Dual-color fluorescence-burst analysis to probe protein efflux through the mechanosensitive channel MscL.
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 labeled compounds through MscL. The method correlates the fluctuations in intensity of fluorescent labeled membranes and encapsulated (macro)molecules (labeled 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.
van den Bogaart
Geert
G
Biochemistry Department, Groningen Biomolecular Science and Biotechnology Institute & Materials Science Centre, University of Groningen, Groningen, The Netherlands.
Krasnikov
Victor
V
Poolman
Bert
B
D000818:Animals; D007611:Aprotinin / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D001692:Biological Transport; D001920:Bradykinin / Q000737:chemistry; D002417:Cattle; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D007328:Insulin / Q000737:chemistry; D007473:Ion Channels / Q000737:chemistry*; D007768:Lactalbumin / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D040542:Mechanotransduction, Cellular; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D007328:Insulin; D007473:Ion Channels; D008081:Liposomes; C086596:MscL protein, E coli; D011506:Proteins; D013879:Thioredoxins; D007768:Lactalbumin; D007611:Aprotinin; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D005978:Glutathione; D001920:Bradykinin