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

• Autorzy: Buevich A V , Lundberg S. , Sethson I. , Edlund U. , Backman L.
• Języki publikacji: EN

Abstrakty

EN

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.

Słowa kluczowe

EN

man; EF-hand; site-specific mutagenesis; alpha-spectrin; nuclear magnetic resonance

• Wydawca: -
• Czasopismo: Cellular and Molecular Biology Letters
• Rocznik: 2004
• Tom: 09
• Numer: 1
• Opis fizyczny: p.167-186,fig.,ref.

Twórcy

autor

Buevich A V

• Umea University, SE-901 87 Umea, Sweden

autor

Lundberg S.

autor

Sethson I.

autor

Edlund U.

autor

Backman L.

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