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A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

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Resolving inhomogeneity using lifetime-weighted fluorescence correlation spectroscopy.

Kunihiko Ishii¹, Tahei Tahara

¹Molecular Spectroscopy Laboratory, Advanced Science Institute (ASI), RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan.

The Journal of Physical Chemistry. B

|September 4, 2010

Summary

This summary is machine-generated.

Lifetime-weighted fluorescence correlation spectroscopy (FCS) detects complex system inhomogeneity by analyzing fluorescence lifetime fluctuations. This advanced method reveals conformational changes in biopolymers, offering insights into their dynamic behavior.

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Area of Science:

Biophysics
Physical Chemistry
Spectroscopy

Background:

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying molecular dynamics.
Analyzing fluorescence intensity fluctuations provides information about diffusion and concentration.
Limitations exist in resolving complex system dynamics and heterogeneity.

Purpose of the Study:

To introduce and validate lifetime-weighted FCS for detecting fluorescence lifetime fluctuations.
To demonstrate the capability of lifetime-weighted FCS in resolving inhomogeneity in complex systems.
To investigate environment-dependent conformational dynamics of biopolymers.

Main Methods:

Development of lifetime-weighted FCS by integrating fluorescence lifetime information into FCS.
Measurement of a mixture of two dyes with distinct fluorescence lifetimes to assess system inhomogeneity.
Application of lifetime-weighted FCS to a dye-labeled polypeptide to study conformational dynamics.

Main Results:

Lifetime-weighted correlation analysis deviates from standard intensity correlation in inhomogeneous systems.
Lifetime-weighted FCS successfully detects ensemble-averaged fluorescence decay profile inhomogeneity without prior knowledge.
Changes in the ratio of lifetime-weighted to ordinary intensity correlation indicate environment-dependent conformational inhomogeneity in polypeptides.
The observed inhomogeneity in polypeptides is persistent across the nano- to millisecond timescale.

Conclusions:

Lifetime-weighted FCS is a novel approach for resolving fluorescence lifetime fluctuations.
This method effectively detects and characterizes inhomogeneity in complex systems.
Lifetime-weighted FCS provides insights into conformational dynamics of biopolymers in response to environmental changes.