Characterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity
15 p.
- Autores:
-
Hussain, Syed-Rehan A.
Florez, Alvaro M.
Osorio, Cristina
Dean, Donald H.
Alzate, Oscar
- Tipo de recurso:
- Article of journal
- Fecha de publicación:
- 2011
- Institución:
- Universidad de Santander
- Repositorio:
- Repositorio Universidad de Santander
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.udes.edu.co:001/3644
- Acceso en línea:
- https://repositorio.udes.edu.co/handle/001/3644
- Palabra clave:
- a-helix 5
Circular dichroism
Molecular modeling
Site-directed mutagenesis
Thermal stability
Bacillus thuringiensis
Dicroísmo circular
Modelamiento molecular
Mutagénesis sitio dirigida
Estabilidad térmica
- Rights
- openAccess
- License
- Derechos Reservados - Universidad de Santander, 2011
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Characterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity |
| title |
Characterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity |
| spellingShingle |
Characterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity a-helix 5 Circular dichroism Molecular modeling Site-directed mutagenesis Thermal stability Bacillus thuringiensis Dicroísmo circular Modelamiento molecular Mutagénesis sitio dirigida Estabilidad térmica |
| title_short |
Characterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity |
| title_full |
Characterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity |
| title_fullStr |
Characterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity |
| title_full_unstemmed |
Characterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity |
| title_sort |
Characterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity |
| dc.creator.fl_str_mv |
Hussain, Syed-Rehan A. Florez, Alvaro M. Osorio, Cristina Dean, Donald H. Alzate, Oscar |
| dc.contributor.author.spa.fl_str_mv |
Hussain, Syed-Rehan A. Florez, Alvaro M. Osorio, Cristina Dean, Donald H. Alzate, Oscar |
| dc.subject.proposal.eng.fl_str_mv |
a-helix 5 Circular dichroism Molecular modeling Site-directed mutagenesis Thermal stability Bacillus thuringiensis |
| topic |
a-helix 5 Circular dichroism Molecular modeling Site-directed mutagenesis Thermal stability Bacillus thuringiensis Dicroísmo circular Modelamiento molecular Mutagénesis sitio dirigida Estabilidad térmica |
| dc.subject.proposal.spa.fl_str_mv |
Dicroísmo circular Modelamiento molecular Mutagénesis sitio dirigida Estabilidad térmica |
| description |
15 p. |
| publishDate |
2011 |
| dc.date.issued.spa.fl_str_mv |
2011-10 |
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2019-08-26T19:13:44Z |
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2019-08-26T19:13:44Z |
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http://purl.org/coar/resource_type/c_2df8fbb1 |
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1909-8758 0123-3475 |
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https://repositorio.udes.edu.co/handle/001/3644 |
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spa |
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spa |
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Revista Colombiana de Biotecnología |
| dc.relation.ispartofseries.spa.fl_str_mv |
Revista Colombiana de Biotecnología;Volumen 13, Número 2, p. 144-154, 2011 |
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Derechos Reservados - Universidad de Santander, 2011 |
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Atribución-NoComercial 4.0 Internacional (CC BY-NC 4.0) |
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https://creativecommons.org/licenses/by-nc/4.0/ |
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openAccess |
