Near-UV circular dichroism of band 3. Evidence for intradomain conformational changes and interdomain interactions.
Near-UV circular dichroism (CD) was used to identify differences in the tertiary structure of human erythrocyte band 3, the chloride/bicarbonate exchange protein, consequent to covalent binding of anion transport inhibitors to the intramonomeric stilbenedisulfonate (ISD) site. Isolated intact band 3 and its membrane domain (B3MD) were compared. Spectral differences were observed which involved intradomain effects, in that they were seen both with intact band 3 and with B3MD, or interdomain effects, in that they were observed only for B3MD, but were inhibited when the cytoplasmic domain was attached. The intradomain effect involved a significant loss in optical activity in the Phe/Tyr region of the spectrum below 280 nm. It was seen only when the ISD site had stilbenedisulfonates bound covalently at pH 7.4. Raising the pH to 9.6 after adduct formation "normalized" this spectral change irreversibly. The interdomain effect was identified in the Trp spectral region at 292 nm. There was a significant increase in optical activity at 292 nm when bulky covalent ligands such as DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) were bound to B3MD, but not when the same ligands were bound to intact band 3. These latter results offer evidence that certain aspects of the conformational response of the integral domain are inhibited by the presence of an attached cytoplasmic domain. The potential significance of interdomain interactions to band 3 function is discussed briefly.
Batenjany
M M
MM
Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202.
Mizukami
H
H
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; D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D006801:Humans; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet; D013267:Stilbenes / Q000737:chemistry
D001457:Anion Exchange Protein 1, Erythrocyte; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid