Abstract
Cobra cardiotoxin (CTX) is the major component of cobra snake venom. The three-dimensional structure of a CTX molecule consists of a rigid core region stabilized by four disulfide bonds and three protruding finger loops—loop I, II and III,formed by five anti-parallel β-sheets. It is a basic polypeptide with a slightly concave disc-like shape. CTXs were classified into two classes by their distinct CD spectra.There are different distribution pattern in Eastern and Western Taiwan cobra –Naja atra, the most significant one is the amount of CTX A6, a recently identified component of Taiwan cobra venom. In this thesis, we take this new CTX as a modelto study the loop I conformation of class I cardiotoxin. We showed that all results indicated the existence of a cis-peptide bond between Pro8-Pro9 by NMR, CD and analysis of the newly solved X-ray structure of CTX A6. This solves the discrepancy between the conformation of loop I in the NMR and X-ray structures of Tγ, the only class I CTX with both NMR and X-ray structures. According to our results, we conclude that the conformation of loop I of class I CTX will be the cis-conformerrather than the trans-conformer of class II CTX.It has been suggested that the binding of nucleotide triphosphate to CTXs may account for its in vitro activities of blocking the enzymatic activity of phospholipids protein kinase and Na+ K+-ATPase. Herein, we demonstrate by using ITC, NMRchemical shift perturbation analysis and 1D selective excitation NOESY spectroscopic studies at pH ~6.0 that dATP binds to the convex side of CTX A1 & A3, CTX homologues from Naja atra, in sharp contrast to the previous suggestion of the dATP binding to the concave side of CTX A2 at pH 3.0. The ITC studies on the binding of ATP to 9 CTX homologues, shows the important role of positively charged residues of the 5th position in modulating CTX-ATP interaction.. It suggests that intracellular ATP may compete with the phospholipids of the inner leaflet of the membrane bilayer for binding on CTXs to facilitate their internalization.In this thesis, we characterized the loop I conformation of class I CTX, shown an anionic binding pocket on the convex side of CTX, and role of Gln5 (Lys5 or Arg5), the biological meanings of loop I CTX and binding between ATP and CTXsalso were proposed. These will help further studies on the structure-function relationship of CTX.