Protein directed assembly of lipids

Transcription

1 Protein directed assembly of lipids D. Nordin, O. Yarkoni, L. Donlon, N. Savinykh, and D.J. Frankel SUPPLEMENTARY MATERIAL Materials and Methods Supported bilayer preparation 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (Avanti Polar Lipids) were dissolved in chloroform in a 3 to 1 molar ratio and dried under nitrogen flow. The lipid mixtures were then re-suspended in ultrapure water at a concentration of 1 mg/ml and intensively vortexed at ambient temperature. Uni-lamellar vesicles were formed by ten freeze-thaw cycles followed by extrusion at 45 C (ten times) through a polycarbonate membrane with pores size of 100 nm by means of a mini-extruder (Avanti Polar Lipids). Solution containing the extruded vesicles was pipetted on to freshly cleaved mica which was then placed on a thin metal puck situated on a hotplate. The hotplate was heated to 45 C and the sample annealed for 20 minutes, after which it was removed for AFM imaging at ambient temperature (20 o C). Fibronectin adsorption For protein adsorption experiments, fibronectin from bovine plasma (Sigma-Aldrich, UK) was dissolved in phosphate buffered saline (PBS) (138 mm NaCl, 2.7 mm KCl, ph 7.4 at 25 o C) to acquire a 50 µg/ml stock solution of protein, which was stored at 4 o C. The fibronectin solution was then diluted to the desired concentration. To form the ring structures, a 3:1 molar solution of extruded DOPC/DPPC vesicles was pipetted on to freshly cleaved mica and annealed for 5 minutes as described previously. Fibronectin solution (5µg/mL) was then added to the lipid bilayer (20 x 20 mm) and the sample annealed for a further minutes at 45 o C. The sample was then removed for AFM imaging at ambient temperature (20 o C). Topographic AFM imaging AFM experiments were conducted on an Agilent 5500 AFM/SPM microscope in a liquid environment at 20 C. Images were obtained in tapping mode using silicon tips (Nanosensors, series PPP-NCH) with a resonance frequency of ~330 khz and a force constant of 42 N/m. Standard nitrogen doped silicon tips with a nominal force constant of N/m were used for contact mode imaging and forces were minimized during the scans. Typical scan rates were in the range of khz at 512 points/line resolution. Imaging forces were kept below 1 nn. All measurements were performed in ultrapure water.

2 Electronic Supplementary Material (ESI) for Chemical Communications Force spectroscopy In force spectroscopy experiments, backside aluminium coated silicon cantilevers (PPP-CONTR, Nanosensors, Switzerland) with a nominal spring constant of N/m and typical tip radius of less than 7 nm were used. The spring constants were calibrated using the equipartition theorem (Thermal K).1 Approximately, force-distance curves were obtained per sample with an applied load in the range of 9 10 nn, constant tip velocity of 1 µm/s and curves length of 1 µm. Force- distance curves were analyzed using a Scanning Probe Image Processor (Image Metrology, Lyngby, Denmark). Only force curves that could be fitted to the wormlike chain model were accepted to represent single molecule pulling events. Determination of rupture forces from force-distance spectra was carried out according to previously reported procedures.2-3 (a) (b) (c) (d) Fig. 1 Atomic force microscopy of lipid bilayers composed of DOPC and DPPC (3:1 molar ratio). a, b, c and d show the same sample but at progressively higher resolution scans. The whole image area is bilayer with the brown contrast being liquid disordered DOPC and the bright features DPPC liquid ordered (gel) domains. It is clear that the surface is uniform with islands of phase separated DPPC regions. Images were obtained in tapping mode in an aqueous environment (ultrapure water) at 20 C.

3 Fig. 2 Histograms illustrating the distribution of ring diameter (a), width of annulus (b) and height profiles (c). The rings range in size from 2 to 12 µm, with an average diameter of 5.31 ± 0.2 µm (mean ± S.E; SD = 1.96 µm; Data = 100). The annulus of the rings is measured as 0.55 ± 0.01 µm (mean ± S.E; SD = 0.06 µm; Data = 100) and corresponds to a single circular structure of DPPC islands. The measured height of the lipid rings is 1.03 ± 0.01 nm (mean ± S.E; SD = 0.05 nm, Data = 100) and is consistent with that of DPPC islands in phase separated bilayers.

4 Electronic Supplementary Material (ESI) for Chemical Communications Fig. 3 Atomic force microscopy images of lipid bilayers composed of DOPC and DPPC (3:1 molar ratio) with addition of the ECM protein fibronectin at a concentration of 5µg/mL at 45 C. This sample was imaged in tapping mode in a liquid environment (ultrapure water) at 20 C at successively higher magnifications. It is clear from image d that individual lipid domains assemble to form the rings (this is particularly evident in the top right hand corner where the individual segments forming the curvature of the circle fit together like pieces in a jigsaw puzzle.)

5 (a) (b) Fig. 4 Atomic force microscopy images illustrating fibronectin molecules at the boundary between the DPPC gel phase and DOPC liquid disordered domains. Bilayers composed of 3:1 molar ratio of DOPC:DPPC with fibronectin at a concentration of 5 µg/ml. 1 J. Hutter and J. Beschhoefer, Rev. Sci. Instrum., 1993, 64, P.Y. Meadows, J.E. Bemis and G.C. Walker, Langmuir, 2003, 19, D. Nordin, O. Yarkoni, N. Savinykh, L. Donlon and D.J. Frankel, Soft Matter, 2011, accepted.