Anion exchanger 1 (AE1, Band 3) is the predominant
membrane protein of erythrocytes. Its 52 kDa C-terminal domain functions as a
chloride-bicarbonate exchanger, while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the cytoskeleton to the membrane. Several
proteins bind to cdb3, including
protein 4.2, a cytoskeletal
protein. Three mutations in cdb3 are associated with
hereditary spherocytosis (HS) and decreased levels of
protein 4.2 in erythrocytes. In this study, these cdb3 mutants (E40K, G130R, and P327R) were expressed in and purified from Escherichia coli. Sedimentation experiments showed that the wild-type and
mutant proteins are dimers. No difference in secondary structure between mutant and wild-type
proteins was detected using circular dichroism (CD) analysis. The wild-type and
mutant proteins underwent similar pH-dependent conformational changes when monitored by intrinsic
tryptophan fluorescence.
Urea denaturation of
proteins monitored by intrinsic fluorescence showed no significant differences in the sensitivity of the
proteins to this chemical denaturant. Thermal denaturation monitored by CD and by calorimetry revealed that only the P327R mutant had a significantly lower midpoint of transition (approximately 5 degrees C) than the wild-type
protein, suggesting a modest decrease in stability. The results show that the HS mutant cdb3
proteins do not differ to any great extent in structure from the wild-type
protein, suggesting that the HS mutations may directly affect
protein 4.2 binding.