Blood, 1 July 2002, Vol. 100, No. 1, pp. 283-288
RED CELLS
Nuclear magnetic resonance studies of mutations at the
tetramerization region of human alpha spectrin
Sunghyouk Park,
Michael E. Johnson, and
Leslie W.-M. Fung
From the Center for Pharmaceutical Biotechnology,
University of Illinois at Chicago; and the Department of Chemistry,
Loyola University of Chicago, IL.
Many spectrin mutations that destabilize tetramer formation and
lead to hereditary hemolytic anemias are located at the N-terminal region of
-spectrin, with the Arg28 position considered to
be a mutation hot spot. We have introduced mutations at positions 28 and 45 into a model peptide, Sp
1-156, consisting of the first 156 residues in the N-terminal region of
-spectrin (
N). The association of these
-spectrin peptides that have single amino acid
replacements with a
-spectrin model peptide, consisting of the
C-terminal region of
-spectrin (
C), was determined, and structural changes due to amino acid replacements were monitored by
nuclear magnetic resonance (NMR). We found evidence for similar and
very localized structural changes in Sp
1-156Arg45Thr and Sp
1-156Arg45Ser, although these 2 mutant peptides associated with
-spectrin peptide with significantly differing affinities. The
Sp
1-156Arg28Ser peptide showed an affinity for the
-spectrin peptide comparable to that of Sp
1-156Arg45Ser, but it exhibited substantial and widespread spectral changes. Our results suggest that
both Arg45 replacements induce only minor structural perturbations in
the first helix of Sp
1-156, but the Arg28Ser replacement affects both the first helix and the following structural domain. Our results
also indicate that the mechanism for reduced spectrin tetramerization
is through mutation-induced changes in molecular recognition at the

-tetramerization site, rather than through conformational
disruption, as has been suggested in prior literature.