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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/33400
Title: Importance of polarity of the α4-α5 loop residue - Asn 166 in the pore-forming domain of the Bacillus thuringiensis Cry4Ba toxin: Implications for ion permeation and pore opening
Authors: Thanate Juntadech
Yodsoi Kanintronkul
Chalermpol Kanchanawarin
Gerd Katzenmeier
Chanan Angsuthanasombat
Mahidol University
Biophysics Institute for Research and Development (BIRD)
Kasetsart University
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 1-Jan-2014
Citation: Biochimica et Biophysica Acta - Biomembranes. Vol.1838, No.1 PARTB (2014), 319-327
Abstract: Bacillus thuringiensis Cry4Ba toxin is lethal to mosquito-larvae by forming ion-permeable pores in the target midgut cell membrane. Previously, the polarity of Asn166located within the α4-α5 loop composing the Cry4Ba pore-forming domain was shown to be crucial for larvicidal activity. Here, structurally stable-mutant toxins of both larvicidal-active (N166D) and inactive (N166A and N166I) mutants were FPLC-purified and characterized for their relative activities in liposomal-membrane permeation and single-channel formation. Similar to the 65-kDa trypsin-activated wild-type toxin, the N166D bio-active mutant toxin was still capable of releasing entrapped calcein from lipid vesicles. Conversely, the two other bio-inactive mutants showed a dramatic decrease in causing membrane permeation. When the N166D mutant was incorporated into planar lipid bilayers (under symmetrical conditions at 150 mM KCl, pH 8.5), it produced single-channel currents with a maximum conductance of about 425 pS comparable to the wild-type toxin. However, maximum conductances for single K+-channels formed by both bio-inactive mutants (N166I and N166A) were reduced to approximately 165-205 pS. Structural dynamics of 60-ns simulations of a trimeric α4-α5 pore model in a fully hydrated-DMPC system revealed that an open-pore structure could be observed only for the simulated pores of the wild type and N166D. Additionally, the number of lipid molecules interacting with both wild-type and N166D pores is relatively higher than those of N166A and N166I pores. Altogether, our results further signify that the polarity at the α4-α5 loop residue - Asn166is directly involved in ion permeation through the Cry4Ba toxin-induced ionic pore and pore opening at the membrane-water interface. © 2013 Elsevier B.V.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84887866677&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/33400
ISSN: 18792642
00052736
Appears in Collections:Scopus 2011-2015

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