Monitoring detergent-mediated solubilization and reconstitution of lipid membranes by isothermal titration calorimetry

Abstract

The solubilization and reconstitution of biological or liposomal membranes by detergents and biomolecules with detergent-like properties play a major role for technical applications (e.g., the isolation of membrane proteins) and biological phenomena (of, e.g., amphiphilic peptides). It is therefore important to know and understand the amounts of a given detergent required for the onset and completion of membrane solubilization and the detergent-lipid interactions in general. Lipid-detergent systems can form a variety of aggregate structures, which can be grouped into two pseudophases (lamellae and micelles) so that solubilization can be approximately described as a phase transition. Here we present a protocol for establishing the phase diagram and a detailed thermodynamic description of a lipid-detergent system based on isothermal titration calorimetry (ITC). The protocol can also be used to detect additive-induced membrane destabilization, permeabilization, domain formation and lipid-dependent transitions between rod-like and spherical micelles. A minimal protocol consisting of all sample preparation procedures and a single solubilization experiment can be accomplished within 2 days; a more extensive series comprising both solubilization and reconstitution experiments requires several days to a few weeks, depending on the number of titrations performed.

Filter gate closure inhibits ion but not water transport through potassium channels

“…Sonicated unilamellar vesicles composed of E. coli polar lipid extract (Avanti Polar Lipids) at a lipid concentration of 28 mg/mL buffer (150 mM KCl, 25 mM Tris, 25 mM Hepes, pH 7.50) were added to the solubilized protein in small steps until reconstitution was complete (44,45). This process was followed by buffer exchange via ultracentrifugation, resuspension of the vesicles, and a final dialysis step.…”

Membrane solubilisation and reconstitution by octylglucoside: Comparison of synthetic lipid and natural lipid extract by isothermal titration calorimetry

“…Solubilisation and reconstitution titrations were carried out essentially as described elsewhere [26]. For solubilisation, small aliquots of a concentrated detergent solution (100 mM) were injected into the ITC sample cell containing lipid vesicles (1-6.5 mM) and a submicellar concentration of OG (16-21 mM).…”

“…During the last two decades, isothermal titration calorimetry (ITC) has been established as an exceptionally powerful technique for monitoring the compositiondependent phase behaviour of detergent/lipid mixtures (see [12,[17][18][19][20][21][22][23] for examples, [24,25] for reviews, and [26] for a step-by-step protocol). More recently, it has also been applied to characterising the lipid interactions of lipopeptides with detergent-like properties [27][28][29][30].…”

High-precision, automated integration of multiple isothermal titration calorimetric thermograms: New features of NITPIC

“…The only expectation that NITPIC builds into its analysis is that of a smoothly varying isotherm, which should be fulfilled for most applications of the ITC. Even if it is not, NITPIC still performs well [7]. With the introduction of the Jackrabbit code changes (Section 4.1.1), speed is no longer an issue.…”

“…For example, protein-protein [1], protein-DNA [2], and protein-ligand [3] binding can be thermodynamically characterized with this method. Thermodynamic parameters for detergent demicellization [4], the insertion of proteins into membranes [5], and other aspects of membrane biology, like protein or peptide uptake/release [6] and membrane solubilization and reconstitution [7] may also be studied with ITC. The rich information content and the label-free nature of the experiment make it the method of choice for many researchers.…”