Mechanistic Mutational Scanning to Uncover the Secret Life of Proteins

Deep mutational scanning (DMS) has emerged as a transformative tool for dissecting individual protein function and broader cell biology. DMS methods enable comprehensive interrogation of sequence-function relationships by systematically testing thousands of genetic variants within a pool for their effect(s) on diverse phenotypes. This review focuses on how these approaches are revolutionizing mechanistic protein biology, cell biology, and pharmacological research. We present a conceptual framework to organize genetic perturbations with phenotypic readouts along a nested cellular continuum-from protein folding and biogenesis to trafficking, posttranslational modification, protein-protein interactions, and downstream signaling. We highlight recent advances in mapping allosteric networks, pharmacologic mechanisms, and multiphenotype screening technologies. These mechanistic insights are reshaping our understanding of protein function at the residue level and informing our understanding of drug action, allostery, the interpretation of rare variants, and protein engineering strategies.