Real-Time Monitoring of Membrane-Protein Reconstitution by Isothermal Titration Calorimetry

Abstract

Phase diagrams offer a wealth of thermodynamic information on aqueous mixtures of bilayer-forming lipids and micelle-forming detergents, providing a straightforward means of monitoring and adjusting the supramolecular state of such systems. However, equilibrium phase diagrams are of very limited use for the reconstitution of membrane proteins because of the occurrence of irreversible, unproductive processes such as aggregation and precipitation that compete with productive reconstitution. Here, we exemplify this by dissecting the effects of the K+ channel KcsA on the process of bilayer self-assembly in a mixture of Escherichia coli polar lipid extract and the nonionic detergent octyl-β-d-glucopyranoside. Even at starting concentrations in the low micromolar range, KcsA has a tremendous impact on the supramolecular organization of the system, shifting the critical lipid/detergent ratios at the onset and completion of vesicle formation by more than 2-fold. Thus, equilibrium phase diagrams obtained for protein-free lipid/detergent mixtures would be misleading when used to guide the reconstitution process. To address this issue, we demonstrate that, even under such nonequilibrium conditions, high-sensitivity isothermal titration calorimetry can be exploited to monitor the progress of membrane-protein reconstitution in real time, in a noninvasive manner, and at high resolution to yield functional proteoliposomes with a narrow size distribution for further downstream applications.

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy

“…With further developments, this FP method may be used to determine changes in the tumbling rate of membrane proteins under native (e.g., in lipid-bound detergent micelles) and denaturing (e.g., in the presence of Gdm-HCl) conditions. FP experiments with specific detergent-lipid solubilization mixtures 9 might potentially generate an understanding of the structural role of these lipids in the stabilization of membrane proteins.…”

“…7,8 Isothermal titration calorimetry (ITC) was also employed for the determination of thermodynamic phase diagrams of ternary lipid-detergent-protein systems and their deviation from data obtained in protein-free lipid-detergent mixtures. 9 Moreover, nuclear magnetic resonance (NMR) spectroscopy was extensively used for the examination of the interactions of membrane proteins with detergent micelles. 10,11 In particular, solid-state NMR is a versatile approach for the investigation of membrane proteins in various detergent and lipid environments, including cell membranes.…”

Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids

“…The disadvantage of the method is its reliance on fluorescence, which requires fluorescent labelling of the protein and the lipids, and limits the time resolution to avoid bleaching. Yet, another approach reported by Keller and collaborators allows monitoring the process the phase changes concurrent with the reconstitution4. While the method is ideal to optimize the conditions of detergent-mediated reconstitution, it still relies on the use of additional techniques to confirm proper protein reconstitution and functionality.…”

“…Their reconstitution from a detergent-stabilized state into a well-defined artificial lipid model system of controlled lipid composition for in vitro studies23 can be achieved by dilution or detergent removal using dialysis, gel filtration, hydrophobic absorption using Bio-beads, or complexation by cyclodextrins. Regardless of the method used, the process itself remains most often intractable (with the exception of an indirect Isothermal Titration Calorimetry approach reported by S. Keller and coworkers based on phase transition monitoring4). Herein we demonstrate that Plasmon Waveguide Resonance (PWR) spectroscopy can be used to directly follow the reconstitution of a detergent-solubilized GPCR into a planar solid-supported lipid membrane5.…”

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

“…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.…”