Abstract
We used cysteine scanning, isothermal titration calorimetry (ITC) and spin label EPR methods to study the two regions that flank the partial domain Helix C of the N-terminal end of α-spectrin (residues 14-20 and residues 44-54) in the absence and presence of a model protein of the β-spectrin C-terminal end. In the absence of β-spectrin, residues 14-20 and 46-52 were known to be unstructured. The EPR spectral values of the inverse line width (ΔH-1) and of the width between the low field peak and the central peak (aZ) of residues in part of the first unstructured region (residues 17-20) and of most residues in the second unstructured junction region (residues 46-52) changed dramatically upon association with β-spectrin, suggesting that the two regions undergo a conformational change, becoming more rigid and likely becoming helical. ITC results showed that three of the seven residues in the junction region (residues 46-52) were very important in its association with β-spectrin, in the following order: L49 > G46 > K48. In general, our results suggest that any mutations that affect the propensity of helical formation in the region spanning residues 17-52 in α-spectrin, or that affect hydrophobic clustering and/or salt-bridge stabilization of the bundled helices, would affect spectrin tetramer formation, and may lead to blood disorders.
Original language | English |
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Pages (from-to) | 10765-10772 |
Number of pages | 8 |
Journal | Biochemistry |
Volume | 47 |
Issue number | 40 |
DOIs | |
Publication status | Published - 7 Oct 2008 |