Factors determining the conformation and quaternary structure of isolated human erythrocyte band 3 in detergent solution.
Fluorescence spectroscopy was used to follow the kinetics of covalent binding of DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) to isolated band 3 in C12E8. We have discovered a dilution-induced loss in the ability of band 3 monomer to form a covalent adduct with DIDS. The loss in DIDS reactivity with dilution followed a 50:50 biphasic time course despite the use of a homogeneous preparation of band 3 oligomers. The loss in reactivity generally correlated with the association of band 3 dimers and tetramers to higher oligomeric structures. The final aggregated product was capable of binding BADS (4-benzamido-4'-amino-2,2'-stilbenedisulfonate) reversibly, but with an affinity nearly 30-fold lower than that of the starting material. Removal of the cytoplasmic domain of band 3 slowed the conformational interconversion of the integral domain by about 5-fold and inhibited the aggregation process. The conformational interconversion was slowed in the presence of 150 mM chloride but not in 90 mM sulfate. Covalent binding of DIDS inhibited the aggregation of band 3. Addition of 250 microM lipid inhibited both the loss of DIDS reactivity and the protein aggregation process. While several types of lipid offer protection, phosphatidic acid accelerated the decay process by eliminating the biphasicity. We conclude that the conformation of the integral domain of band 3 can be modulated allosterically by the addition of ligands, including various lipids. The results offer direct evidence for cooperative interactions between band 3 subunits during loss of activity, and they show that the cytoplasmic domain participates in the control of this transition.
Schopfer
L M
LM
Veterans Administration Medical Center, Omaha, Nebraska 68105.
Salhany
J M
JM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D005454:Fluorescence Polarization; D006801:Humans; D010712:Phosphatidic Acids / Q000737:chemistry; D011487:Protein Conformation; D012996:Solutions
D001457:Anion Exchange Protein 1, Erythrocyte; D003902:Detergents; D010712:Phosphatidic Acids; D012996:Solutions; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid