Studying protein variation in evolution, disease, and biology through deep insertion, deletion, and missense mutational scanning

Insertions and deletions (indels) are a major source of genetic variation in evolution and the cause of nearly 30% of Mendelian disease. Despite their importance, indels have been left out of nearly every systematic mutational scan to date, limiting our understanding of indels in disease, protein biology, and evolution. We developed a library generation method, DIMPLE, that generates deletions, insertions, and missense at even rates within any gene with low cost, low bias and low error rates. Using this protocol, we have generated libraries within a multitude of genes of varying function, length, evolutionary origin and type. Here we present results from a massive mutational scans of the mammalian potassium channel Kir2.1, measuring mutational effects on trafficking, and identifying fundamental differences in insertions, deletions, and missense types across secondary structure. We further find connections between indel tolerance, genomic data and evolutionary conservation. These results suggest a tantalizing connection between flexibility, folding, and function in this model membrane protein.