Detergent interaction with band 3, a model polytopic membrane protein.
The interaction of band 3, the 95-kDa anion-exchange protein of the human erythrocyte membrane, with a variety of nonionic detergents was studied. Band 3 dimers (Stokes radius = 76 A) prepared in octaethylene glycol monododecyl ether (C12E8) could be exchanged into a variety of detergents by size-exclusion high-performance liquid chromatography (HPLC), with complete removal of C12E8 from band 3 being confirmed using radiolabeled detergent. Critical micellar concentration (cmc) values, determined for all detergents in the buffer used for HPLC analysis, ranged from 0.47 microM to 223 mM. Band 3 was found to aggregate in all detergents below their cmc, and concentrations of detergents 2-200 times the cmc were required to prevent aggregation. For detergents with a low cmc, it was important to ensure that the concentration of detergent micelles minimally equalled the concentration of protein. Hydrodynamic measurements and cross-linking studies showed that band 3 remained dimeric in most detergents above their cmc. Furthermore, circular dichroism and inhibitor binding studies supported the view that band 3 can retain its native structure after detergent exchange. Detergents with short alkyl chains (C8) denature band 3, while detergents with longer alkyl chains (C12) maintained the native structure of band 3. The ability to exchange band 3 into a variety of detergents with the maintenance of native structure is an essential prerequisite for crystallization trials. The results obtained in this study of band 3, a model polytopic (multispanning) membrane protein, may be generally applicable to other membrane proteins.
Casey
J R
JR
Department of Medicine, University of Toronto, Ontario, Canada.
Reithmeier
R A
RA
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry*; D006801:Humans
D001457:Anion Exchange Protein 1, Erythrocyte; D003902:Detergents