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High Precision FRET at Single-molecule Level for Biomolecule Structure Determination
Published on: May 13, 2017
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Distance measurements in the F0F1-ATP synthase from E. coli using smFRET and PELDOR spectroscopy
Markus Burger1, Stephan Rein1, Stefan Weber1
1Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstraße 21, 79104, Freiburg, Germany.
European Biophysics Journal : EBJ
|November 10, 2019
Summary
Single-molecule FRET (smFRET) and pulsed electron-electron double resonance (PELDOR) accurately measure distances in biomolecules. Both techniques, when applied to H+ -ATPase, yield comparable distance distributions, validating their use in structural biology.
Area of Science:
- Biophysics
- Structural Biology
- Biochemistry
Background:
- Fluorescence resonance energy transfer (FRET) and pulsed electron-electron double resonance (PELDOR) are powerful techniques for measuring distances within biomolecules.
- Both methods utilize reporter groups (fluorophores for FRET, spin labels for PELDOR) attached to proteins to probe molecular structure.
Purpose of the Study:
- To compare the distance measurements obtained from single-molecule FRET (smFRET) and pulsed electron-electron double resonance (PELDOR) on a well-characterized enzyme.
- To validate the accuracy and consistency of both smFRET and PELDOR in determining multicomponent distance distributions in complex biomolecules.
Main Methods:
- Site-directed mutagenesis was used to introduce specific cysteine residues into H+-ATPase (EF0F1) for optimal labeling.
- Both smFRET (single-molecule measurement) and PELDOR (ensemble measurement) were employed using attached fluorophores and spin labels, respectively.
- Distance distributions from both methods were compared against the known crystal structure of H+-ATPase.
Main Results:
- Both smFRET and PELDOR successfully generated similar multicomponent distance distributions for H+-ATPase.
- The dominant distance distribution (over 50%) corresponded to the introduced cysteine residues and agreed with the crystal structure.
- Additional distance distributions were assigned to endogenous cysteine sites, with PELDOR detecting one distribution not observed with smFRET.
Conclusions:
- smFRET and PELDOR are complementary techniques that provide consistent distance information for complex biomolecules like H+-ATPase under optimal conditions.
- PELDOR offers the advantage of obtaining distance distributions from ensemble measurements, while smFRET requires single-molecule analysis.
- The study validates the application of both smFRET and PELDOR for detailed structural investigations of enzyme complexes.
