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Biophys. J. BioFAST: First Published December 1, 2006. doi:10.1529/biophysj.106.088708
© 2006 by the Biophysical Society.

A more recent version of this article appeared on February 15, 2007.
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CHANNELS, RECEPTORS, AND ELECTRICAL SIGNALING

Dual-color fluorescence-burst analysis to probe protein efflux through the mechanosensitive channel MscL

Geert van den Bogaart 1, Victor Krasnikov 1 and Bert Poolman 1*

1 University of Groningen

* To whom correspondence should be addressed. E-mail: b.poolman{at}rug.nl.

Submitted on May 9, 2006
Revised on July 12, 2006
Accepted on 1 November 2006


  Abstract
The mechanosensitive channel protein of large conductance, MscL, from Escherichia coli has been implicated in protein efflux, but the passage of proteins through the channel has never been demonstrated. We used dual-color fluorescence-burst analysis to evaluate the efflux of fluorescent labelled compounds through MscL. The method correlates the fluctuations in intensity of fluorescent labelled membranes and encapsulated (macro)molecules (labelled with second fluorophore) for each liposome diffusing through the observation volume. The analysis provides quantitative information on the concentration of macromolecules inside the liposomes and the fraction of functional channel proteins. For MscL, reconstituted in large unilamellar vesicles, we show that insulin, bovine pancreas trypsin inhibitor and other compounds smaller than 6.5 kDa can pass through MscL, whereas larger macromolecules cannot.

Key Words: MscL, dual-color fluorescence burst analysis, fluorescence correlation spectroscopy, mechanosensitive channel, membrane reconstitution, protein diffusion




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Copyright © 2006 by the Biophysical Society.