Alanine-scanning mutagenesis identifies the critical residues within the ankyrin-binding domain of erythroid βI-spectrin. (A) Sequence alignment of the ankyrin-binding region of βI spectrin²³  (codons 1768-1898) with βII, βIII, and βIV spectrin. The red arrows above the alignment labeled KP (Kennedy peptide) represent the limits of ankyrin-binding activity previously identified, ie, deletion of sequences outside this region did not perturb ankyrin-binding activity.²³  The histogram summarizes the strength of homology among these spectrins; red, conserved in all 4 spectrins; green conserved in 3 of 4; light blue, conserved in 2 of 4; and dark blue, not conserved. The putative structural feature of each region of sequence also is depicted. Arrows below the alignment indicate the position of each of the 42 mutants examined in this study. Red arrows highlight amino acids that abrogated ankyrin binding; the length of the arrow approximates the inhibitory potency of the mutation. Black arrows indicate amino acid changes that had no effect. All amino acids mutations were to alanine unless otherwise indicated by the bold letters. The W1779R mutant explored the effect of deletion of the highly conserved trp at that locus; L1812W replaced the “invariant” trp that characterizes most spectrin repreats; L1848H restored the H that is conserved in other spectrin repeats; A1867P was designed to mimic the pro at this position in βV spectrin (sequence not shown); and A1884V explored the impact of the Saõ Paolo mutation. (B) Western blot using anti-HIS antibody to detect his-ankR (top) and Coomassie blue stained SDS-PAGE (bottom) show the results of a single pull-down assay of 17 representative peptides. The first lane is the wt βI-14,15 peptide; lane 2 is αI-14,15 spectrin peptide that does not bind ankyrin. The next 12 lanes represent mutations that diminished ankyrin binding; their position is as labeled. Lane 15 is representative of an alanine substitution that has no effect on ankyrin binding; the last 2 lanes are the 2 peptides incorporating tryptophan at position 1812 and histidine at position 1848. (C) CD and thermal-melt analysis of selected peptides. The panel on the left shows the CD analysis of 3 peptides. The wt peptide in blue is nearly super-imposable with L1793A (red), a mutant peptide that abrogates ankyrin binding, and the Trp/His double mutant (black) that binds ankyrin normally. Each of these peptides had a calculated α-helix content of ≈70%. Thermal melt analysis (right panel) of the same peptides reveals a loss of cooperative unfolding of the L1793A mutant. Other peptides selected for CD and thermal melt analysis were similar to the wt peptide.