Rotational mobility of an erythrocyte membrane integral protein band 3 in dimyristoylphosphatidylcholine reconstituted vesicles and effect of binding of cytoskeletal peripheral proteins

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Abstract
Band 3 protein was isolated from human erythrocyte membranes, purified and reconstituted into a well-defined phospholipid bilayer matrix (dimyristoylphosphatidylcholine). The preparation yielded uniform single-bilayered vesicles of the diameter 40-80 nm. The rotational motion and band 3 was studied by saturation transfer ESR spectroscopy of covalently attached maleimide spin-labels. The rotational mobility changed in response to the host lipid phase transition. The rotational correlation time was in a range from 73 (37.degree. C) to 94 .mu.s (26.degree. C) in the fluid phase and from 240 (15.degree. C) to 420 .mu.s (5.degree. C) in the solid phase. The motion was analyzed based on the anisotropic rotation of band 3 in the reconstituted vesicles. To obtain information on the rotational diffusion constant around the axis parallel to the membrane normal, an attempt was made to measure the angle between the spin-label magnetic axis and the membrane normal. The result gave 3.9 .times. 104 s-1 at 37.degree. C as a rough estimate for the diffusion constant. This is compatible to anisotropic rotation of a cylinder of radius 3.3 nm in a 2 dimensional with matrix with inner viscosity 2 P [poise] and inner thickness 4 nm. The cytoskeletal peripheral proteins caused a definite increase in the rotational correlation time (from 73-180 .mu.s at 37.degree. C, for example). The restriction of the rotational mobility was due to the ankyrin-linked interaction between band 3 and spectrin-actin-band 4.1 proteins in the reconstituted membranes.