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Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET
Published on: August 17, 2022
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Visualizing conformational dynamics of proteins in solution and at the cell membrane
Sharona E Gordon1, Mika Munari1, William N Zagotta1
1Department of Physiology and Biophysics, University of Washington, Seattle, United States.
Elife
|June 21, 2018
Summary
We developed ACCuRET, a novel method using fluorescent amino acids and copper to precisely measure protein movements. This technique accurately tracks conformational dynamics in soluble and membrane proteins within cells.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- Enzyme function relies on conformational dynamics, which are difficult to study with traditional methods.
- Fluorescence Resonance Energy Transfer (FRET) can measure these dynamics but faces accuracy limitations, especially for membrane proteins.
Purpose of the Study:
- To develop a new, accurate method for measuring protein conformational dynamics.
- To overcome technical barriers limiting FRET accuracy, particularly in membrane proteins.
Main Methods:
- Combined amber codon suppression for fluorescent noncanonical amino acid incorporation.
- Developed ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer) for labeling proteins with acceptor transition metals.
- Utilized cell unroofing for in-situ membrane protein analysis and computational distance correction.
Main Results:
- ACCuRET accurately measures absolute distances and distance changes with high precision.
- Demonstrated accurate measurement of protein rearrangements in native membranes using cell unroofing.
- Implemented computational correction for distance distributions, enhancing accuracy.
Conclusions:
- ACCuRET offers a flexible and powerful approach for studying conformational dynamics.
- The method is applicable to both soluble and membrane proteins.
- ACCuRET overcomes previous limitations in measuring protein dynamics, especially in cellular environments.
Keywords:
FRETMBPbiochemistrychemical biologymembrane proteinmolecular biophysicsnoncanonical amino acidnoneprotein dynamicsstructural biologytransition metalMore Related Videos
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