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NMR studies of calcium-binding to mutant alpha-spectrin EF-hands

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Buevich A. V., Lundberg S., Sethson I., Edlund U., Backman L.

Cellular and Molecular Biology Letters

2004

tom 09

nr 1

The co-operative calcium binding mechanism of the two C-terminal EF-hands of human αII-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 α-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 (~2470 μM and ~240 μM, 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.

Ograniczanie wyników

1 Cellular and Molecular Biology Letters

1 Backman L.

1 Buevich A. V.

1 Edlund U.

1 Lundberg S.

1 Sethson I.

1 2004

Wyniki wyszukiwania

NMR studies of calcium-binding to mutant alpha-spectrin EF-hands