
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	article
author	William N Zagotta Brandon S Sim Anthony K Nhim Marium M Raza Eric GB Evans Yarra Venkatesh Chloe M Jones Ryan A Mehl E James Petersson Sharona E Gordon
author_facet	William N Zagotta Brandon S Sim Anthony K Nhim Marium M Raza Eric GB Evans Yarra Venkatesh Chloe M Jones Ryan A Mehl E James Petersson Sharona E Gordon
author_sort	William N Zagotta
title	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_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_sort	improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion fret
publisher	eLife Sciences Publications Ltd
publishDate	2021
url	https://doaj.org/article/93b61d38288c44e393a7774bb956124a
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An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET

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