Crystals of P2 myelin protein in lipid-bound form.

Abstract

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.

• Authors: Jan Sedzik, Giulia Carlone, Anna Fasano, Grazia Maria Liuzzi, Paolo Riccio
• Journal: Journal of structural biology (J Struct Biol) Vol. 142 Issue 2 Pg. 292-300 (May 2003) ISSN: 1047-8477 [Print] United States
• PMID: 12713957 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Lipids
• Membrane Proteins
• Myelin P2 Protein
• Histidine

Topics

• Animals
• Crystallization (methods)
• Crystallography, X-Ray
• Histidine
• Lipids (chemistry, isolation & purification)
• Membrane Microdomains (chemistry)
• Membrane Proteins (chemistry)
• Myelin P2 Protein (chemistry, isolation & purification)
• Protein Binding
• Spectrometry, Mass, Electrospray Ionization
• Spinal Nerve Roots (chemistry)