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Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time
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Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET.
William N Zagotta1, Eric G B Evans2, Pierce Eggan1
1Department of Physiology and Biophysics, University of Washington, Seattle, Washington.
Biophysical Journal
|February 2, 2024
Summary
This study introduces time-resolved transition metal Förster resonance energy transfer (tmFRET) to precisely measure protein conformational changes and dynamics, advancing our understanding of protein allostery.
Area of Science:
- Biophysics
- Structural Biology
- Biochemistry
Background:
- Proteins undergo conformational changes crucial for biological functions.
- Protein allostery involves ligand-induced rearrangements.
- Transition metal Förster resonance energy transfer (tmFRET) can probe these changes.
Purpose of the Study:
- To combine tmFRET with fluorescence lifetime measurements for detailed analysis of protein allostery.
- To investigate the conformational landscape and energetics of maltose-binding protein.
- To validate a novel approach for measuring long intramolecular distances in proteins.
Main Methods:
- Utilized time-resolved tmFRET with novel metal-bipyridyl FRET acceptors.
- Employed fluorescence lifetime measurements to capture protein dynamics.
- Applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy with Cu(phen)2+ as a spin label.
Main Results:
- Accurately determined distance distributions and conformational heterogeneity of proteins.
- Demonstrated the sensitivity of time-resolved tmFRET in detecting subtle conformational and energetic changes.
- Validated tmFRET measurements using EPR spectroscopy, confirming Cu(phen)2+ as a useful spin label.
Conclusions:
- Time-resolved tmFRET provides insights into protein dynamics and conformational heterogeneity.
- The developed tmFRET system is sensitive to subtle changes in protein conformations crucial for allostery.
- This versatile approach aids in deciphering protein conformational landscapes and regulatory mechanisms.
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