AGCDA. Artículos de Investigación

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Examinando AGCDA. Artículos de Investigación por Autor "Dean, Donald H."

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  • Publicación
    Acceso abierto
    Characterization 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, Oscar
    The 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.
  • Publicación
    Acceso abierto
    Participation 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.
  • Publicación
    Acceso abierto
    Two disulfide mutants in domain I of Bacillus thuringiensis Cry3Aa δ-endotoxin increase stability with no effect on toxicity
    (2012-05) Wu, Sheng Jiun; Florez, Alvaro M.; Homoelle, Bradley J.; Dean, Donald H.; Alzate, Oscar
    To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteinefree Cry3Aa δ-endotoxin from Bacillus thuringiensis. These mutations were designed to create disulfide bonds between α-helices 2 and 5 (positions 110 - 193), and α-helices 5 and 7 (positions 195 - 276 and 198 - 276). Comparison of the CD spectra of the wild-type and the double-cysteine mutant proteins indicates a tighter helical packing consistent with formation of at least two of the disulfide bonds between the central and the outer helices. Thermal stability analysis indicates that potential covalent linkages between the central α-helix 5 and the other helices increase resistance to thermal denaturation by 10˚C to 14˚C compared to the thermal stability of the wild-type protein. Spectroscopic analysis of the disulfide-specific absorbance band indicates that the double mutant proteins are more stable to temperature and denaturant (guanidine hydrochloride) than the wild-type protein, as a result of the formation of two of the disulfide bridges. These results indicate that the double mutations M110C/F193C and A198C/V276C successfully established disulfide bonds, resulting in a more stable structure of the entire toxin. Despite the increase in stability and structural changes introduced by the disulfide bonds, no effect on toxicity was observed. A possible mechanism involving the insertion of all of domain I of Cry3Aa toxin into the target membrane accounts for these observations.