Crystals of P2 myelin protein in lipid-bound form Jan Sedzik, a,b,1 Giulia Carlone, a Anna Fasano, a Grazia Maria Liuzzi, a andPaoloRiccio c, * a Department of Biochemistry and Molecular Biology, Bari University, Bari, Italy b Institute for Protein Research, Osaka University, Osaka, Japan c Department of Biology, D.B.A.F., University of Basilicata, Potenza, Italy Received 12 November 2002, and in revised form 27 January 2003 Abstract The P2 protein of peripheral nervous system myelin induces experimental allergic neuritis in rats, a model of Guillain–Barr e syndrome in humans. Previous purification procedures have used acid extraction to obtain the protein in lipid-free form (LF-P2). Here, we have purified the P2 protein in lipid-bound form (LB-P2) by extracting myelin with the detergent CHAPS, followed by Cu 2þ -affinity column chromatography. All myelin lipids were present in the preparation as shown by high-performance thin-layer chromatography and mass spectrometry. The LB-P2 preparation, which differs from LF-P2 in solubility and in the secondary- structure composition, was dialyzed to remove unbound lipids and excess detergent and crystallized using the hanging-drop vapor diffusion technique. Crystals of lipid-bound P2 appeared usually very reproducibly within 2 weeks at pH 5.7 in polyethylene glycol 6000 (PEG6000) at concentrations of 20–30% (w/v), and larger crystals were obtained by additional sitting-drop crystallization. X- raydiffractionshowedreflectionsupto2.7  A.Thecrystallizationconditions(25–30%PEG6000,pH5.0)andtheunitcelldimensions (a ¼ 94:5  A, b ¼ 94:5  A, c ¼ 74:2  A, a ¼ b ¼ 90°, c ¼ 120°) of LB-P2 were different from those earlier described for LF-P2 (10% PEG4000, pH 3, and unit cell dimensions a ¼ 91:8  A, b ¼ 99:5  A, c ¼ 56:5  A, a ¼ b ¼ c ¼ 90:0°). It is important that P2 has been crystallized with specifically bound lipids; therefore, solving this new crystal structure will reveal details of this proteinÕs behavior and role in the myelin sheath. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Protein P2; Myelin; Crystallization 1. Introduction The P2 protein of peripheral nervous system (PNS) myelinisasmallbasicproteinwithamolecularmassof 14.8kDa. The protein has been sequenced: it has 146 amino acid residues and its pI is 9.6 (SWISS-PROT Accession No. P02690) (Kitamura et al., 1980). P2 protein is expressed in Schwann cells and is located on thecytoplasmicsideofcompactmyelin,beingincontact with the membrane leaflets from two successive turns of myelinaroundtheaxon.Itconstitutesupto15%oftotal myelin proteins in PNS nerves of different species (Greenfield et al., 1973). The function of P2 has not yet beenestablished,butitisknownthatithasneuritogenic properties and can induce experimental allergic neuritis (EAN) in Lewis rats, providing an animal model of Guillain–Barr e syndrome, an immuno-mediated PNS injury in humans (Cavaletti et al., 2000; Whitaker, 1981). P2 protein is a member of a family of fatty acid- binding proteins (FABPs) (Martenson and Uyemura, 1992). In vitro binding studies have indicated high af- finity for oleic acid, retinoic acid, and retinol (Uyemura et al., 1984). To date, at least three different protocols for extrac- tion of P2 protein from PNS myelin have been pub- lished: (1) extraction at low pH (Kadlubowski et al., 1980;Sedziketal.,1988),(2)extractionwithhighsaltat or above neutral pH (Riccio et al., 1998), and (3) extraction from the membrane with detergents (Riccio et al., 1998). Journal of Structural Biology 142 (2003) 292–300 www.elsevier.com/locate/yjsbi Journal of Structural Biology * Corresponding author. Fax: +39-0971-20-5687. E-mail addresses: sedzik@swipnet.se (J. Sedzik), riccio@unibas.it (P. Riccio). URL: http://www2.unibas.it/utenti/riccio. 1 Present address: Department of Biosciences at NOVUM, Karo- linska Institutet, H€ alsov€ agen 7, 141-57 Huddinge, Sweden. 1047-8477/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1047-8477(03)00031-5