linking domains I and II in Cry1Aa were abolished individually in ␣-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp 242 -Arg 265 bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.During sporulation, Bacillus thuringiensis produces a parasporal crystal body composed of one or more proteins that are toxic to a number of insect larvae (1) or to other invertebrates (2). After solubilization in the insect midgut and activation by intestinal proteases, these proteins bind to specific receptors at the surface of the apical brush border membrane of epithelial columnar cells, insert into the membrane, and form pores that disrupt midgut cellular functions (3-5).Elucidation of the crystal structure of the coleopteranspecific Cry3A toxin (6) and the lepidopteran-specific Cry1Aa toxin (7) revealed a similar three-domain structure for both proteins. Domain I, composed of eight amphipathic ␣-helices, is thought to be involved in membrane insertion and pore formation (8 -15). Domain II, composed of three -sheets and two short ␣-helices, is involved in the binding of the toxin to its receptor on the epithelial cell surface (16 -23). Domain III, composed of two -sheets forming a face-to-face -sandwich, appears to be involved in the stability (6), specificity (24 -26), and binding (27-34) of the toxin.These domains are closely packed together with the largest number of interdomain contacts found between domains I and II (6, 7). In Cry1Aa, domains I and II are linked by four salt bridges: Asp
A potential-sensitive fluorescent probe, 3,3'-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K(+) ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K(+) ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.