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Nat Methods. 2017 Dec;14(12):1213-1221. doi: 10.1038/nmeth.4464. Epub 2017 Oct 16.

Protein interaction perturbation profiling at amino-acid resolution.

Author information

1
Otto-Warburg Laboratory, Max-Planck Institute for Molecular Genetics (MPIMG), Berlin, Germany.
2
Institute of Pharmaceutical Sciences, University of Graz and BioTechMed-Graz, Graz, Austria.

Abstract

The identification of genomic variants in healthy and diseased individuals continues to rapidly outpace our ability to functionally annotate these variants. 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, this method enables systematic profiling of protein-interaction determinants at 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 and thus highlight how the impact of disease variation on interactome networks can be systematically assessed.

PMID:
29039417
DOI:
10.1038/nmeth.4464
[Indexed for MEDLINE]

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