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Zn2+ binding to human calbindin D(28k) and the role of histidine residues.

Författare

Summary, in English

We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn2+. The binding of Zn2+ is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.

Publiceringsår

2008

Språk

Engelska

Sidor

760-767

Publikation/Tidskrift/Serie

Protein Science

Volym

17

Issue

4

Dokumenttyp

Artikel i tidskrift

Förlag

The Protein Society

Ämne

  • Medicinal Chemistry

Nyckelord

  • histidine
  • EF-hand
  • zinc
  • mutagenesis

Status

Published

Forskningsgrupp

  • Clinical Chemistry, Malmö

ISBN/ISSN/Övrigt

  • ISSN: 1469-896X