Properties of normal and mutant polypeptide fragments from the dimer self-association sites of human red cell spectrin
| Autor: | J. C. Pinder, W. B. Gratzer, Gaël Nicolas, Didier Dhermy, Marie-Christine Lecomte |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Circular dichroism
Erythrocytes Protein Conformation Dimer Mutant Biophysics Biophysical Phenomena chemistry.chemical_compound Humans Point Mutation Spectrin chemistry.chemical_classification Binding Sites Chemistry Circular Dichroism General Medicine Peptide Fragments Amino acid Biochemistry Sedimentation equilibrium Helix Mutagenesis Site-Directed Dimerization Alpha helix |
| Zdroj: | European Biophysics Journal. 28:208-215 |
| ISSN: | 1432-1017 0175-7571 |
| DOI: | 10.1007/s002490050201 |
| Popis: | We have examined the properties and interactions of expressed polypeptide fragments from the N-terminus of the alpha-chain and the C-terminus of the beta-chain of human erythroid spectrin. Each polypeptide comprises one complete structural repeating unit, together with the incomplete repeat that interacts with its partner when spectrin tetramers are formed. The shared repeat thus generated is made up of two helices from the C-terminal part of the beta-chain and one helix from the N-terminus of the alpha-chain. Three mutant beta-chain fragments with amino acid substitutions in the incomplete terminal repeat were also studied. The alpha- and beta-chain fragments were both substantially monomeric, as shown by sedimentation equilibrium. Circular dichroism analysis and thermal denaturation profiles revealed that the complete repeat present in each fragment had entered the stable tertiary fold. Unexpectedly, the conformational stability of the folded beta-chain repeat was found to be grossly perturbed by the mutations, all of them well beyond its C-terminal boundary; possible explanations for this phenomenon are considered. Sedimentation equilibrium showed that in equimolar mixtures the wildtype alpha- and beta-chain peptides formed a 1:1 complex. Mixing curves, observed by circular dichroism, revealed that association was accompanied by an increase in alpha-helicity. From continuous-variation profiles an association constant in the range 1-2 x 10(6) M-1 was inferred. The association was unaffected by the apparently unstructured anionic tail of 54 residues, found at the C-terminus of the spectrin beta-chain. Of the three mutations in the beta-chain fragment, one (an Ala--Val replacement in the A helix segment of the incomplete repeat) had a relatively small effect on the association with the alpha-chain fragment, whereas Trp--Arg mutations in the A and in the remote B helix segments were much more deleterious. These observations are consistent with the relative severities of the haemolytic conditions associated with the mutations. |
| Databáze: | OpenAIRE |
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