Soluble oligomerization provides a beneficial fitness effect on destabilizing mutations

Abstract

Mutations create the genetic diversity on which selective pressures can act, yet also create structural instability in proteins. How, then, is it possible for organisms to ameliorate mutation-induced perturbations of protein stability while maintaining biological fitness and gaining a selective advantage? Here we used site-specific chromosomal mutagenesis to introduce a selected set of mostly destabilizing mutations into folA--an essential chromosomal gene of Escherichia coli encoding dihydrofolate reductase (DHFR)--to determine how changes in protein stability, activity, and abundance affect fitness. In total, 27 E. coli strains carrying mutant DHFR were created. We found no significant correlation between protein stability and its catalytic activity nor between catalytic activity and fitness in a limited range of variation of catalytic activity observed in mutants. The stability of these mutants is strongly correlated with their intracellular abundance, suggesting that protein homeostatic machinery plays an active role in maintaining intracellular concentrations of proteins. Fitness also shows a significant correlation with intracellular abundance of soluble DHFR in cells growing at 30 °C. At 42 °C, the picture was mixed, yet remarkable: A few strains carrying mutant DHFR proteins aggregated, rendering them nonviable, but, intriguingly, the majority exhibited fitness higher than wild type. We found that mutational destabilization of DHFR proteins in E. coli is counterbalanced at 42 °C by their soluble oligomerization, thereby restoring structural stability and protecting against aggregation.

Fig. 1.

The experimental approach. (1) 10 DHFR residues, predominantly from the hydrophobic core and at least 4 Å from the NADPH and dihydrofolate binding sites, were chosen for mutagenesis based on the structural and phylogenetic predictions and published biophysical and biochemical data. (2) Sixteen single mutants (Table 1) were generated, cloned into pET vector, expressed, and purified (see SI Materials and Methods). (3) Gibbs free energy difference between folded and unfolded states (ΔG), apparent midtransition temperature of unfolding (), and catalytic parameters (k_cat, K_m) were measured (Table 1 and SI Materials and Methods). (4) A site-directed chromosomal mutagenesis method was developed to introduce in vitro characterized mutations into chromosomal folA gene of E. coli’s MG1655 strain without perturbing the gene’s regulatory region. In addition to 16 single mutant strains, 11 multiple mutant strains were generated by combining exhaustively four most destabilizing mutations (V75H, I91L, W133F, I155A) (Table S1). (5) Fitness effects of the introduced mutations (in total, 27 strains) were measured by growth competition of mutant strains with WT DHFR strain.

Fig. 2.

Correlating fitness at 30 °C with molecular properties. (A) Fitness measured at 30 °C by competing 27 DHFR mutant strains with a reference WT DHFR strain is plotted against insoluble fraction of DHFR proteins determined in cell lysates by Western blot. Fitness of the WT DHFR strain and its abundance in the insoluble fraction is set to one. (B) Soluble protein abundance (blue circles) determined for single DHFR mutant strains is plotted against apparent midtransition temperature of unfolding () measured in vitro by DSC. Protein abundance predicted at 30 °C by Eq. 1 is depicted in red. Predicted fraction somewhat deviates from a plateau because of the experimental imprecision of ΔG values derived from the urea unfolding under two-state assumption and values inferred from the DSC thermograms (Fig. S4). (C) Fitness measured at 30 °C by competing 27 DHFR mutant strains with a reference WT DHFR strain is plotted against soluble fraction of DHFR proteins determined in cell lysates by Western blot. Fitness of the WT DHFR strain and its abundance in the soluble fraction is set to one.

Fig. 3.

Distribution of fitness effects and correlation with stability. (A–C) Histograms of distributions of fitness effects at 30 °C, 37 °C, and 42 °C for all mutant DHFR strains (16 single and 11 multiple mutants) as measured by competition with a reference WT DHFR strain. Fitness of the reference strain is set to 1. Individual fitness values can be found at Table S2. (D–F) Correlation of fitness values measured for single mutant DHFR strains by competition at 30 °C, 37 °C, and 42 °C to in vitro measured .

Fig. 4.

In vitro oligomerization assay. (A) A representative cross-linking experiment. 11 μM of WT DHFR, W133V, or I155A purified proteins in 25 mM potassium-phosphate buffer (pH 7.8) and 100 μM NADPH were incubated for 45 min at room temperature (RT) or 42 °C with or without 2 mM of cross-linking (c-l) agent (glutaraldehyde). Shown is the SDS/PAGE analysis of 10 μL of protein samples after Coomassie staining. Red arrows indicate molecular weight equivalent of monomeric and oligomeric protein species. Smears at high molecular weight seen for W133V protein at 37 °C and 42 °C in the presence of c-l are due to extensive aggregation. (B) The in vitro propensity to oligomerize is correlated to fitness values measured by competition at 42 °C. Oligomerization propensity was determined by measuring the relative density of the SDS/PAGE bands corresponding to all DHFR oligomerized species for each of the purified mutant (Fig. S8). The degree of oligomerization for every mutant is normalized to WT DHFR. (C) Correlation of oligomerization propensity (as measured in B) to mutant’s .