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Abstract

Limited proteolysis of the core domain of the 36-kDa protein p36 by trypsin gives a first insight into the structural organization of the four annexin repeats. Trypsin opens only a single peptide bond, situated between residues 204 and 205. The two fragments (of 20 kDa and 15 kDa), each containing two annexin repeats, remain as a tight complex (nicked core), which binds phospholipids in a Ca2(+)-dependent manner. After denaturation by 9 M urea, the nicked core is again formed upon renaturation provided both fragments are present. If the fragments are separated by chromatography in urea prior to renaturation, they show different behaviour. The 15-kDa C-terminal repeats aggregate, while the 20-kDa N-terminal repeats stay in solution. In comparison to p36, fragments with two (20-kDa fragment) or one (N-terminal CNBr fragment) annexin repeats show a conformational alteration in CD spectroscopy and hydrodynamics and display an increased susceptibility to proteases. In line with these differences, their Ca2(+)-dependent affinity to phospholipids is more than 10-20-fold decreased. Thus the four annexin repeats form together an integrated domain with multiple contacts between the repeats. Although stable derivatives with less than four repeats can be obtained, their Ca2+/phospholipid binding affinities are noticeably reduced.