Stable monomeric form of an originally dimeric serine proteinase inhibitor, ecotin, was constructed via site directed mutagenesis.
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 beta-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 Met84Arg 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.
Pál
G
G
Department of Biochemistry, Eötvös University, Budapest, Hungary.
Szilágyi
L
L
Gráf
L
L
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D005453:Fluorescence; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D033903:Periplasmic Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D017931:DNA Primers; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D014364:Tryptophan; D014357:Trypsin