Evaluation of Structural Interdependence of Membrane-Spanning and Cytoplasmic Domains of Band 3

The structural interdependence of the membrane-spanning (55000 dalton) and cytoplasmic (40 000 dalton) domains of the erythrocyte membrane protein band 3 was investigated. The influence of the membrane-spanning domain on the behavior of the cytoplasmic domain was initially examined by comparing the structural properties of the cytoplasmic domain in situ with its properties in isolated form, i.e., after proteolytic removal from the membrane. The previously described calorimetric and fluorescence properties of the isolated cytoplasmic domain [Appell, K. C., & Low, P. S. (1981) J. Biol. Chem. 256, 11104-11111] were used as the basis for this comparison. The pH titration of the domain's intrinsic fluorescence was essentially identical before and after cleavage from the membrane. The thermal denaturation temperatures (T_m) of the uncleaved and the isolated cytoplasmic domains were also very similar, decreasing more than 15°C between pH 6 and pH 8.5. However, the cytoplasmic domain in situ was slightly less stable at all pHs than its isolated counterpart. In contrast, the thermal stability of the membrane-spanning domain of band 3 was essentially unaltered by proteolytic removal of the cytoplasmic domain. These observations suggest that the structures of the membrane-spanning and the cytoplasmic domains of band 3 are not significantly altered by separation after proteolytic cleavage. The effect of denaturation of one domain of band 3 on the thermal stability of the other was also investigated. At pH 6.5, the integral domain denatures at a lower temperature than the cytoplasmic domain. However, in membranes prepared from 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-labeled cells, the integral domain is stabilized so that it unfolds only after the thermal transition of the cytoplasmic domain. Regardless of whether the integral domain is native or denatured at the onset of thermal unfolding of the cytoplasmic domain, the calorimetric behavior of the latter remains unaltered. Furthermore, by choosing the appropriate membrane stripping procedure (i.e., NaOH or acetic acid), it is possible to selectively denature one of the two major domains of band 3 without significantly perturbing the other. Taken together, the above observations suggest that the extent of interaction between the two major domains of band 3 is minimal.