title: Phenotypic selection and characterization of randomly produced non-haemolytic mutants of the toxic sea anemone protein sticholysin II.
abstract: A rapid screening method for haemolytic activity, using blood agar plates, has been developed to analyze randomly produced mutant variants of the pore-forming protein sticholysin II (Stn II). Those exhibiting a reduced activity were selected and the DNA corresponding to each Stn II variant sequenced. Once the mutation produced was determined, protein variants were isolated and characterized in terms of structure (circular dichroism spectra and thermal stability) and haemolytic activity. Three single mutation protein variants, at residues K19, F106 and Y111, showed a significantly decreased haemolytic activity while their thermostability was identical to that of the wild-type protein. Considering the obtained data and based on the three-dimensional structure of the protein, the role of these residues on the mechanism of haemolysis has been analyzed.
authors.affiliation: Facultad de Química, Departamento de Bioquímica y Biología Molecular, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Alegre-Cebollada
authors.forename: Valle
authors.initials: V
authors.lastname: Lacadena
authors.forename: Mercedes
authors.initials: M
authors.lastname: Oñaderra
authors.forename: José M
authors.initials: JM
authors.lastname: Mancheño
authors.forename: José G
authors.initials: JG
authors.lastname: Gavilanes
authors.forename: Alvaro Martínez
authors.initials: AM
authors.lastname: del Pozo
mesh_terms: D000818:Animals; D003064:Cnidarian Venoms / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006461:Hemolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012615:Sea Anemones / Q000737:chemistry*; D013696:Temperature
chemical_list: D003064:Cnidarian Venoms; D006460:Hemolysin Proteins; C404794:sticholysin II