Protein origin
Natural Lysenin from earthworm, Eisenia foetida, was obtained from Peptide Institute (Osaka, Japan). Lysenin mutant (E92, 94, 97Q) cDNA fragment was ordered from GenScript, USA. The cDNA fragments were subcloned into a pET28a vector at BamHI and Hind III sites. This vector was transformed into BL21 (DE3) strain (New England BioLabs France, Evry, France). The transformed cells were inoculated into 1 liter of LB medium containing 50μg/ml kanamycin sulfate, and incubated at 37°C while shaking at 220rpm until the OD600 value reached 0.6. For induction of lysenin expression, isopropyl β-D-1thiogalactopyranoside (IPTG) was added to the bacterial medium (0.5 mM, final concentration) while shaking at 20°C and 200rpm. The bacteria were collected by centrifugation at 2000g for 10 minutes. The activity of proteases was inhibited by the addition of 1mM phenylmethane sulfonyl fluoride. To disrupt the bacteria we perform 3 sonication cycles with intervals of 30 seconds on ice. The resulted suspension was shaken at 4°C for 30 minutes in presence of 0.1% Triton X-100 and RNase/DNase at 10µg/ml concentration. The crude extract was then centrifuged at 10,000 x g for 30 minutes. The supernatant (volume; typically up to 10 ml) was collected and mixed with 1ml of chelating Nickel Ni-NTA Affinity Resin (Generon, United Kingdom) in Phosphatebuffered saline (PBS) at pH7.5. Lysenin binding to the Ni-NTA resin was performed through 1 hour incubation at 4°C with gentle shaking, and the resin was washed with PBS containing 100 mM imidazole-HCl, pH 7.5 for 3 times. The lysenin was eluted with 2 ml of 250mM imidazole-HCl. To eliminate the imidazole, the eluate was dialyzed against 1 liter of PBS at pH7.5 for overnight.
Sample preparation for High-speed atomic force microscopy observation
Egg Sphingomyelin (SM) and Cholesterol (chol) (Avanti Polar Lipids, Alabama, USA) at a molar ratio SM/Chol (1:1) were used to form giant unilamellar vesicles (GUVs) through electroswelling 32. Of each lipid 10µl at 3mM dissolved in chloroform/methanol (3:1) were deposited in a glass plate coated with indium tin oxide with about 100Ω resistivity (Sigma-Aldrich) and placed 60 minutes in the desiccator for complete solvent evaporation. A U-shaped rubber piece of ∼1mm thickness was sandwiched between two indium tin oxide coated slides. The so-formed chamber was filled with ∼400µl of 200mM sucrose solution and exposed to 1.5V sinusoidal 10Hz AC current for 3 hours followed by squared 5Hz AC current for 15 minutes, at 55°C. GUVs were harvested from the chamber. To form the supported lipid bilayers (SLBs) for HS-AFM, 1µl of GUV solution was placed on a 1.5mm-diameter freshly cleaved mica disk covered with 1µl of Phosphate-buffered saline (PBS) and incubated for 30 minutes. To remove lipid that was not firmly attached the SLB was intensely rinsed with PBS. Once the bilayer was formed, 1µl of purified Lysenin was incubated for 15 minutes. Excess protein was again rinsed with PBS or physiological buffer.
High-speed atomic force microscopy
HS-AFM movies were acquired with an Ando-type set-up33 equipped with a super luminescent diode (emission wavelength: 750 nm; EXS 7505-B001, Exalos, Schlieren, Switzerland) and a digital high-speed lock-in Amplifier (Hinstra, Transcommers, Budapest, Hungary).34 8µm-long cantilevers with spring constant k = 0.15 Nm-1, resonance frequency f(r) = 500-700 kHz and quality factor Q ≈ 1 in liquid (USC-1.2, NanoWorld, Neuchâtel, Switzerland), featuring an electron beam deposition (EBD) tip, were used. For high-resolution imaging the tip was sharpened by helium plasma etching using a plasma cleaner (Diener electronic, Ebhausen, Germany), resulting in a final tip radius tip of ∼2 nm, as judged from analysis of the indentation inside the Lysenin rings. Amplitude modulation was used for imaging with free amplitude of ∼1.2 nm and operating set point amplitude of ~0.9 nm. Under this conditions we calculate the apply force following F = (𝑘𝑘𝑐𝑐/𝑄𝑄𝑐𝑐)*((1- α)*𝐴𝐴0(𝐴𝐴0−As2/A0)1/2 where A0 is the free amplitude, As is the setpoint amplitude, and α ≈0.5 for short cantilevers, α being the ratio (0<α<1) of amplitude reduction caused by the cantilever resonance frequency shift over the total amplitude reduction. Under our imaging conditions F=84pN.35 All experiments were performed at room temperature and in PBS or physiological buffer (20mM HEPES, pH7.5, 120mM NaCl)
High-speed atomic force microscopy image treatment
Image treatment was limited to the correction of a first-order XY plane fit and XY drift correction of the HS-AFM movie.36
Structure analysis
Molecular graphics and analyses were performed with the UCSF Chimera package.37