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...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2021-10-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/70236 |
| _version_ | 1811201487676637184 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-12T02:22:28Z |
| format | Article |
| id | doaj.art-93b61d38288c44e393a7774bb956124a |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T02:22:28Z |
| publishDate | 2021-10-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-93b61d38288c44e393a7774bb956124a2022-12-22T03:52:05ZengeLife Sciences Publications LtdeLife2050-084X2021-10-011010.7554/eLife.70236An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRETWilliam N Zagotta0https://orcid.org/0000-0002-7631-8168Brandon S Sim1Anthony K Nhim2Marium M Raza3Eric GB Evans4Yarra Venkatesh5https://orcid.org/0000-0002-4478-1553Chloe M Jones6Ryan A Mehl7https://orcid.org/0000-0003-2932-4941E James Petersson8https://orcid.org/0000-0003-3854-9210Sharona E Gordon9https://orcid.org/0000-0002-0914-3361Department of Physiology and Biophysics, University of Washington, Seattle, United StatesDepartment of Physiology and Biophysics, University of Washington, Seattle, United StatesDepartment of Physiology and Biophysics, University of Washington, Seattle, United StatesDepartment of Physiology and Biophysics, University of Washington, Seattle, United StatesDepartment of Physiology and Biophysics, University of Washington, Seattle, United StatesDepartment of Chemistry, University of Pennsylvania, Philadelphia, United StatesDepartment of Chemistry, University of Pennsylvania, Philadelphia, United States; Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania, Philadelphia, United StatesDepartment of Biochemistry and Biophysics, Oregon State University, Corvallis, United StatesDepartment of Chemistry, University of Pennsylvania, Philadelphia, United StatesDepartment of Physiology and Biophysics, University of Washington, Seattle, United StatesWith 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.https://elifesciences.org/articles/70236FluorescenceFRETprotein dynamicsnoncanonical amino acidmaltose binding proteinfluorescence lifetime |
| spellingShingle | 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 eLife Fluorescence FRET protein dynamics noncanonical amino acid maltose binding protein fluorescence lifetime |
| 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 | Fluorescence FRET protein dynamics noncanonical amino acid maltose binding protein fluorescence lifetime |
| url | https://elifesciences.org/articles/70236 |
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