Atomic mutations at the single tryptophan residue of human recombinant annexin V: Effects on structure, stability, and activity

The single tryptophan residue (Trp187) of human recombinant annexin V, cont aining 320 residues and 5328 atoms, was replaced with three different isost eric analogues where hydrogen atoms at positions 4, 5, and 6 in the indole ring were exchanged with fluorine. Such single atom exchanges of H --> F re present atomic mutations that result in slightly increased covalent bond le ngths and inverted polarities in the residue side-chain structure. These mi nimal changes in the local geometry do not affect the secondary and tertiar y structures of the mutants, which were identical to those of wild-type pro tein in the crystal form. But the mutants exhibit significant differences i n stability, folding cooperativity, biological activity, and fluorescence p roperties if compared to the wild-type protein. These rather large global e ffects, resulting from the minimal local changes, have to be attributed eit her to the relatively strong changes in polar interactions of the indole ri ng or to differences in the van der Waals radii or to a combination of both facts, The changes in local geometry that are below resolution of protein X-ray crystallographic studies are probably of secondary importance in comp arison to the strong electronegativity introduced by the fluorine atom. Cor respondingly, these types of mutations provide an interesting approach to s tudy cooperative functions of integrated residues and modulation of particu lar physicochemical properties, in the present case of electronegativity, i n a uniquely structured and hierarchically organized protein molecule.