1. Stable monomeric form of an originally dimeric serine proteinase inhibitor, ecotin, was constructed via site directed mutagenesis
- Author
-
László Szilágyi, László Gráf, and Gábor Pál
- Subjects
Protein Conformation ,Stereochemistry ,Dimer ,Molecular Sequence Data ,Biophysics ,Antiparallel (biochemistry) ,Biochemistry ,Chromatography, Affinity ,Fluorescence ,Serine ,chemistry.chemical_compound ,Bacterial Proteins ,Structural Biology ,Escherichia coli ,Genetics ,Trypsin ,Binding site ,Site-directed mutagenesis ,Molecular Biology ,DNA Primers ,Sequence Deletion ,Electrophoresis, Agar Gel ,Base Sequence ,Chemistry ,Escherichia coli Proteins ,Serine proteinase inhibitor ,Tryptophan ,Cell Biology ,Ecotin ,Heterotetramer ,Recombinant Proteins ,Kinetics ,Chromatography, Gel ,Mutagenesis, Site-Directed ,Serine Proteinase Inhibitors ,Periplasmic Proteins ,Trypsin Inhibitors ,Site directed mutagenesis - Abstract
Ecotin, a homodimer protein of E. coli, is a unique member of canonical serine proteinase inhibitors, since it is a potent agent against a variety of serine proteinases having different substrate specificity. Monomers of ecotin are held together mostly by their long C-terminal strands that are arranged as a two-stranded antiparallel β-sheet in the functional dimer. One ecotin dimer can chelate two proteinase molecules, each of them bound to both subunits of ecotin at two different sites, namely the specific primary and the non-specific secondary binding sites. In this study the genes of wild type ecotin and its Met84 Arg P1 site mutant were truncated resulting in new forms of ecotin that lack 10 amino acid residues at their C-terminus. These mutants do not dimerize spontaneously, though in combination with trypsin they assemble into the familiar heterotetramer. Our data suggest that this heterotetramer exists even in extremely diluted solutions, and the interaction, which is responsible for the dimerization of ecotin, contributes to the stability of the heterotetrameric complex.
- Published
- 1996
- Full Text
- View/download PDF