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Method Article
Jonathan Woodsmith
Stelzl Lab, University of Graz
Corresponding Author
Ulrich Stelzl
Stelzl Lab, University of Graz
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Identification of genomic variants in healthy and diseased individuals continues to rapidly outpace our ability to functionally annotate them. Techniques that both systematically assay the functional consequences of nucleotide resolution variation and can scale to hundreds of genes are urgently required. We designed a sensitive yeast two-hybrid based “off switch” for positive selection of interaction disruptive variants from complex genetic libraries. Combined with massively parallel programmed mutagenesis and a sequencing readout, it enables systematic profiling of protein interaction determinants at an amino acid resolution. We defined >1,000 interaction disrupting amino acid mutations across eight subunits of the BBSome, the major human cilia protein complex associated with the pleiotropic genetic disorder Bardet-Biedl-Syndrome. These high-resolution interaction perturbation profiles provide a framework for interpreting patient derived mutations across the entire protein complex, highlighting how the impact of disease variation on interactome networks can be assessed systematically.
Keywords
Protein-protein interactions, massively parallel programmed mutagenesis, reverse yeast two hybrid
The authors declare no competing financial interests.
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Associated Publications
Protein interaction perturbation profiling at amino-acid resolution
by Jonathan Woodsmith, Luise Apelt, Victoria Casado-Medrano, +3
Nature Methods (17 October, 2017)
Method Article
Jonathan Woodsmith
Stelzl Lab, University of Graz
Corresponding Author
Ulrich Stelzl
Stelzl Lab, University of Graz
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 17 Oct, 2017
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Identification of genomic variants in healthy and diseased individuals continues to rapidly outpace our ability to functionally annotate them. Techniques that both systematically assay the functional consequences of nucleotide resolution variation and can scale to hundreds of genes are urgently required. We designed a sensitive yeast two-hybrid based “off switch” for positive selection of interaction disruptive variants from complex genetic libraries. Combined with massively parallel programmed mutagenesis and a sequencing readout, it enables systematic profiling of protein interaction determinants at an amino acid resolution. We defined >1,000 interaction disrupting amino acid mutations across eight subunits of the BBSome, the major human cilia protein complex associated with the pleiotropic genetic disorder Bardet-Biedl-Syndrome. These high-resolution interaction perturbation profiles provide a framework for interpreting patient derived mutations across the entire protein complex, highlighting how the impact of disease variation on interactome networks can be assessed systematically.
Loading...
Associated Publications
Protein interaction perturbation profiling at amino-acid resolution
by Jonathan Woodsmith, Luise Apelt, Victoria Casado-Medrano, +3
Nature Methods (17 October, 2017)