Probing Protein Sequence Function Relationships using Deep Mutational Scanning

Abstract

Deep Mutational Scanning (DMS) approaches help elucidate sequence-function-phenotype relationships in proteins, which ultimately improves our understanding of residue (or nucleotide)-specific contributions to protein function and organismal fitness. Such comprehensive knowledge finds use in reliable prediction of consequences of mutations, as well as in protein design and engineering. We have investigated the molecular mechanisms of how mutations at surface exposed sites, away from the active sites in a model protein, CcdB produce drastic defects on the protein s activity and organismal phenotype. Subsequently, we have surveyed double mutant libraries of CcdB, using a DMS approach, to identify mutants that can suppress the inactive phenotypes of exposed non active-site mutants in CcdB. These studies provide insights into the generally overlooked mutations that alter the fraction of active protein expressed, without affecting the activity of the folded fraction. We next describe a facile DMS method to accurately estimate binding energetics of protein-protein interactions (PPIs) and have used this methodology to probe residue specific contributions to partner binding in an intrinsically disordered protein CcdA. We also developed a model based on the CcdA mutational landscape to predict mutational effects on binding affinities in other IDPs. Using the insights from the CcdA mutational study, we also describe how Aspartate Scanning can be used to predict interface residues and local secondary structures for the MazF6 interacting, intrinsically disordered domain of MazE6 protein. This rapid and inexpensive methodology is readily applicable to experimentally unexplored, protein-interacting, intrinsically disordered domains. Finally, we also investigate how mutations in the ccdA gene can affect the protein s activity and phenotype of the bacterial cell harboring it, in its native operonic context, and found a surprisingly high sensitivity to mutations (including synonymous mutations) in a manner dependent on the codo...