Purified lens major intrinsic protein (MIP) forms highly ordered tetragonal two-dimensional arrays by reconstitution

被引:63
作者
Hasler, L
Walz, T
Tittmann, P
Gross, H
Kistler, J
Engel, A
机构
[1] Univ Basel, Bioctr, ME Muller Inst Microscopy, CH-4056 Basel, Switzerland
[2] Univ Sheffield, Krebs Inst Biomolec Res, Dept Mol Biol & Biotechnol, Sheffield S10 2TN, S Yorkshire, England
[3] Swiss Fed Inst Technol, Inst Cell Biol, CH-8093 Zurich, Switzerland
[4] Univ Auckland, Sch Biol Sci, Auckland 1, New Zealand
关键词
major intrinsic protein; membrane protein; 2D crystallization; electron crystallography; STEM;
D O I
10.1006/jmbi.1998.1796
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Lens major intrinsic protein (MIP) is the founding member of the MIP family of membrane channel proteins. Its isolation from ovine lens fibre cell membranes and its two-dimensional crystallization are described. Membranes were solubilized with n-octyl-beta-D-glucoside and proteins fractionated by sucrose gradient centrifugation containing decyl-beta-D-maltoside. MIP was purified by cation exchange chromatography, and homogeneity was assessed by mass analysis in the scanning transmission electron microscope. Purified MIP reconstituted into a lipid bilayer at a low lipid-to-protein ratio formed highly ordered tetragonal two-dimensional crystals. The square unit cell had a side length of 6.4 nm, and exhibited in negative stain four stain-excluding elongated domains surrounding a central stain-filled depression. Projection maps of freeze-dried crystals exhibited a resolution of 9 Angstrom, and revealed a monomer structure of MIP consisting of distinct densities. Despite significant differences in the packing of tetramers in the crystals, the projection map of the MIP monomer was similar to that of aquaporin-1 (AQP1), the first member of the MIP family which had its structure resolved to 6 Angstrom. Our protocols for the purification and reconstitution of MIP establish the feasibility for future work to visualize structure elements which determine the diverse functional properties of the MIP family members. (C) 1998 Academic Press Limited.
引用
收藏
页码:855 / 864
页数:10
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