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 2.7 Å. The crystallization conditions (25–30% PEG6000, pH 5.0) and the unit cell dimensions ( a=94.5 A ̊ , b=94.5 A ̊ , c=74.2 A ̊ , α= β=90°, γ=120°) of LB-P2 were different from those earlier described for LF-P2 (10% PEG4000, pH 3, and unit cell dimensions a=91.8 A ̊ , b=99.5 A ̊ , c=56.5 A ̊ , α= β= γ=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 protein’s behavior and role in the myelin sheath.