Dissection of Calbindin D9k into two Ca2+-binding subdomains by a combination of mutagenesis and chemical cleavage

Abstract

Calbindin D9k is a 75-residue globular protein made up of two Ca2+ binding subdomains of the EF-hand type. In order to examine the subdomains independently, a method was devised to selectively cleave the loop between them. Using site-directed mutagenesis, a unique methionine was substituted for Pro43 in the loop, thus allowing cleavage using cyanogen bromide. Agarose gel electrophoresis shows that the fragments have a high affinity for one another, although less so in the absence of calcium. H-1-NMR spectra of the fragments indicate that the structures of the heterodimers are changed little from that of the intact protein. However, the Ca2+ binding constants of the individual subdomains are several orders of magnitude lower than for the corresponding sites in the uncleaved protein.

Keywords

NMR; SITE-DIRECTED MUTAGENESIS, CYANOGEN BROMIDE, CALBINDIN-D(9K)

Published in

FEBS Letters
1992, volume: 298, number: 2-3, pages: 211-214

SLU Authors

• Finn, Bryan

  • Lund University

UKÄ Subject classification

Physical Chemistry
Biochemistry and Molecular Biology

Publication identifier

• DOI: https://doi.org/10.1016/0014-5793(92)80059-P

Permanent link to this page (URI)

https://res.slu.se/id/publ/132602