Expression, purification, and reconstitution of the voltage-sensing domain from Ci-VSP

Abstract

The voltage-sensing domain (VSD) is the common scaffold responsible for the functional behavior of voltage-gated ion channels, voltage sensitive enzymes, and proton channels. Because of the position of the voltage dependence of the available VSD structures, at present, they all represent the activated state of the sensor. Yet in the absence of a consensus resting state structure, the mechanistic details of voltage sensing remain controversial. The voltage dependence of the VSD from Ci-VSP (Ci-VSD) is dramatically right shifted, so that at 0 mV it presumably populates the putative resting state. Appropriate biochemical methods are an essential prerequisite for generating sufficient amounts of Ci-VSD protein for high-resolution structural studies. Here, we present a simple and robust protocol for the expression of eukaryotic Ci-VSD in Escherichia coli at milligram levels. The protein is pure, homogeneous, monodisperse, and well-folded after solubilization in Anzergent 3-14 at the analyzed concentration (~0.3 mg/mL). Ci-VSD can be reconstituted into liposomes of various compositions, and initial site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopic measurements indicate its first transmembrane segment folds into an α-helix, in agreement with the homologous region of other VSDs. On the basis of our results and enhanced relaxation EPR spectroscopy measurement, Ci-VSD reconstitutes essentially randomly in proteoliposomes, precluding straightforward application of transmembrane voltages in combination with spectroscopic methods. Nevertheless, these results represent an initial step that makes the resting state of a VSD accessible to a variety of biophysical and structural approaches, including X-ray crystallography, spectroscopic methods, and electrophysiology in lipid bilayers.

Figure 1

Sequence alignment and function comparison of Ci-VSD with existing VSDs. (A) Gene of Ci-VSP consists of N-terminal, voltage sensing domain, a linker and phosphatase domain. The first 260 residues, indicated by the arrow on the top, is our current interest for biochemical preparation. Sequence of the fourth transmembrane segment (S4) of Ci-VSP was aligned with homologous sequences from voltage-gated ion channels. The positive charged residues (R/K) were distributed at every third position, and primarily responsible for voltage sensing and highly conserved. (B) Relationship between the charge movement and the test pulse amplitude (Q-V curve) of VSDs from a typical potassium channel Shaker (black) and Ci-VSP (red). The curves was simulated with V_1/2 = −48 mV and z = 4.7 for Shaker (38), and V_1/2 = 71.8 mV and z = 1.1 for Ci-VSP (3). The dotted line at 0 mV indicates the potential states of VSDs under biochemical conditions in absence of asymmetric voltage across the proteins: Shaker’s VSD at activated state (or Up state); Ci-VSD at resting state (or Down state).

Figure 2

Detergent screen. (A) Western blot of solubilization test: Top, supernatant fractions extracted by different detergents after ultra-centrifugation; Bottom: pellet fraction dissolved by SDS. FC-10, FC-12, FC-14, Anzergent 3-12 and Anzergent 3-14 were able to extract Ci-VSD efficiently out of crude cell membrane. FC-12 and Anzergent 3-14 were chosen for subsequent purification test. (B) Representative elution profile of crude eluate from cobalt affinity chromatography on to SEC with Anzergent 3-14. Its elution volume is 13.7 mL on the Superdex 200 HR 10/30 column. Homogeneity of main peak was calculated from the ratio of weighted main peak (grey area) over weighted whole region (black arrow on bottom). (C) Homogeneity probability of Ci-VSD eluted in eighteen detergents. Eight detergents (Anzergent 3-12, Anzergent 3-14, C₈E₄, FC-14, LDAO, Mega-10, NG and OG), whose homogeneity probability is higher than the reference line (dotted), were chosen for the further stability test.

Figure 3

Ci-VSD characterization. (A) SDS-PAGE gel of Ci-VSD-1-260. The sample was overloaded to amplify the impurities. (B) Circular Dichroism spectra in molar ellipticity of Ci-VSD in Anzergent 3-14. The calculated helicity is 52%, which is consistent with high helical contents for the expected four transmembrane helices. It suggests that Ci-VSD maintained its secondary structure in the tested detergent and the N-terminal region 1–100 is most likely unstructured. (C) Ci-VSD’s molecular weight determined by multiple angle light scattering methods in Anzergent 3-14. The molecular weight (black line) is evenly distributed throughout the peak region (blue line) indicating a homogeneous protein-detergent complex.

Figure 4

Ci-VSD reconstitution. (A) Cartoon representation of FRET assay to evaluate the aggregation behavior. Ci-VSD was individually labeled with fluorescence donor and acceptor, then mixed at 1:1 molar ratio and reconstituted into liposomes. FRET signal in the range of 560–580 nm indicates of closeness of fluorephores in liposome, thus the degree of Ci-VSD aggregation. (B) FRET signal of Ci-VSD in five different liposomes at 1:2000 protein to lipids ratio 24 hours after reconstitution. The signals from two liposomes POPC:POPG = 3:1 and Asolectin were significantly lower than other three compositions. (C) CW-EPR spectra of Ci-VSD in POPC:POPG (red) and Asolectin (black) at seven residues 123–128 in the first transmembrane segment (S1). Spectra features are distinctly different among residues, but essentially the same in both liposomes.

Figure 5

Direction of Ci-VSD in liposome. (A) Cartoon representation of Chromium (III) quenching methods to access the direction of Ci-VSD in liposome. Addition of 30 mM CrOx in the exterior solution will quench spin label signal at the outside surface of liposome. Percent of quenching illustrates the direction of the labeled position in reference to liposome: inside, outside, or randomly distributed. (B) Spectra of spin labeled Ci-VSD 141C in POPC:POPG liposome (black) and in the presence of 30 mM CrOx (red). Percent of amplitude reduction is 44%.

Figure 6

Structure of S1 of Ci-VSD in liposome studied by EPR spectroscopy. (A) Cartoon representation of VSD’s scaffold of four helices. Relative position of membrane was shown in grey, where it is solvent inaccessibility. The positions 110 and 141 are outside of lipid bilayer and on the opposite sides. (B) Ni-EDDA accessibility (ΠNi, blue), Oxygen accessibility (ΠO₂, red) and Mobility (ΔH_o⁻¹, black) for the S1 of Ci-VSD. The grey region represents the transmembrane region identified by ΠNi. The ΠO₂ of KvAP in the homologous region were plotted in grey as a reference. Two sets of ΠO₂ values oscillate in the same range with almost the same pattern, with maximum values at every three or four residues. They indicate the S1 of Ci-VSD folds into α-helix in liposome as the same way as KvAP. Clearly, Ci-VSD folds into the same scaffold as existing VSDs. (C) Periodic probability analysis for ΠO₂ of the transmembrane region 113–138 shows P_max = 105° and αPI = 3.5. It suggests α-helical conformation as 3.4 residues per turn (360°/105°) with high probability (αPI > 2).