Identification and quantification of within-burst dynamics in singly labeled single-molecule fluorescence lifetime...

Area of Science:

• Molecular Biophysics
• Single-Molecule Spectroscopy
• Biochemistry

Background:

• Single-molecule spectroscopy enables probing biomolecular structural reorganization across various timescales.
• Existing methods using fluorescence lifetimes focus on local dynamics near labeled residues.
• A framework for quantifying micro- to millisecond dynamics within single-molecule fluorescence bursts is lacking.

Purpose of the Study:

• To develop and validate an analytical framework for quantifying within-burst lifetime-based dynamics.
• To analyze conformational dynamics using single-molecule photo-isomerization-related fluorescence enhancement.
• To investigate local structural dynamics in unbound alpha-synuclein monomers.

Main Methods:

• Development of a novel analytical framework for analyzing single-molecule fluorescence lifetime data.
• Testing the framework's capabilities using simulated data.
• Application of the framework to experimental data from unbound alpha-synuclein.

Main Results:

• Successful identification and quantification of within-burst lifetime-based dynamics.
• Demonstration of millisecond-timescale local structural dynamics in unbound alpha-synuclein.
• Validation of the analytical framework's effectiveness.

Conclusions:

• The proposed analytical framework enables quantification of micro- to millisecond dynamics from single-molecule fluorescence bursts.
• This framework reveals local structural dynamics in unbound alpha-synuclein monomers.
• It provides a pathway to map energy landscapes probed by fluorescence lifetime measurements.