title: Scorpion toxins as natural scaffolds for protein engineering.
abstract: A compact, well-organized, and natural motif, stabilized by three disulfide bonds, is proposed as a basic scaffold for protein engineering. This motif contains 37 amino acids only and is formed by a short helix on one face and an antiparallel triple-stranded beta-sheet on the opposite face. It has been adopted by scorpions as a unique scaffold to express a wide variety of powerful toxic ligands with tuned specificity for different ion channels. We further tested the potential of this fold by engineering a metal binding site on it, taking the carbonic anhydrase site as a model. By chemical synthesis we introduced nine residues, including three histidines, as compared to the original amino acid sequence of the natural charybdotoxin and found that the new protein maintains the original fold, as revealed by CD and 1H NMR analysis. Cu2+ ions are bound with Kd = 4.2 x 10(-8) M and other metals are bound with affinities in an order mirroring that observed in carbonic anhydrase. The alpha/beta scorpion motif, small in size, easily amenable to chemical synthesis, highly stable, and tolerant for sequence mutations represents, therefore, an appropriate scaffold onto which polypeptide sequences may be introduced in a predetermined conformation, providing an additional means for design and engineering of small proteins.
authors.affiliation: Département d'Ingénierie et d'Etudes des Protéines, Commissariat à l'Energie Atomique, Gif-sur-Yvette, France.
authors.forename: C
authors.initials: C
authors.lastname: Vita
authors.forename: C
authors.initials: C
authors.lastname: Roumestand
authors.forename: F
authors.initials: F
authors.lastname: Toma
authors.forename: A
authors.initials: A
authors.lastname: Ménez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D018999:Charybdotoxin; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008670:Metals; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry*; D017354:Point Mutation; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D012604:Scorpion Venoms / Q000096:biosynthesis / Q000737:chemistry; D012605:Scorpions
chemical_list: D002352:Carrier Proteins; D008670:Metals; D010455:Peptides; D012604:Scorpion Venoms; D018999:Charybdotoxin; D003300:Copper; D002256:Carbonic Anhydrases