CHANGING THE STRUCTURAL CONTEXT OF A FUNCTIONAL BETA-HAIRPIN - SYNTHESIS AND CHARACTERIZATION OF A CHIMERA CONTAINING THE CURAREMIMETIC LOOP OF A SNAKE TOXIN IN THE SCORPION ALPHA BETA SCAFFOLD/

An approach to obtain new active proteins is the incorporation of all or a part of a well defined active site onto a natural structure actin g as a structural scaffold. According to this strategy we tentatively engineered a new curaremimetic molecule by transferring the functional central loop of a snake toxin, sequence 26-37, sandwiched between two hairpins, onto the structurally similar beta-hairpin of the scorpion toxin charybdotoxin, stabilized by a short helix. The resulting chimer ic molecule, only 31 amino acids long, was produced by solid phase syn thesis, refolded, and purified to homogeneity. As shown by structural analysis performed by CD and NMR spectroscopy, the chimera maintained the expected alpha/beta fold characteristic of scorpion toxins and pre sented a remarkable structural stability. The chimera competitively di splaces the snake curaremimetic toxin alpha from the acetylcholine rec eptor at 10(-5) M concentrations. Antibodies, elicited in rabbits agai nst the chimera, recognize the parent snake toxin and prevent its bind ing to the acetylcholine receptor, thus neutralizing its toxic functio n. All these data demonstrate that the strategy of active site transfe r to the charybdotoxin scaffold has general applications in the engine ering of novel ligands for membrane receptors and in vaccine design.