SCORPION TOXINS AS NATURAL SCAFFOLDS FOR PROTEIN ENGINEERING

A compact, well-organized, and natural motif, stabilized by three disu lfide 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 opp osite face, It has been adopted by scorpions as a unique scaffold to e xpress a wide variety of powerful toxic ligands with tuned specificity for different ion channels, We further tested the potential of this f old by engineering a metal binding site on it, taking the carbonic anh ydrase site as a model, By chemical synthesis we introduced nine resid ues, including three histidines, as compared to the original amino aci d sequence of the natural charybdotoxin and found that the new protein maintains the original fold, as revealed by CD and H-1 NMR analysis, CU2+ ions are bound with K-d = 4.2 X 10(-8) M and other metals are bou nd with affinities in an order mirroring that observed in carbonic anh ydrase, The alpha/beta scorpion motif, small in size, easily amenable to chemical synthesis, highly stable, and tolerant for sequence mutati ons represents, therefore, an appropriate scaffold onto which polypept ide sequences may be introduced in a predetermined conformation, provi ding an additional means for design and engineering of small proteins.