title: Dissection of calbindin D9k into two Ca(2+)-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. 1H-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.
authors.affiliation: Department of Physical Chemistry 2, University of Lund, Sweden.
authors.forename: B E
authors.initials: BE
authors.lastname: Finn
authors.forename: J
authors.initials: J
authors.lastname: Kördel
authors.forename: E
authors.initials: E
authors.lastname: Thulin
authors.forename: P
authors.initials: P
authors.lastname: Sellers
authors.forename: S
authors.initials: S
authors.lastname: Forsén
mesh_terms: D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D003488:Cyanogen Bromide / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D064026:Calbindins; D010446:Peptide Fragments; D064030:S100 Calcium Binding Protein G; D003488:Cyanogen Bromide; D002118:Calcium