Cholestane spin label

DMPC (14:0–14:0 PC), SOPC (18:0–18:1 PC), DOPC (18:1–18:1 PC), PAPC (16:0–20:4 PC), and Cholesterol (Chol) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The cholesterol analog cholestane spin-label (CSL) and bovine eye lens α-crystallin were purchased from Sigma Aldrich (St. Louis, MO, USA), where α-crystallin was used without further purification. Based on the information (αA = 19.8 kDa, αB = 22 kDa, and αA:αB = 3:1), the average molecular weight for the α-crystallin subunit was estimated to be 20.35 kDa. Preparation of membranes, α-crystallin, and α-crystallin membrane association studies was performed in HEPES buffer (10 mM HEPES, 100 mM NaCl, pH = 7.4).

Cholesterol (Chol), sphingomyelin (SM), and phospholipids (PLs): 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylCholine (POPC), 1-palmditoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), were obtained dissolved in chloroform from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Cholesterol analog cholestane spin label (CSL), HEPES, Tris-HCl, NaN₃, sodium chloride (NaCl) lysozyme, deoxycholic acid, and DNase I were obtained from Sigma Aldrich (St. Louis, MO, USA). Isopropyl-1-thio-β-D-galactopyranoside (IPTG), ampicillin, phenylmethylsulfonyl fluoride, and polyethyleneimine were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Recombinant human αAc and αBc were expressed and purified, and the reconstituted 3:1 heteromeric complex of αAc to αBc (i.e., αABc) was prepared using the methods described in Section 4.2 and stored in HEPES buffer (10 mM HEPES, 100 mM NaCl, pH = 7.4). All preparations of α-crystallin (αAc, αBc, and αABc) and the Chol/MHLL membranes, as well as associated binding studies, were performed in HEPES buffer (10 mM HEPES, 100 mM NaCl, pH = 7.4).