Visualizing conformational dynamics of proteins in solution and at the cell membrane
Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2018-06-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/37248 |
| _version_ | 1811252958301519872 |
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| author | Sharona E Gordon Mika Munari William N Zagotta |
| author_facet | Sharona E Gordon Mika Munari William N Zagotta |
| author_sort | Sharona E Gordon |
| collection | DOAJ |
| description | Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins. |
| first_indexed | 2024-04-12T16:43:36Z |
| format | Article |
| id | doaj.art-bfcbb8ec25cc4864913c6b989584adb9 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T16:43:36Z |
| publishDate | 2018-06-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-bfcbb8ec25cc4864913c6b989584adb92022-12-22T03:24:41ZengeLife Sciences Publications LtdeLife2050-084X2018-06-01710.7554/eLife.37248Visualizing conformational dynamics of proteins in solution and at the cell membraneSharona E Gordon0https://orcid.org/0000-0002-0914-3361Mika Munari1https://orcid.org/0000-0003-3598-6116William N Zagotta2https://orcid.org/0000-0002-7631-8168Department 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 StatesConformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.https://elifesciences.org/articles/37248FRETMBPprotein dynamicsnoncanonical amino acidtransition metalmembrane protein |
| spellingShingle | Sharona E Gordon Mika Munari William N Zagotta Visualizing conformational dynamics of proteins in solution and at the cell membrane eLife FRET MBP protein dynamics noncanonical amino acid transition metal membrane protein |
| title | Visualizing conformational dynamics of proteins in solution and at the cell membrane |
| title_full | Visualizing conformational dynamics of proteins in solution and at the cell membrane |
| title_fullStr | Visualizing conformational dynamics of proteins in solution and at the cell membrane |
| title_full_unstemmed | Visualizing conformational dynamics of proteins in solution and at the cell membrane |
| title_short | Visualizing conformational dynamics of proteins in solution and at the cell membrane |
| title_sort | visualizing conformational dynamics of proteins in solution and at the cell membrane |
| topic | FRET MBP protein dynamics noncanonical amino acid transition metal membrane protein |
| url | https://elifesciences.org/articles/37248 |
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