| Deep mutational scanning to probe function of coagulation proteins Laura Haynes. |
Abstract
As whole genome sequencing becomes increasingly more common clinical practice, the number of identified missense mutations in coding regions of the genome has grown exponentially. Only a fraction of these protein variants can be definitively classified as pathogenic or benign, approximately 70% classified as variants of unknown significance (VUSs). One approach to address this unmet need is deep mutational scanning (DMS). DMS is a powerful approach that couples the capabilities of modern high-throughput DNA sequencing with classical biochemical and cell biology techniques. At its most fundamental level, DMS identifies the effects of all amino acid substitutions at every position within a protein simultaneously by linking the phenotype being investigated with the cDNA encoding the protein of interest. We have extensively applied DMS to study plasminogen activator inhibitor-1 (PAI-1) as a critical regulator of fibrinolysis. By applying DMS to PAI-1, we have cataloged the effects of amino acid substitutions on its spontaneous transition to a latent state, protease inhibition, and interactions with its cofactor vitronectin.
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