Abstract
The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.
Highlights
Detergent-induced membrane solubilization is critical for applications including membrane-protein purification[1,2], and targeted drug delivery, where vesicle rupture enables release of encapsulated therapeutics[3]
In this work we demonstrate the combination of single-molecule fluorescence and Förster resonance energy transfer (FRET) to monitor, in real-time, the detergent-induced solubilization of large unilamellar vesicles (LUVs) with sizes smaller than the diffraction limit
Steady-state fluorescence measurements were carried out as an initial step to characterize the interaction between Triton X 100 (TX-100) and labelled vesicles
Summary
Detergent-induced membrane solubilization is critical for applications including membrane-protein purification[1,2], and targeted drug delivery, where vesicle rupture enables release of encapsulated therapeutics[3]. In this work we demonstrate the combination of single-molecule fluorescence and Förster resonance energy transfer (FRET) to monitor, in real-time, the detergent-induced solubilization of large unilamellar vesicles (LUVs) with sizes smaller than the diffraction limit.
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