Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP)

• DOI: 10.1039/c0cp01911a
• Publication Date: 2011-01-07T15:00:21Z
• Authors: Armin Hoffmann; Daniel Nettels; Jennifer Clark; Alessandro Borgia; Sheena E. Radford; Jane Clarke; Benjamin Schuler
• Keywords: Single-molecule FRET; Single-molecule experiment; Energy landscape; Fluorescence Correlation Spectroscopy

Single molecule Förster resonance energy transfer (FRET) experiments are a versatile method for investigating the conformational distributions and dynamics of biological macromolecules. In common type experiment, fluorescence bursts from individual molecules freely diffusing in solution detected as they pass through observation volume confocal microscope. Correlation analysis shows that under typical experimental conditions, time scales up to several tens milliseconds, probability return is greater than new being detected. Here we present RASP (recurrence single particles), based on this recurrence behavior allows us significantly extend information can be extracted FRET experiments. The number peak shapes subpopulations within sample identified essentially model-free way by constructing efficiency histograms. These obtained first selecting photon small range (initial bursts), then building histogram only short (the interval) after initial bursts. Systematic variation interval kinetics interconversion between determined ~50 μs ~100 ms equilibrium measurements. We demonstrate applicability measurements peptides proteins with different degrees heterogeneity folding dynamics. concepts presented here extended other observables available