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Updated: Jul 3, 2025
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High Precision FRET at Single-molecule Level for Biomolecule Structure Determination
Published on: May 13, 2017
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Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands
Sharona E Gordon1, Eric G B Evans2, Shauna C Otto1
1Department of Physiology and Biophysics, University of Washington, Seattle, Washington.
Biophysical Journal
|February 13, 2024
Summary
Researchers developed new transition metal Förster resonance energy transfer (tmFRET) probes to measure protein dynamics. These probes extend the measurable distance range, enabling detailed studies of protein conformational changes under physiological conditions.
Area of Science:
- Biophysics
- Structural Biology
- Biochemistry
Background:
- Protein structure determination has advanced significantly, but understanding protein function requires integrating dynamics and energetics.
- Fluorescence spectroscopy, particularly Förster resonance energy transfer (FRET), offers insights into molecular dynamics at the ångström scale and physiological temperatures.
Purpose of the Study:
- To develop novel transition metal FRET (tmFRET) probes with an expanded working distance for studying protein dynamics.
- To enhance the ability to measure small, physiologically relevant conformational changes in proteins.
Main Methods:
- Utilized cysteine-reactive bipyridyl and phenanthroline compounds as chelators for Fe²⁺ and Ru²⁺ to create new tmFRET acceptors.
- Paired novel acceptors with the noncanonical amino acid Acd as a donor to achieve a working distance of up to 50 Å.
- Compared measured FRET efficiencies with predictions from rotameric ensemble models.
Main Results:
- Successfully developed tmFRET probes with an extended working distance up to 50 Å.
- Demonstrated the ability to resolve small, maltose-dependent distance changes with high accuracy (∼1.5 Å).
- Validated the probes' effectiveness by comparing experimental FRET efficiencies to computational models.
Conclusions:
- The novel tmFRET probes significantly expand the utility of tmFRET for studying protein dynamics.
- Steady-state tmFRET measurements with these new probes provide unprecedented resolution for conformational rearrangements under physiological conditions.
- This advancement facilitates a deeper understanding of allosteric regulation and other dynamic processes in proteins.
