Structural changes induced by nucleotide binding to porcine liver annexin VI (AnxVI) were probed by reaction-induced difference spectroscopy (RIDS). Photorelease of the nucleotide from ATP[Et(PhNO2)] produced RIDS of AnxVI characterized by reproducible changes in the amide I region. The magnitude of the infrared change was comparable to RIDS of other ATP-binding proteins, such as Ca(2+)-ATPase and creatine and arginine kinases. Analysis of RIDS revealed the existence of ATP-binding site(s) (K(d) < 1 microM) within the AnxVI molecule, comprising five to six amino acid residues located in the C-terminal portion of the protein molecule. The binding stoichiometry of ATP:AnxVI was determined as 1:1 (mol/mol). ATP, in the presence of Ca2+, induced changes in protein secondary structure reflected by a 5% decrease in alpha-helix content of the protein in favor of unordered structure. Such changes may influence the affinity of AnxVI for Ca2+ and modulate its interaction with membranes.
Copyright 1999 Academic Press.