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.

Figure 1

ITC reconstitution isotherms in (a) the absence or (b) the presence of 1.5 μM KcsA tetramer at 8 °C. Both isotherms depict the heats of reaction, Q (circles), obtained upon titration of 35 mM OG with 50 mM E. coli polar lipid extract. Also shown are the SOL and SAT boundaries (red and blue lines, respectively) and the uncertainty in the SAT boundary in the presence of KcsA (light blue band). Inset: Thermogram displaying differential heating power, Δp, versus time, t. The discontinuity at t ≈ 10 h is due to an increase in injection volume.

Figure 2

Thermograms (left) and isotherms (right) of reconstitution titrations performed at various lipid and detergent concentrations in the presence of 1.5 μM KcsA at 8 °C. (a) 40 mM lipid into 35 mM OG. (b) 30 mM lipid into 35 mM OG. (c) 20 mM lipid into 35 mM OG. (d) 20 mM lipid into 32.5 mM OG. (e) 20 mM lipid into 30 mM OG. Discontinuities in thermogram amplitudes are due to changes in injection volume. See Figure 1 for details.

Figure 3

Critical lipid/detergent concentration pairs in the presence of KcsA (open symbols and dotted lines) and phase diagram of E. coli polar lipid extract and OG (colored areas) at 8 °C. Experimental data from Figures 1 and 2 (red triangles and blue circles) and linear regressions (red and blue dotted lines) denoting, respectively, the SOL and SAT boundaries in the presence of 1.5 μM KcsA tetramer. Also shown are the micellar, coexistence, and vesicular ranges for protein-free E. coli polar lipid extract/OG mixtures (red, white, and blue hatched areas, respectively), the uncertainties in the SAT boundary in the presence of protein (blue error bars), and the trajectory of the titration depicted in Figure 1b (dashed arrow).

Figure 4

Size characterization of reconstituted proteoliposomes and protein-free lipid vesicles by DLS. (a) Normalized autocorrelation function, C(τ), versus delay time, τ, as determined for sonicated E. coli polar lipid vesicles before reconstitution (black) and the reconstitution mixture after calorimetrically monitored lipid addition to OG-solubilized KcsA (red). (b) Intensity-weighted distribution functions, f(d), of the hydrodynamic diameter, d, of sonicated E. coli polar lipid vesicles before reconstitution (black) and proteoliposomes after reconstitution and removal of aggregates and OG by dilution, centrifugation, and extrusion (blue).

Figure 5

Confirmation of proteoliposome formation and channel activity after reconstitution. (a) FCS autocorrelation functions, G(τ), versus delay time, τ, before and after resolubilization of proteoliposomes with 68 mM OG. Experimental data (colors) and fits (dashed). (b) Single-channel currents recorded after transfer of KcsA from proteoliposomes into planar membranes composed of E. coli polar lipid extract. pH 4.0 on both sides, voltage as indicated.