SYNTHESIS OF A METAL-BINDING PROTEIN DESIGNED ON THE ALPHA BETA SCAFFOLD OF CHARYBDOTOXIN/

The alpha/beta scaffold of the scorpion toxin charybdotoxin has been u sed for the engineering of a metal binding site. Nine substitutions, i ncluding three histidines as metal ligands, have been introduced into the original toxin sequence. The newly designed sequence, 37 amino aci ds long, has been assembled by solid-phase synthesis and HBTU -(1H-ben zotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) coupli ng of Fmoc-protected amino acids. Formation of the three disulfide bon ds occurred efficiently and rapidly in the presence of glutathione, an d this post-synthesis modification has facilitated the purification ta sk enormously. The process of synthesis and purification was performed in less than a week with an overall 10.2% yield. Circular dichroism a nalysis showed that the newly designed protein is folded in a alpha/be ta structure, similarly to the parent toxin. Electronic absorption spe ctroscopy, circular dichroism and gel filtration experiments have been used to show that CU2+ and Zn2+ ions bind with high affinity to the n ewly engineered protein. These results demonstrate that the alp fold, common to all scorpion toxins, is a very versatile basic structure, to lerant for substitutions and able to present new sequences in a predet ermined conformation. The chemical approach is shown to be effective, rapid and practical for the production of novel designed small protein s. (C) Munksgaard 1995.