Conformational Change and Inactivation of Membrane Phospholipid-Related Activity of Cardiotoxin V from Taiwan Cobra Venom at Acidic pH

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• 作者：Chien-Min Chiang ; Kun-Yi Chien ; Hai-jui Lin ; Ji-Fu Lin ; Hsien-Chi Yeh ; Pei-li Ho ; and Wen-guey Wu
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：July 16, 1996
• 年：1996
• 卷：35
• 期：28
• 页码：9167 - 9176
• 全文大小：470K
• 年卷期：v.35,no.28(July 16, 1996)
• ISSN：1520-4995

文摘

The phospholipid binding activity of cardiotoxin V from Najanaja atra (CTX A5) was studiedby use of Langmuir monolayers and found to exhibit pH-dependence inbinding to phosphatidylcholinemembrane with an apparent pK_a around 6.0.Proton NMR investigation of the CTX A5 molecule inthepresence of phosphatidylcholine micelles reveals a decrease inassociation of CTX A5 with membranesat low pH as a result of the protonation of His-4 near the membranebinding site of loop I region of CTX.The pH-dependent binding can be attributed mainly, but not solely,to the change in charge content of theCTX molecule upon His-4 protonation at the membrane/water interface.This is shown by analyzing thepH- and ionic strength dependence of binding of CTXs to phospholipidmonolayers according to Gouy-Chapman theory. The protonation of the His-4 residue also resultsin a local conformational change inthe loop I region since the chemical shifts of amide protons for theamino acid residues from Cys-3 toThr-14 are all found to vary as a function of pH with an apparentpK_a similar to that of His-4.Interestingly,the effect is relayed to other amino acid residues in the structuralcore of the protein such as those inC-terminal (Lys-60, Cys-61, and Asn-62) and triple-strandedantiparallel mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-sheet (Cys-22, Lys-24, Ala-25, Arg-38, and Ala-41) regions. An additional local conformationalchange in the molecule results aroundpH 5 as evidenced by circular dichroism spectroscopic studies, althoughthis change does not affect thecharacteristic mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-sheet and three-finger loop structure of CTXmolecule as revealed by two-dimensionalNOESY ¹H NMR study. The latter conformational changeat acidic pH, however, completely inactivatesCTX-induced aggregation/fusion activity of sphingomyelin vesicles.The results suggest that decipheringthe functional sites of CTXs on the basis of structure and dynamicsdetermined at low pH should be donewith caution. Since 19 out of 44 CTX homologues with known aminoacid sequence contain His-4, theeffect of His-4 on the structure and function of CTX molecules isimportant and is discussed in terms ofthe diverse membrane targets of CTX subtypes. Also discussed isthe pH-induced activation of snakevenom proteins in the victim.