[Solid-phase synthesis and biological characterization of S12A-HNTX-IV and R29A-HNTX-IV: two mutants of hainantoxin-IV].
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.
Xu
Xia
X
College of Life Science, Hunan Normal University, Changsha 410081, China.
Xiong
Xia
X
Li
Dong-Ling
DL
Xiao
Yu-Cheng
YC
Wang
Xian-Chun
XC
Liang
Song-Ping
SP
D019943:Amino Acid Substitution; D000818:Animals; D009154:Mutation; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000187:drug effects* / Q000502:physiology; D013111:Spider Venoms / Q000138:chemical synthesis* / Q000235:genetics; D013329:Structure-Activity Relationship; D013779:Tetrodotoxin / Q000494:pharmacology
D026941:Sodium Channel Blockers; D015222:Sodium Channels; D013111:Spider Venoms; C474037:hainantoxin-IV, Seleconosmia hainana; D013779:Tetrodotoxin