N 1 pyrene maleimide

Bis(maleimido)hexane (BMH) and 1,11-bismaleimido-triethyleneglycol (BM(PEG)₃) were obtained from Thermo Scientific (Rockford, IL), N-(1-pyrene)maleimide (NPM), 5-((((2-iodoacetyl)amino)ethyl) amino)napthalene-1-sulfonic acid (IAEDANS) and tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) from Invitrogen (Molecular Probes, Eugene, OR), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol from Avanti Polar Lipids (Birmingham, AL), dithiothreitol (DTT) 99% purity from Acros Organics (Morris Plains, NJ), guanidine hydrochloride (GdnHCl) (99% purity) from Fisher Scientific (Fair Lawn, NJ). All solvents used were of analytical grade.

Rosetta 2 DE3 Escherichia coli were used for the expression of INF2-FFC. After breaking the cells and getting the supernatant, a GST (glutathione S-transferase) column was first used to purify INF2. ProTEV was used after that for cleavage of GST. A gel filtration column was then used to purify INF2. The purified protein was dialyzed vs Hepes buffer (10mM Hepes pH=7.4, 50mM KCl, 1mM MgCl₂, 1mM EGTA) and was stored at −81°C for further use. Rabbit skeletal muscle actin (RSA) was purified from acetone powder of muscles [20 (link)] and Mical oxidized actin (Mox-Actin) was generated as described by [18 (link)] N-(1-pyrene)-maleimide was obtained from Molecular Probes (Eugene, OR) or AnaSpec Inc. (San Jose, CA). RSA labeling with pyrene maleimide was carried out in thiol-free GB2 supplemented with 2 mM MgCl₂ and 100 mM KCl, at 1:2.5 (actin:dye) molar ratio, for 1 h on ice. The resulting pyrene-labeled F-actin was pelleted, depolymerized (GB2), and gel-filtered on Superdex S200 16/60 column [32 (link)]. Mical-oxidized RSA (Mox-actin) was prepared and purified according to the published protocol [18 (link)].