Examinando por Autor "Osorio, Cristina"
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- PublicaciónAcceso abiertoCharacterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity(2011-10) Hussain, Syed-Rehan A.; Florez, Alvaro M.; Osorio, Cristina; Dean, Donald H.; Alzate, OscarThe centrally located a-helix 5 of Bacillus thuringiensis d-endotoxins is critical for insect toxicity through ion-channel formation. We analyzed the role of the highly conserved residue Histidine 168 (H168) using molecular biology, electrophysiology and biophysical techniques. Toxin H168R was ~3-fold more toxic than the wild type (wt) protein whereas H168Q was 3 times less toxic against Manduca sexta. Spectroscopic analysis revealed that the H168Q and H168R mutations did not produce gross structural alterations, and that H168R (Tm= 59 °C) was more stable than H168Q (Tm= 57.5 °C) or than the wt (Tm= 56 °C) toxins. These three toxins had similar binding affinities for larval midgut vesicles (Kcom) suggesting that the differences in toxicity did not result from changes in initial receptor binding. Dissociation binding assays and voltage clamping analysis suggest that the reduced toxicity of the H168Q toxin may result from reduced insertion and/or ion channel formation. In contrast, the H168R toxin had a greater inhibition of the short circuit current than the wt toxin and an increased rate of irreversible binding (kobs), consistent with its lower LC50 value. Molecular modeling analysis suggested that both the H168Q and H168R toxins could form additional hydrogen bonds that could account for their greater thermal stability. In addition to this, it is likely that H168R has an extra positive charge exposed to the surface which could increase its rate of insertion into susceptible membranes.
- PublicaciónAcceso abiertoParticipation of valine 171 in α-helix 5 of Bacillus thuringiensis Cry1Ab δ-endotoxin in translocation of toxin into Lymantria dispar midgut membranes(2010-10-01) Alzate, Oscar; Osorio, Cristina; Florez, Alvaro M.; Dean, Donald H.The Cry1Ab δ-endotoxin V171C mutant protein exhibits a 25-fold increase in toxicity against Lymantria dispar, which correlates with a faster rate of partitioning into the midgut membrane and slightly decreased protein stability. This is an insect-specific mechanism; similar results were not observed in Manduca sexta, another Cry1Ab δ-endotoxin-susceptible insect.
- PublicaciónAcceso abiertoProtein Engineering of Bacillus thuringiensis δ-Endotoxins(2012-03) Florez, Alvaro M.; Osorio, Cristina; Alzate, OscarProtein engineering of insecticidal Bt δ-endotoxins is a powerful tool for designing novel Cry toxins with altered properties, including changing the toxin’s specificity. By following some elementary rules governing the structure/function relationship, it has been possible to create new toxins with modified properties including increased toxicity and binding affinity, enhanced ion-transport activity, and changes in insect specificity. These methods have also produced valuable information and have led to an improved understanding of the mode of action of these important biopesticides. The results discussed in this chapter derive from rational molecular design where protein structure is modified by incorporating single or multiple amino acid substitutions aimed at modifying specific protein functions. In this review, we analyze several protein modifications that have been successfully used for creating stable, functional proteins with minimal structural alterations. The understanding and proper use of protein engineering approaches may help in implementing appropriate pest management strategies by improving the efficacy of these toxins against insect pests.