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Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence
Benjamin Schuler1, Everett A Lipman, Peter J Steinbach
1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Building 5, Room 104, National Institutes of Health, Bethesda, MD 20892, USA. schuler@bioc.unizh.ch
Summary
Forster resonance energy transfer (FRET) measurements provide distance information for polyproline. Deviations from rigid rod predictions highlight the importance of chain bending and dye size in FRET analysis.
Area of Science:
- Biophysics
- Physical Chemistry
- Polymer Science
Background:
- Forster resonance energy transfer (FRET) is a technique used to measure distances.
- Accurate distance measurements are crucial for understanding molecular behavior.
- Polypeptides flexibility can influence FRET measurements.
Purpose of the Study:
- To assess the quantitative accuracy of FRET for determining distances in single-molecule experiments.
- To investigate FRET efficiency in polyproline chains of varying lengths.
- To compare experimental FRET data with theoretical predictions.
Main Methods:
- Single-molecule fluorescence experiments measuring FRET efficiency distributions.
- Utilizing donor and acceptor dyes attached to polyproline ends.
- Employing Langevin molecular dynamics simulations for distance analysis.
Main Results:
- Observed mean FRET efficiencies align with ensemble lifetime measurements.
- Significant deviations from Forster theory predictions for rigid rods were observed.
- Lower efficiencies at short distances and higher efficiencies at longer distances were noted.
- Chain bending in longer polyprolines was identified as a key factor.
Conclusions:
- FRET measurements can offer distance insights but require careful interpretation.
- Deviations from theoretical models are influenced by orientational averaging and dye size.
- Polymer chain dynamics, such as bending, significantly impact FRET-derived distances.
- Langevin dynamics simulations aid in explaining experimental FRET discrepancies.