Crystals of P2 myelin protein in lipid-bound form.
The P2 protein of peripheral nervous system myelin induces experimental allergic neuritis in rats, a model of Guillain-Barré 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(2+)-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-ray diffraction showed reflections up to 2.7A. The crystallization conditions (25-30% PEG6000, pH 5.0) and the unit cell dimensions (a = 94.5A, b = 94.5A, c=74.2A, alpha = beta = 90 degrees, gamma = 120 degrees ) of LB-P2 were different from those earlier described for LF-P2 (10% PEG4000, pH 3, and unit cell dimensions a = 91.8A, b = 99.5A, c = 56.5A, alpha = beta = gamma = 90.0 degrees ). It is important that P2 has been crystallized with specifically bound lipids; therefore, solving this new crystal structure will reveal details of this protein's behavior and role in the myelin sheath.
Sedzik
Jan
J
Department of Biochemistry and Molecular Biology, Bari University, Bari, Italy. sedzik@swipnet.se
Carlone
Giulia
G
Fasano
Anna
A
Liuzzi
Grazia Maria
GM
Riccio
Paolo
P
D000818:Animals; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray; D006639:Histidine; D008055:Lipids / Q000737:chemistry* / Q000302:isolation & purification; D021962:Membrane Microdomains / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D018996:Myelin P2 Protein / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization; D013126:Spinal Nerve Roots / Q000737:chemistry
D008055:Lipids; D008565:Membrane Proteins; D018996:Myelin P2 Protein; D006639:Histidine