Abstract
我是賈亞曼Cardiotoxin II (CTX II), isolated from the venom of Taiwan Cobra(Najanaja atra) is a small molecular weight all beta-sheetprotein, fortifiedby four disulfide bridges. In the presentstudy, the stability and nucleotide binding capabilities of CTXII are investigated using variousspectroscopic techniques.Studies on the stability of CTX II provide manyinterestingresults. (1) The protein was quite stable in the pH range of2.0 - 8.0. Beyond this range of pH, the stability of theprotein decreasedsignificantly. As evidenced from the estimatedamide temperature coefficientvalues, using 2D NMR techniques,the structure in solution of CTX II at pH3.0 and 7.0 appear tobe grossly similar. (2) Equilibrium hydrogen-deuteriumexchangeexperiments indicate that the double stranded beta-sheet segmentisrelatively less stable than the triple stranded beta-sheetdomain in the protein (CTX II) molecule. The most stableregions in the protein includethe hydrophobic patch comprised ofLeu20, Cys21, Tyr22, Ile39, Val41 and Val52. Thus, theseresidues constitute the initiation sites during the folding ofthe CTX II molecule. (4) The difference in the Gibb'sfreeenergy of the folded and unfolded state(s) of the CTX IImolecule is estimated to be 4.813 kcalmol-1. (5) TFE, a helix-inducing solvent, is found to induce helical conformation in CTXII at higher concentration (>70% v/v). Analysis of the resultsobtained on the TFE-CTX II titrationsunder a variety ofconditions reveal that TFE is possibly not a specifichelix-inducing solvent, as is normally believed. It has beenshown that dATP binds with CTX II with the dissociation constantin the micromolar range. The observed intermolecular NOEs wereutilized in modeling the CTX II-dATP complex structure. It isfound that the nucleotide binds at the N- and C- terminal endsof the CTX II molecule. Thestability of the CTX II molecule isfound to increase upon complexing withdATP. Titrations withother nucleotides revealed that CTX II is a generalNTP bindingprotein and that the binding is significantly influenced by thelength of the pyrophosphate tail of the nucleotide. Theprobable physiol-ogical relevance of the nucleotide binding (toCTX II) is also discussed.