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Hussain, Syed-Rehan A.af346681-389f-42aa-ada3-bd368aa0c9d1-1Florez, Alvaro M.03b37924-311e-4ca6-bae7-fb60528ecb1c-1Osorio, Cristinad1053838-b1f0-4825-ab49-673f10e9521d-1Dean, Donald H.e9be6363-31b6-435b-8ab0-ad1ca994c335-1Alzate, Oscar73209183-39f5-47d8-b148-c89b5056c6bb-12019-08-26T19:13:44Z2019-08-26T19:13:44Z2011-1015 p.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.La a-Hélice 5 del domino I de las d-endotoxinas de Bacillus thuringiensis, es crítica para la toxicidad de las toxinas contra insectos al participar en la formación de canales iónicos. La participación en la función tóxica del residuo Histidina 168 (H168) –el cual es altamente conservado– fue estudiada mediante técnicas de biología molecular, electrofisiología y biofísica. La toxina mutante H168R fue ~ 3 veces más tóxica que la toxina silvestre (ts) en Manduca sexta, mientras que H168Q fue 3 veces menos tóxica. Los análisis espectroscópicos indicaron que las mutaciones no producen alteraciones estructurales significativas y que la toxina H168R (Tm= 59 °C) es más estable que las toxinas H168Q (Tm= 57.5 °C) y wt (Tm= 56 °C). Las tres toxinas exhibieron uniones de afinidad similares (Kcom) en vesículas de intestino de larvas de insecto, indicando que las diferencias en la toxicidad no se deben a cambios en la unión inicial al receptor. Los ensayos de unión/disociación y fijación de voltaje mostraron que la reducción de la toxicidad de la toxina H168Q se puede atribuir a una disminución en la inserción y/o en la formación de canales iónicos. De otro lado, H168R mostró una inhibición a la corriente de corto circuito mayor que la ts y un aumento en unión irreversible (kobs), lo cual es consistente con un menor valor de CL50. La modelación molecular sugiere que H168Q y H168R forman puentes de hidrógeno adicionales, lo que les confiere mayor estabilidad térmica. Adicionalmente, es probable que H168R tenga una carga positiva extra expuesta en la superficie, lo cual aumentaría su tasa de inserción en membranas susceptibles.application/pdf1909-87580123-3475https://repositorio.udes.edu.co/handle/001/3644spaRevista Colombiana de BiotecnologíaRevista Colombiana de Biotecnología;Volumen 13, Número 2, p. 144-154, 2011Derechos Reservados - Universidad de Santander, 2011info:eu-repo/semantics/openAccessAtribución-NoComercial 4.0 Internacional (CC BY-NC 4.0)https://creativecommons.org/licenses/by-nc/4.0/http://purl.org/coar/access_right/c_abf2https://revistas.unal.edu.co/index.php/biotecnologia/article/view/27964/28220a-helix 5Circular dichroismMolecular modelingSite-directed mutagenesisThermal stabilityBacillus thuringiensisDicroísmo circularModelamiento molecularMutagénesis sitio dirigidaEstabilidad térmicaCharacterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicityArtículo de revistahttp://purl.org/coar/resource_type/c_6501http://purl.org/coar/resource_type/c_2df8fbb1Textinfo:eu-repo/semantics/articlehttp://purl.org/redcol/resource_type/ARTinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/version/c_970fb48d4fbd8a85PublicationTEXTCharacterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity.pdf.txtCharacterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity.pdf.txtExtracted texttext/plain46968https://repositorio.udes.edu.co/bitstreams/8c36c487-ba13-4626-85a8-f11b22a29903/downloadfbeabc78da6dc0b16ecdf4f924129255MD53THUMBNAILCharacterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity.pdf.jpgCharacterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity.pdf.jpgGenerated Thumbnailimage/jpeg1540https://repositorio.udes.edu.co/bitstreams/9f125d25-1028-441a-bb39-efec5999b2be/download3d1b53fb082ba27c0b6e8901a4428d57MD54ORIGINALCharacterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity.pdfCharacterization of a mutant bacillus thuringiensis delta endotoxin with enhanced stability and toxicity.pdfapplication/pdf270500https://repositorio.udes.edu.co/bitstreams/b57c9c04-f9bd-4252-bc07-95101628f0ef/downloadf1df1c0ab8c22ca5c4885dcdbbde4eaeMD55LICENSElicense.txtlicense.txttext/plain; charset=utf-859https://repositorio.udes.edu.co/bitstreams/d4ae7c06-ff7e-434d-92fc-c706e8fde1ba/download38d94cf55aa1bf2dac1a736ac45c881cMD52001/3644oai:repositorio.udes.edu.co:001/36442023-10-11 11:08:42.945https://creativecommons.org/licenses/by-nc/4.0/Derechos Reservados - Universidad de Santander, 2011https://repositorio.udes.edu.coRepositorio Universidad de Santandersoporte@metabiblioteca.comTGljZW5jaWEgZGUgUHVibGljYWNpw7NuIFVERVMKRGlyZWN0cmljZXMgZGUgVVNPIHkgQUNDRVNPCgo= |