Structural-functional studies of peptides derived from a long-chain snake neurotoxin Naja naja oxiana

The design and structural characterization of mini-proteins with a compact, folded structure provide insight into the complex architecture of proteins today and has long been a challenging issue in structural- functional studies. Alpha neurotoxins from snake venom have a distinct folded structure comprised of a disulphide core and three loops or “fingers”; each of these loops are considered as a separate functional domain. Because of selectivity and specificity of snake alpha neurotoxins, they are ideal candidates for structural-functional studies. With the assumption that each "loop" in the structure of alpha neurotoxin is able to fold as a structurally independent unit and could possibly have functional properties, we have minimized the structure of a long-chain alpha neurotoxin into 18 and 31 amino acid peptides using solid-phase synthesis and cloning methods, respectively. The molecules are structurally studied using circular dichroism spectroscopy and also in vitro using organ bath apparatus and chick biventer cervicis muscle (CBCM). The 18 and 31-mer peptides form predominant beta structures (-turn and -sheet/alpha helix) in aqueous solutions which vary with solvent ionic strength. Data from in vitro and in silico studies indicate that these minimized structures block the twitches in chick biventer cervices muscle with concentrations higher than 0.5 M; and this effect is absolutely dose dependent. On the other hand, the long-chain alpha neurotoxin completely and irreversibly blocks the CBCM even at 50 nanomolar concentration and both effects are post synaptic. These data support the primary assumption that the peptides derived from the second loop of snake alpha neurotoxin can have a distinct folded structure and furthermore, exist as an independent biological unit.