Non-vesicular transfer of membrane proteins from nanoparticles to lipid bilayers

Abstract

Discoidal lipoproteins are a novel class of nanoparticles for studying membrane proteins (MPs) in a soluble, native lipid environment, using assays that have not been traditionally applied to transmembrane proteins. Here, we report the successful delivery of an ion channel from these particles, called nanoscale apolipoprotein-bound bilayers (NABBs), to a distinct, continuous lipid bilayer that will allow both ensemble assays, made possible by the soluble NABB platform, and single-molecule assays, to be performed from the same biochemical preparation. We optimized the incorporation and verified the homogeneity of NABBs containing a prototypical potassium channel, KcsA. We also evaluated the transfer of KcsA from the NABBs to lipid bilayers using single-channel electrophysiology and found that the functional properties of the channel remained intact. NABBs containing KcsA were stable, homogeneous, and able to spontaneously deliver the channel to black lipid membranes without measurably affecting the electrical properties of the bilayer. Our results are the first to demonstrate the transfer of a MP from NABBs to a different lipid bilayer without involving vesicle fusion.

Figure 1.

Tetrameric KcsA incorporates efficiently into NABBs. (A) Gel filtration of empty NABBs and 1K-NABB and 4K-NABB samples. Solid (280-nm) and dashed (460-nm) curves represent protein and NBD lipid absorbance, respectively. (Inset) Coomassie-stained SDS-PAGE of gel filtration–purified samples of 4K-NABB, 1K-NABB, and KcsA in PE/PG vesicles, with (Δ) or without (−) heating at 95°C before loading. The bands represent KcsA tetramer (68 kD), monomer (17 kD), and Zap1 (31 kD). (B) Negative-stain EM image of 4K-NABBs. Some particles are seen characteristically “stacking” together, oriented with the plane of the disc and perpendicular to EM grid. Bar, 100 nm. (C) Class averages of single particles picked from EM micrographs of 4K-NABBs (panels 1–4) show density within the discs oriented at different angles relative to the plane of the disc. Cartoon representations below each class average show the likely orientation of the densities emerging from the discs. Class averages of empty NABBs (panels 5 and 6) lack these densities. Each class average was computed from ∼200 isolated single particles. The side of each panel is 30 nm.

Figure 2.

KcsA E71A channels transfer from NABBs to lipid bilayers. (A) Cartoon representation of KcsA transfer from NABBs into lipid bilayers. The NABB model was produced using the University of California, San Francisco, Chimera package using HDL particle coordinates provided by J.P. Segrest [University of Alabama at Birmingham, Birmingham, AL] and KcsA coordinates deposited in the Protein Data Bank (accession no. 3EFF). The BLM was constructed using Visual Molecular Dynamics software. (B) Spontaneous transfer of channels as a function of time from NABBs after adding NABBs with or without KcsA E71A to BLMs. The same amount of KcsA, from a 2-µg/ml stock, was added in each case.

Figure 3.

KcsA E71A functional properties are preserved after transfer from NABBs to BLMs. (A) Representative single-channel traces of KcsA E71A transferred from 1K-NABBs to BLMs in the absence (top) or presence (bottom) of 5 µM BaCl₂ added to the trans chamber. The electrophysiology recording conditions were 100 mM of symmetric K⁺ across the BLM (V_m = +100 mV; low-pass filter at 1 kHz). (B) Single-channel I-V plot of KcsA E71A transferred from NABBs. (C) Open probability (Po) of the transferred channel as a function of voltage. The change in Po in the case of Ba²⁺ block was fit to the equation:$P_o\left(V\right)=\frac{1}{1+\frac{B}{K_0\cdot e^{\frac{-zFV}{RT}}}},$where B is the blocker concentration (5 µM), K₀ is the apparent blocker affinity at 0 mV, and z is the voltage dependence. The fit values for K₀ and z were 48.2 ± 5.6 µM and 0.54 ± 0.02, respectively. Symbols represent averages ± SD from three separate experiments.