Conformation and stability of the anion transport protein of human erythrocyte membranes

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Abstract
The conformation and stability of purified preparations of band 3, the anion transport protein of human erythrocyte membranes, and its constituent proteolytic subfragments were studied by circular dichroism. Band 3, purified in the presence of the nonionic detergent n-dodecyl octaethylene glycol monoether (C12E8), had an .alpha.-helical content of 46%. Denaturation of purified band 3 with guanidine hydrochloride occurred in 2 phases, one reflecting much more resistance to denaturation than the other. Band 3 can be separated into 2 domains by limited in situ proteolytic cleavage. The carboxyl-terminal membrane-associated domain (MW 55,000) purified in C12E8 contained 58% .alpha.-helix and was very resistant to denaturation by guanidine hydrochloride. The purified amino-terminal, cytoplasmic domain (MW 41,000) contained 27% .alpha.-helix and was completely converted to a random-coil conformation by 3 M guanidine hydrochloride. The 2 phases of denaturation observed for intact band 3 corresponded to the 2 domains of the protein. Irreversible heat denaturation of purified band 3 occurred with half-maximal change in .theta.222.5 at 48.degree. C. Covalent attachment of the anion transport inhibitor 4,4''-diisothiocyanostilbene-2,2''-disulfonate to band 3 had little effect on the circular dichroism spectra of band 3 or the membrane-associated domain but resulted in stabilization of band 3 to heat denaturation (half-maximal change in .theta.222.5 = 61.degree. C). Circular dichroism studies of membranes that had been digested extensively with proteolytic enzymes and stripped of all extrinsic fragments revealed that the portions of red cell membrane proteins that are embedded in the lipid bilayer contain a very high (86-94%) content of .alpha.-helix.