Conformational studies of the tetramerization site of human erythroid spectrin by cysteine-scanning spin-labeling EPR methods

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We used cysteine-scanning and spin-labeling methods to prepare singly spin labeled recombinant peptides for electron paramagnetic resonance studies of the partial domain regions at the tetramerization site (N-terminal end of α and C-terminal end of β) of erythroid spectrin. The values of the inverse line width parameter (ΔH0-1) from a family of SpoI-1-368Δ peptides scanning residues 21-30 exhibited a periodicity of ∼3.5-4. We used molecular dynamics calculations to show that the asymmetric mobility of this helix is not necessarily due to tertiary contacts, but is likely due to intrinsic properties of helix C′, a helix with a heptad pattern sequence. The residues with low ΔH0-1 values (residues at positions 21, 25, and 28/29) were those on the hydrophobic side of this amphipathic helix. Native gel electrophoresis results showed that these residues were functionally important and are involved in the tetramerization process. Thus, EPR results readily identified functionally important residues in the α spectrin partial domain region. Mutations at these positions may lead to clinical symptoms. Similarly, the ΔH0-1 values from a family of spin-labeled SpβI-1898-2083Δ peptides also exhibited a periodicity of ∼3.5-4, indicating a helical conformation in the two scanned regions (residues 2008-2018 and residues 2060-2070). However, the region consisting of residues 2071-2076 was in a disordered conformation. Both helical regions include a hydrophilic side with high ΔH0-1 values and a hydrophobic side with low ΔH0-1 values, demonstrating the amphipathic nature of the helical regions. Residues 2008, 2011, 2014, and 2018 in the first scanned region and residues 2061, 2065, and 2068 in the second scanned region were on the hydrophobic side. These residues were critical in αβ spectrin association at the tetramerization site. Mutations at some of these positions have been reported to be detrimental in clinical studies. © 2005 American Chemical Society.

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