ABSTRACT
Hainantoxin-IV (HNTX-IV) purified from the venom of the spider Selenocosmia hainana is a potent antagonist that acts on tetrodotoxin-sensitive (TrX-S) sodium channels. It is a 35-residue polypeptide and includes three disulfide bridges. In order to investigate the structure-function relationship of HNTX-IV, two mutants (S12A-HNTX-IV and R29A-HNTX-IV) of HNTX-TV in which Ser12 and Arg29 were replaced by Ala respectively, were synthesized by solid-phase Fmoc chemistry, followed by oxidative refolding of purified peptides under the optimal conditions. The synthetic mutants were analyzed by MALDI-TOF mass spectrometry, nuclear magnetic resonance spectroscopy (NMR) and electrophysiological experiments for molecular weight, conformation and physiological activity, respectively. The results show that the mutants and native HNTX-IV (nHNTX-IV) have almost identical three-dimensional structures. The bioactivity level of S12A-HNTX-IV is also about the same as that of nHNTX-IV, suggesting that Ser12 does not play any important role for the bioactivity of this toxin. The bioactivity of R29A-HNTX-IV is reduced by at last 155 times, indicating that Arg29 is a key residue relative to the bioactivity of HNTX-IV. It is presumed that the decrease in activity of R29A-HNTX-IV is due to the changes of the property in the binding site rather than the change in the basic conformation of the molecule.