Neutravidin

All lipids were purchased from Avanti Polar Lipids. For binding of NeutrAvidin, 5% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) was introduced. Vesicles were prepared by drying the lipids onto the interior of a flask for 30 min, followed by hydration in buffer and extrusion 11 times through 30 nm pores (1 bar). The I-BAR domain of IRSp53 was produced as described previously (Saarikangas et al., 2009 (link)). Standard chemicals for buffer preparation were from Sigma. NeutrAvidin was from ThermoFisher.
Nanowells were prepared as described previously (Junesch et al., 2012 (link), 2015 (link); Malekian et al., 2017 (link); Ferhan et al., 2018 (link)) using 107 nm polystyrene colloids on Nb₂O₅ and 158 nm on SiO₂ (Microparticles). Nanowells in SiO₂ were prepared on fused silica to enable direct etching of the solid support (Malekian et al., 2017 (link)). Nanowells in Nb₂O₅ were prepared on borosilicate glass (which cannot be easily etched) onto which Nb₂O₅ was first deposited by reactive sputter coating with O₂ and Ar (Junesch et al., 2012 (link)) (Nordiko). A 20 nm thick SiO₂ layer was deposited by plasma enhanced chemical vapor deposition (Surface Technology Systems). Recipes aiming for stochiometric SiO₂ or Si₃N₄ were used.
For bilayer formation with negative lipids, a 20 mM citric acid buffer was used with 150 mM KCl at a pH of 4.8. IRSp53 binding was performed in a buffer with 20 mM tris(hydroxymethyl) aminomethane and 150 mM NaCl with pH adjusted to 7.4 unless stated otherwise. The pH values were adjusted with concentrated HCl and NaOH.
The setup for extinction spectroscopy with high resolution and tracking of multiple resonance features has been described previously (Junesch et al., 2015 (link); Ferhan et al., 2018 (link)). Extinction is presented using the natural logarithm of the ratio between reference and measured intensities.

AFM cantilevers (PNP‐TR‐20, Nanoworld, Switzerland) were used to measure neutravidin‐biotin binding forces. Prior to measurements, cantilevers were functionalized with PEG‐biotin (QBD10200, Sigma‐Aldrich, MO, USA)^[82
] and treated using oxygen plasma at 600 mTorr and 100 W for 10 min. For forming amino groups, cantilevers were immersed in a 5% APTMS and absolute ethanol solution for 12 h. After rinsing with toluene, cantilevers were incubated in 1 mg ml⁻¹ PEG‐biotin in chloroform with 0.5% trimethylamine catalysts for 2 h. Neutravidin‐biotin binding forces were measured using an AFM (NX‐10, Park systems) under 0.1 µm ⁻¹s in a PBS more than 50 points.

The solution in the 1% Eu bead (Thermo Fisher Scientific, Waltham, MA) stock was replaced with 500 μl of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer via three rounds of centrifugation at 15,000g for 15 min. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were dissolved at a concentration of 15 and 50 mg/ml in 0.1 M MES (DCN, Carlsbad, CA) at pH 6 before use. MES (0.1 M) at pH 6 was added to the Eu bead solution followed by the addition of 5 μl of EDC and 100 μl of NHS. The reaction mixture was rotated on an orbital shaker with 500 rpm for 30 min at room temperature. The mixture was further purified with three rounds of centrifugation at 15,000g for 15 min. After removing the supernatant, 200 μl of 200 mM phosphate buffer and 250 μl of 1 mg/ml Neutravidin (Thermo Fisher Scientific, Waltham, MA) in 10 mM phosphate buffer at pH 7.2 (DCN, Carlsbad, CA) were added. The solution was mixed on an orbital shaker (500 rpm) for 3 hours at room temperature, followed by the addition of 5 μl of 1 M ethanolamine (TCI America, Portland, OR) and continue to shake overnight. The Eu-Neutravidin label was then resuspended in 50 mM tris, 1% casein, 5 mM EDTA, 0.2% Tween 20 of pH 8.0 (DCN, Carlsbad, CA) via three rounds of centrifugation at 15,000g for 15 min. This allowed the blocking of Eu-Neutravidin label with casein.