Rotational diffusion and self-association of band 3 in reconstituted lipid vesicles

Abstract

Band 3, the anion transporter of the human erythrocyte membrane, has been purified and reconstituted into phospholipid vesicles of varying composition. Rotational diffusion of band 3 in these vesicles was measured by observing flash-induced transient dichroism of an eosin triplet probe covalently bound to the protein. In egg phosphatidylcholine vesicles of high lipid/protein ratio at temperatures well above the gel to liquid-crystalline phase transition, the absorption anisotropy decays to a constant value of 12 +/- 1% (expressed as a percentage of the initial anisotropy). However, higher values of the residual anisotropy and a slower decay are observed upon decreasing the temperature, even though the lipids remain in a liquid-crystalline phase. A similar effect is observed upon decreasing the lipid/protein ratio at constant temperature. It is concluded that self-association of band 3 occurs which is dependent on temperature and protein concentration in the bilayer. It is very probable that similar effects occur in the erythrocyte membrane, where a strong temperature dependence of band 3 rotational mobility is also observed. Reconstitution of band 3 into dioleoylphosphatidylcholine vesicles yields results similar to those obtained with egg phosphatidylcholine. When dimyristoylphosphatidylcholine is used for reconstitution, band 3 is immobilized below the lipid phase transition and remains partially associated above the transition. A precise analysis of the anisotropy decay curves is hampered by the presence of multiple rotating species.(ABSTRACT TRUNCATED AT 250 WORDS)