Pi7, an orphan peptide from the scorpion Pandinus imperator: A H-1-NMR analysis using a nano-NMR probe

The three-dimensional solution structure of a novel peptide, Pi7, purified from the venom of the scorpion Pandinus imperator, and for which no specifi c receptor has been found yet, was determined by two-dimensional homonuclea r proton NMR methods from a nanomole amount of compound using a nano-nmr pr obe. Pandinus imperator peptide 7 does not block voltage-dependent Kf-chann els and does not displace labeled noxiustoxin from rat brain synaptosomal m embranes. The toxin has 38 amino acid residues and, similarly to Pi1, is st abilized by four disulfide bridges (Cys6-Cys27, Cys12-Cys32, Cys16-Cys34, a nd Cys22-Cys37). In addition, the lysine at position 26 crucial for potassi um-channel blocking is replaced in Pi7 by an arginine. Tyrosine 34, equival ent to Tyr36 of ChTX is present, but the N-terminal positions 1 and 2 are o ccupied by two acidic residues Asp and Glu, respectively. The dihedral angl es and distance restraints obtained from measured NMR parameters were used in structural calculations in order to determine the conformation of the pe ptide. The disulfide-bridge topology was established using distance restrai nts allowing ambiguous partners between S atoms combined with NMR-derived s tructural information. The structure is organized around a short alpha-heli x spanning residues Thr9 to Thr20/Gly21 and a beta-sheet. These two element s of secondary structure are stabilized by two disulfide bridges, Cys12-Cys 32 and Cys16-Cys34. The antiparallel beta-sheet is composed of two strands extending from Asn22 to Cys34 with a tight turn at Ile28-Asn29 in contact w ith the N-terminal fragment Ile4 to Cys6.