SYNTHESIS AND CHARACTERIZATION OF LEIUROTOXIN-I ANALOGS LACKING ONE DISULFIDE BRIDGE - EVIDENCE THAT DISULFIDE PAIRING-3-21 IS NOT REQUIREDFOR FULL TOXIN ACTIVITY

Leiurotoxin I (Lei-NH2), a toxin isolated from the venom of the scorpi on Leiurus quinquestriatus hebraeus, is a blocker of the apamin-sensit ive Ca2+-activated K+ channels. It is a 31-residue polypeptide cross-l inked by three disulfide bridges which are presumably between Cys(3)-C ys(21), Cys(8)-Cys(26), and Cys(12)-Cys(28). To investigate the role o f these disulfides, analogs of Lei-NH2 lacking one disulfide bridge (i .e., [Abu(3,21)]Lei-NH2, [Abu(8,26)]Lei-NH2, and [Abu(12,28)]Lei-NH2) were chemically synthesized by selective replacement of each pair of h alf-cystines forming a bridge by two alpha-aminobutyrate (Abu) residue s. The two disulfide pairings of the main folded form of the synthetic analogs were established by enzymatic proteolysis. They were as expec ted between Cys(8)-Cys(26) and Cys(12)-Cys(28) for [Abu(3,21)]Lei-NH2 but were unexpectedly between Cys(3)-Cys(12) and Cys(21)-Cys(28) for [ Abu(8.26)]Lei-NH2 and between Cys(3)-Cys(8) and Cys(21)-Cys(26) for [A bu(12,28)]Lei-NH2. The synthetic peptides were tested in vitro for the ir capacity to compete with the binding of [I-125]apamin to rat brain synaptosomes and in vivo for their neurotoxicity in mice. In both assa ys, [Abu(3,21)]Lei-NH2 exhibited full Lei-NH2-like activity whereas [A bu(8,26)]Lei-NH2 and [Abu(12,28)]Lei-NH2 possessed only residual activ ities (<2% native toxin activity). This suggests that disulfide bridge Cys(3)-Cys(21) is not essential per se for high toxin activity. Circu lar dichroism (CD) spectroscopy of the three analogs showed that only [Abu(3,21)]Lei-NH2 exhibited a CD spectrum similar to that of Lei-NH2, suggesting they both adopt closely related conformations, in agreemen t with the pharmacological data. Structural models of the analogs were constructed on the basis of the disulfide pairing assignment and comp ared with that of Lei-NH2.