(27q) Probing the Origins of Collateral Fitness Effects? of Mutations

Our modern understanding of protein evolution is constrained by the ability to determine the origin of fitness effects of mutations. While much is known about how changes in the ability of protein to perform its cellular function affect fitness (primary fitness effects), little is known about fitness effects that are unrelated to the protein’s cellular function (collateral fitness effects). Using deep mutational scanning, our lab has previously determined the collateral fitness effects of all single amino acid substitutions in TEM-1 β-lactamase by measuring fitness effects of mutations in the absence of β-lactam antibiotics. Over 42% of mutations caused deleterious collateral fitness effects. We selected over thirty such mutations for further study. All caused protein misfolding and aggregation, suggesting that aggregation might be the cause of the deleterious effects. Here, we used deep mutational scanning to probe how stabilization and destabilization of the folded state modulates collateral fitness effects. Collateral fitness effects of TEM-1 mutations were measured in the presence of β-lactamase inhibitors avibactam and tazobactam. Avibactam is a reversible binder and stabilizes the folded structure as it is a substrate mimic. Tazobactam is a suicide inhibitor that forms a covalent complex with the enzyme and destabilizes the folded structure. In addition, we use mass spectrometry to examine if mutations with deleterious collateral fitness effects cause TEM-1 to co-aggregate with other E. coli proteins.