Cargando…
Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase
Predicting mutations that enhance protein–protein affinity remains a challenging task, especially for high-affinity complexes. To test our capability to improve the affinity of such complexes, we studied interaction of acetylcholinesterase with the snake toxin, fasciculin. Using the program ORBIT, w...
Autores principales: | , , , , , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2009
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742391/ https://www.ncbi.nlm.nih.gov/pubmed/19643977 http://dx.doi.org/10.1093/protein/gzp045 |
_version_ | 1782171818487971840 |
---|---|
author | Sharabi, Oz Peleg, Yoav Mashiach, Efrat Vardy, Eyal Ashani, Yacov Silman, Israel Sussman, Joel L. Shifman, Julia M. |
author_facet | Sharabi, Oz Peleg, Yoav Mashiach, Efrat Vardy, Eyal Ashani, Yacov Silman, Israel Sussman, Joel L. Shifman, Julia M. |
author_sort | Sharabi, Oz |
collection | PubMed |
description | Predicting mutations that enhance protein–protein affinity remains a challenging task, especially for high-affinity complexes. To test our capability to improve the affinity of such complexes, we studied interaction of acetylcholinesterase with the snake toxin, fasciculin. Using the program ORBIT, we redesigned fasciculin's sequence to enhance its interactions with Torpedo californica acetylcholinesterase. Mutations were predicted in 5 out of 13 interfacial residues on fasciculin, preserving most of the polar inter-molecular contacts seen in the wild-type toxin/enzyme complex. To experimentally characterize fasciculin mutants, we developed an efficient strategy to over-express the toxin in Escherichia coli, followed by refolding to the native conformation. Despite our predictions, a designed quintuple fasciculin mutant displayed reduced affinity for the enzyme. However, removal of a single mutation in the designed sequence produced a quadruple mutant with improved affinity. Moreover, one designed mutation produced 7-fold enhancement in affinity for acetylcholinesterase. This led us to reassess our criteria for enhancing affinity of the toxin for the enzyme. We observed that the change in the predicted inter-molecular energy, rather than in the total energy, correlates well with the change in the experimental free energy of binding, and hence may serve as a criterion for enhancement of affinity in protein–protein complexes. |
format | Text |
id | pubmed-2742391 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-27423912009-09-14 Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase Sharabi, Oz Peleg, Yoav Mashiach, Efrat Vardy, Eyal Ashani, Yacov Silman, Israel Sussman, Joel L. Shifman, Julia M. Protein Eng Des Sel Original articles Predicting mutations that enhance protein–protein affinity remains a challenging task, especially for high-affinity complexes. To test our capability to improve the affinity of such complexes, we studied interaction of acetylcholinesterase with the snake toxin, fasciculin. Using the program ORBIT, we redesigned fasciculin's sequence to enhance its interactions with Torpedo californica acetylcholinesterase. Mutations were predicted in 5 out of 13 interfacial residues on fasciculin, preserving most of the polar inter-molecular contacts seen in the wild-type toxin/enzyme complex. To experimentally characterize fasciculin mutants, we developed an efficient strategy to over-express the toxin in Escherichia coli, followed by refolding to the native conformation. Despite our predictions, a designed quintuple fasciculin mutant displayed reduced affinity for the enzyme. However, removal of a single mutation in the designed sequence produced a quadruple mutant with improved affinity. Moreover, one designed mutation produced 7-fold enhancement in affinity for acetylcholinesterase. This led us to reassess our criteria for enhancing affinity of the toxin for the enzyme. We observed that the change in the predicted inter-molecular energy, rather than in the total energy, correlates well with the change in the experimental free energy of binding, and hence may serve as a criterion for enhancement of affinity in protein–protein complexes. Oxford University Press 2009-10 2009-07-30 /pmc/articles/PMC2742391/ /pubmed/19643977 http://dx.doi.org/10.1093/protein/gzp045 Text en © 2009 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original articles Sharabi, Oz Peleg, Yoav Mashiach, Efrat Vardy, Eyal Ashani, Yacov Silman, Israel Sussman, Joel L. Shifman, Julia M. Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase |
title | Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase |
title_full | Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase |
title_fullStr | Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase |
title_full_unstemmed | Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase |
title_short | Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase |
title_sort | design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase |
topic | Original articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742391/ https://www.ncbi.nlm.nih.gov/pubmed/19643977 http://dx.doi.org/10.1093/protein/gzp045 |
work_keys_str_mv | AT sharabioz designexpressionandcharacterizationofmutantsoffasciculinoptimizedforinteractionwithitstargetacetylcholinesterase AT pelegyoav designexpressionandcharacterizationofmutantsoffasciculinoptimizedforinteractionwithitstargetacetylcholinesterase AT mashiachefrat designexpressionandcharacterizationofmutantsoffasciculinoptimizedforinteractionwithitstargetacetylcholinesterase AT vardyeyal designexpressionandcharacterizationofmutantsoffasciculinoptimizedforinteractionwithitstargetacetylcholinesterase AT ashaniyacov designexpressionandcharacterizationofmutantsoffasciculinoptimizedforinteractionwithitstargetacetylcholinesterase AT silmanisrael designexpressionandcharacterizationofmutantsoffasciculinoptimizedforinteractionwithitstargetacetylcholinesterase AT sussmanjoell designexpressionandcharacterizationofmutantsoffasciculinoptimizedforinteractionwithitstargetacetylcholinesterase AT shifmanjuliam designexpressionandcharacterizationofmutantsoffasciculinoptimizedforinteractionwithitstargetacetylcholinesterase |