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Comparison between whole distribution- and average-based approaches to the determination of fluorescence resonance energy transfer efficiency in ensembles of proteins in living cells.

Abstract：

:Current methods for analysis of data from studies of protein-protein interactions using fluorescence resonance energy transfer (FRET) emerged from several decades of research using wide-field microscopes and spectrofluorometers to measure fluorescence from individual cells or cell populations. Inherent to most measurements is an averaging of the distributions of FRET efficiencies over large populations of protein complexes, which washes out information regarding the stoichiometry and structure of protein complexes. Although the introduction of laser-scanning microscopes in principle could facilitate quantification of the distributions of FRET efficiencies in live cells, only comparatively recently did this potential fully materialize, through development of spectral- or lifetime-based approaches. To exploit this new opportunity in molecular imaging, it is necessary to further develop theoretical models and methods of data analysis. Using Monte Carlo simulations, we investigated FRET in homogenous and inhomogeneous spatial distributions of molecules. Our results indicate that an analysis based on distributions of FRET efficiencies presents significant advantages over the average-based approach, which include allowing for proper identification of biologically relevant FRET. This study provides insights into the effect of molecular crowding on FRET, and it offers a basis for information extraction from distributions of FRET efficiencies using simulations-based data fitting.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Singh DR,Raicu V

doi

10.1016/j.bpj.2010.01.048

subject

Has Abstract

pub_date

2010-05-19 00:00:00

pages

2127-35

issue

10

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(10)00213-4

journal_volume

98

pub_type

杂志文章

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