Previous studies have shown that a single P327R point mutation in the cytoplasmic domain of band 3 (cdb3) protein, known as band 3 Tuscaloosa, leads to a reduction in protein 4.2 content of the erythrocyte membrane and hemolytic anemia. Recent studies have shown that this point mutation does not dissociate the cdb3 dimer, nor does it lead to large-scale rearrangement of the protein structure (Bustos, S. P., and Reithmeier, R. A. F. (2006) Biochemistry 45, 1026-1034). To better define the structural changes in cdb3 that lead to the hemolytic anemia phenotype, site-directed spin labeling (SDSL), in combination with continuous wave electron paramagnetic resonance (EPR) and pulsed double electron-electron resonance (DEER) spectroscopies, has been employed in this study to compare the structure of the R327 variant with wild type P327 cdb3. It is confirmed that the P327R mutation does not dissociate the cdb3 dimer, nor does it change the spatial orientation of the two peripheral domains relative to the dimer interface. However, it does affect the packing of the C-terminal end of helix 10 of the dimerization arms in a subpopulation of cdb3 dimers, it leads to spectral changes at some residues in beta-strand 11 and in the N-terminal end of helix10, and it produces measurable spectral changes at other residues that are near the mutation site. The data indicate that the structural changes are subtle and are localized to one surface of the cdb3 dimer. The spectroscopic description of structural features of the P327R variant provides important clues about the location of one potential protein 4.2 binding surface on cdb3 as well as new insight into the structural basis of the membrane destabilization.