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NMR studies of calcium-binding to mutant alpha-spectrin EF-hands
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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2004 (English)In: CELLULAR & MOLECULAR BIOLOGY LETTERS, ISSN 1425-8153, Vol. 9, no 1, 167-86 p.Article in journal (Refereed) Published
Abstract [en]

The co-operative calcium binding mechanism of the two C-terminal EF-hands of human all-spectrin has been investigated by site-specific mutagenesis and multi-dimensional NMR spectroscopy. To analyse the calcium binding of each EF-hand independently, two mutant structures (E33A and D69S) of wild type alpha-spectrin were prepared. According to NMR analysis both E33A and D69S were properly folded. The unmutated EF-hand in these mutants remained nearly intact and active in calcium binding, whereas the mutated EF-hand lost its affinity for calcium completely. The apparent calcium binding affinity of the E33A mutant was much lower compared to the D39S mutant (similar to2470 muM and similar to240 muM, respectively). When the chemical shift perturbations were followed upon calcium titration, a positive correlation between the D69S mutant and the binding of the first calcium ion to the wild type was revealed. These observations showed that the first EF-hand in spectrin binds the first calcium ion and thereby triggers a conformational change that allows the second calcium ion to bind to the other EF-hand.

Place, publisher, year, edition, pages
2004. Vol. 9, no 1, 167-86 p.
Keyword [en]
EF-hand, site-specific mutagenesis, multidimensional NMR
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-14274OAI: oai:DiVA.org:umu-14274DiVA: diva2:153945
Available from: 2007-05-28 Created: 2007-05-28 Last updated: 2017-10-16Bibliographically approved

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