Purified lens major intrinsic protein (MIP) forms highly ordered tetragonal two-dimensional arrays by reconstitution.
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 A, 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 A. 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.
Hasler
L
L
M. E. Müller-Institute for Microscopy at the Biozentrum, University of Basel, Basel, CH-4056, Switzerland.
Walz
T
T
Tittmann
P
P
Gross
H
H
Kistler
J
J
Engel
A
A
D000818:Animals; D020346:Aquaporins; D003461:Crystallography; D005136:Eye Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D008562:Membrane Glycoproteins; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D012756:Sheep
D020346:Aquaporins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; C030328:aquaporin 0