Ion channels formed in planar lipid bilayers by the dipteran-specific Cry4B Bacillus thuringiensis toxin and its α1-α5 fragment

被引:41
作者
Puntheeranurak, T
Uawithya, P
Potvin, L
Angsuthanasombat, C
Schwartz, JL
机构
[1] Natl Res Council Canada, Biotechnol Res Inst, Montreal, PQ H4P 2R2, Canada
[2] Mahidol Univ, Inst Mol Biol & Genet, Lab Mol Biophys, Nakhon Pathom 73170, Thailand
[3] Univ Montreal, Grp Etud Prot Membranaires, Montreal, PQ H3C 3J7, Canada
基金
加拿大自然科学与工程研究理事会;
关键词
Bacillus thuringiensis; delta-endotoxin; truncated protein; planar lipid bilayer; ion channel;
D O I
10.1080/09687680310001625792
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Trypsin activation of Cry4B, a 130-kDa Bacillus thuringiensis (Bt) protein, produces a 65-kDa toxin active against mosquito larvae. The active toxin is made of two protease-resistant products of ca. 45 kDa and ca. 20 kDa. The cloned 21-kDa fragment consisting of the N-terminal region of the toxin was previously shown to be capable of permeabilizing liposomes. The present study was designed to test the following hypotheses: ( 1) Cry4B, like several other Bt toxins, is a channel-forming toxin in planar lipid bilayers; and (2) the 21-kDa Nterminal region, which maps for the first five helices (alpha1 - alpha5) of domain 1 in other Cry toxins, and which putatively shares a similar tri-dimensional structure, is sufficient to account for the ion channel activity of the whole toxin. Using circular dichroism spectroscopy and planar lipid bilayers, we showed that the 21-kDa polypeptide existed as an alpha-helical structure and that both Cry4B and its alpha1 - alpha5 fragment formed ion channels of 248 +/- 44 pS and 207 +/- 23 pS, respectively. The channels were cation-selective with a potassium-to-chloride permeability ratio of 6.7 for Cry4B and 4.5 for its fragment. However, contrary to the full-length toxin, the alpha1 - alpha5 region formed channels at low dose; they tended to remain locked in their open state and displayed flickering activity bouts. Thus, like the full-length toxin, the alpha1 - alpha5 region is a functional channel former. A pH- dependent, yet undefined region of the toxin may be involved in regulating the channel properties.
引用
收藏
页码:67 / 74
页数:8
相关论文
共 40 条
[1]   Role of α-helix seven of Bacillus thuringiensis Cry1Ab δ-endotoxin in membrane insertion, structural stability, and ion channel activity [J].
Alcantara, EP ;
Alzate, O ;
Lee, MK ;
Curtiss, A ;
Dean, DH .
BIOCHEMISTRY, 2001, 40 (08) :2540-2547
[2]  
ANGSUTHANASOMBAT C, 1992, FEMS MICROBIOL LETT, V94, P63, DOI 10.1111/j.1574-6968.1992.tb05290.x
[3]  
ANGSUTHANASOMBAT C, 1993, FEMS MICROBIOL LETT, V111, P255
[4]  
[Anonymous], 1992, Ionic Channels of Excitable Membranes Sunderland
[5]   The role of a proline-induced broken-helix motif in α-helix 2 of Bacillus thuringiensis δ-endotoxins [J].
Arnold, S ;
Curtiss, A ;
Dean, DH ;
Alzate, O .
FEBS LETTERS, 2001, 490 (1-2) :70-74
[6]  
CHAKRABARTTY A, 1994, PROTEIN SCI, V3, P843
[7]   Amino acid substitution in α-helix 7 of Cry1Ac δ-endotoxin of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera Hubner [J].
Chandra, A ;
Ghosh, P ;
Mandaokar, AD ;
Bera, AK ;
Sharma, RP ;
Das, S ;
Kumar, PA .
FEBS LETTERS, 1999, 458 (02) :175-179
[8]   2-POINT CALIBRATION OF CIRCULAR DICHROMETER WITH D-10-CAMPHORSULFONIC ACID [J].
CHEN, GC ;
YANG, JT .
ANALYTICAL LETTERS, 1977, 10 (14) :1195-1207
[9]   Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac [J].
Coux, F ;
Vachon, V ;
Rang, C ;
Moozar, K ;
Masson, L ;
Royer, M ;
Bes, M ;
Rivest, S ;
Brousseau, R ;
Schwartz, JL ;
Laprade, R ;
Frutos, R .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (38) :35546-35551
[10]   Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins [J].
Crickmore, N ;
Zeigler, DR ;
Feitelson, J ;
Schnepf, E ;
Van Rie, J ;
Lereclus, D ;
Baum, J ;
Dean, DH .
MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, 1998, 62 (03) :807-+