Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers

1993 ◽  
Vol 132 (1) ◽  
Author(s):  
Jean-Louis Schwartz ◽  
Line Garneau ◽  
Diane Savaria ◽  
Luke Masson ◽  
Roland Brousseau ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Planar Lipid Bilayers ◽  
Crystal Toxins

scholarly journals Mutagenic Analysis of a Conserved Region of Domain III in the Cry1Ac Toxin of Bacillus thuringiensis

2002 ◽  
Vol 68 (1) ◽  
pp. 194-200 ◽  
Author(s):  
Luke Masson ◽  
Bruce E. Tabashnik ◽  
Alberto Mazza ◽  
Gabrielle Préfontaine ◽  
Léna Potvin ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Aspartic Acid ◽  
Lipid Bilayers ◽  
Insect Cell Line ◽  
Planar Lipid Bilayers ◽  
Primary Role ◽  
Membrane Pore ◽  
Cry1ac Toxin ◽  
Conserved Region ◽  
Domain Iii

ABSTRACT We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4- to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with Plutella xylostella larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.

scholarly journals Rapid topology probing using fluorescence spectroscopy in planar lipid bilayer: the pore-forming mechanism of the toxin Cry1Aa of Bacillus thuringiensis

2010 ◽  
Vol 136 (5) ◽  
pp. 497-513 ◽  
Author(s):  
Nicolas Groulx ◽  
Marc Juteau ◽  
Rikard Blunck
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Pore Formation ◽  
Time Course ◽  
Structural Information ◽  
Dynamic Change ◽  
Fluorescence Emission ◽  
Aqueous Environment ◽  
Planar Lipid Bilayers ◽  
Forming Mechanism

Pore-forming toxins, many of which are pathogenic to humans, are highly dynamic proteins that adopt a different conformation in aqueous solution than in the lipid environment of the host membrane. Consequently, their crystal structures obtained in aqueous environment do not reflect the active conformation in the membrane, making it difficult to deduce the molecular determinants responsible for pore formation. To obtain structural information directly in the membrane, we introduce a fluorescence technique to probe the native topology of pore-forming toxins in planar lipid bilayers and follow their movement during pore formation. Using a Förster resonance energy transfer (FRET) approach between site-directedly labeled proteins and an absorbing compound (dipicrylamine) in the membrane, we simultaneously recorded the electrical current and fluorescence emission in horizontal planar lipid bilayers formed in plastic chips. With this system, we mapped the topology of the pore-forming domain of Cry1Aa, a biological pesticide from Bacillus thuringiensis, by determining the location of the loops between its seven α helices. We found that the majority of the toxins initially traverse from the cis to the trans leaflet of the membrane. Comparing the topologies of Cry1Aa in the active and inactive state in order to identify the pore-forming mechanism, we established that only the α3–α4 hairpin translocates through the membrane from the trans to the cis leaflet, whereas all other positions remained constant. As toxins are highly dynamic proteins, populations that differ in conformation might be present simultaneously. To test the presence of different populations, we designed double-FRET experiments, where a single donor interacts with two acceptors with very different kinetics (dipicrylamine and oxonol). Due to the nonlinear response of FRET and the dynamic change of the acceptor distribution, we can deduce the distribution of the acceptors in the membrane from the time course of the donor fluorescence. We found that Cry1Aa is present on both membrane leaflets.

scholarly journals A cytolytic δ-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers

FEBS Letters ◽  
1989 ◽  
Vol 244 (2) ◽  
pp. 259-262 ◽  
Author(s):  
Barbara H. Knowles ◽  
Michael R. Blatt ◽  
Mark Tester ◽  
Jane M. Horsnell ◽  
Joe Carroll ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Planar Lipid Bilayers

scholarly journals Estimation of the radius of the pores formed by the Bacillus thuringiensis Cry1C δ-endotoxin in planar lipid bilayers

2002 ◽  
Vol 1567 ◽  
pp. 113-122 ◽  
Author(s):  
Olivier Peyronnet ◽  
Brian Nieman ◽  
Francis Généreux ◽  
Vincent Vachon ◽  
Raynald Laprade ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Planar Lipid Bilayers

scholarly journals Saturation behavior of single, amiloride-sensitive Na+ channels in planar lipid bilayers

1984 ◽  
Vol 46 (6) ◽  
pp. 831-835 ◽  
Author(s):  
L. Olans ◽  
S. Sariban-Sohraby ◽  
D.J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Planar Lipid Bilayers ◽  
Na Channels

scholarly journals Stimulation‐dependent gating of TRPM3 channel in planar lipid bilayers

2015 ◽  
Vol 30 (3) ◽  
pp. 1306-1316 ◽  
Author(s):  
Kunitoshi Uchida ◽  
Lusine Demirkhanyan ◽  
Swapna Asuthkar ◽  
Alejandro Cohen ◽  
Makoto Tominaga ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Planar Lipid Bilayers
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