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Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

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Sequential Two-Photon Delayed Fluorescence Anisotropy for Macromolecular Size Determination.

Yi-Han Lu¹, Matthew C Jenkins¹, Katherine G Richardson¹

¹School of Chemistry and Biochemistry and Petit Institute of Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, United States.

The Journal of Physical Chemistry. B

|April 25, 2023

Summary

This summary is machine-generated.

We developed optically activated delayed fluorescence anisotropy (OADFA) to measure slow molecular dynamics. This technique extends fluorescence anisotropy measurements to large biomolecular complexes and colloidal systems.

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Area of Science:

Biophysics
Biochemistry
Physical Chemistry

Background:

Time-resolved fluorescence anisotropy (FA) is a sensitive technique for studying molecular dynamics.
The short fluorescence lifetime of conventional dyes limits FA applications to small molecules.
Larger biomolecular complexes exhibit slow rotational diffusion, which is not measurable with standard FA.

Purpose of the Study:

To extend the timescale of fluorescence anisotropy measurements.
To enable the study of slow depolarization processes in large macromolecular complexes.
To develop a novel method for analyzing slow molecular dynamics.

Main Methods:

Introduced optically activated delayed fluorescence anisotropy (OADFA) using sequential two-photon excitation.
Utilized triplet shelving and recovery of optical excitation to extend measurement times.
Derived a quantitative rate model and performed Monte Carlo simulations.

Main Results:

OADFA effectively stretches fluorescence anisotropy measurement times from nanoseconds to hundreds of microseconds.
Demonstrated the capability of OADFA for measuring slow depolarization processes in large macromolecular complexes.
Developed a molecular-level understanding of the OADFA depolarization process through simulations.

Conclusions:

OADFA is a powerful new technique for studying slow dynamics in large biomolecular and colloidal systems.
This method overcomes the limitations of conventional FA for large structures.
OADFA opens new avenues for research in biophysics and materials science.