ATP-binding site of annexin VI characterized by photochemical release of nucleotide and infrared difference spectroscopy

Structural changes induced by nucleotide binding to porcine liver annexin V I (AnxVI) were probed by reaction-induced difference spectroscopy (RIDS). P hotorelease of the nucleotide from ATP[Et(PhNO2)] produced RIDS of AnxVI ch aracterized 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 Ca2+-ATPase and creatine and arginine kinases. Analysis of RIDS rev ealed the existence of ATP-binding site(s) (K-d < 1 mu M) within the AnxVI molecule, comprising five to six amino acid residues located in the C-termi nal portion of the protein molecule. The binding stoichiometry of ATP: AnxV I was determined as 1:1 (mol/mol). ATP, in the presence of Ca2+, induced ch anges in protein secondary structure reflected by a 5% decrease in alpha-he lix content of the protein in favor of unordered structure. Such changes ma y influence the affinity of AnxVI for Ca2+ and modulate its interaction wit h membranes. (C) 1999 Academic Press.