Environmental and mutational modulation of collateral fitness effects informs their mechanisms

Fitness effects of mutations that do not arise from changes in a protein's ability to perform its physiological functions (called collateral fitness effects or CFEs) are an understudied aspect of fitness landscapes. We have previously systematically measured the CFEs of all possible single amino acid substitutions in four proteins and found the frequency of deleterious mutations to vary by two orders of magnitude. Of these proteins, TEM-1 β-lactamase had the highest frequency, and deleterious mutations caused TEM-1 aggregation. Here, we systematically measured TEM-1 collateral fitness landscapes in environments and situations expected to alter protein aggregation or protein stability. We found a moderate correlation between deleterious CFEs and predicted thermodynamic stability effects in TEM-1's α-domain. Empirically, we found that the frequency and magnitude of deleterious CFEs can be reduced by altering the growth environment to disfavor aggregation (i.e. reducing the growth temperature or shifting to minimal media) or by stabilizing TEM-1 (via the M182T mutation or the addition of the β-lactamase inhibitor avibactam to the growth medium). However, although raising the growth temperature to favor aggregation exacerbated deleterious CFEs of many mutations, many mutations' effects were reduced. Furthermore, although reductions in CFEs occurred with reductions in TEM-1 aggregation for some mutants, for many mutants, they did not. We propose that mutational destabilization exposes protein motifs that can cause deleterious CFEs, but that these motifs and those that cause aggregation are not necessarily the same motifs.