Abstract
Protein interaction perturbation profiling at amino acid resolution
Highlights
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 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
Summary
Mutagenesis carried out as in Kitzman et al \(Step-by-step protocol available, Nature Methods, 2015) with the following alterations: To create WT template strands, each ORF was amplified from a gateway destination vector using generic att site primers. Each PCR product is flanked with identical short sequences facilitating parallel mutant library synthesis while still allowing direct transfer of the final PCR products into a gateway donor vector via a BP reaction
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