Protein quality control acts on folding intermediates to shape the effects of mutations on organismal fitness

Abstract

What are the molecular properties of proteins that fall on the radar of protein quality control (PQC)? Here we mutate the E. coli's gene encoding dihydrofolate reductase (DHFR) and replace it with bacterial orthologous genes to determine how components of PQC modulate fitness effects of these genetic changes. We find that chaperonins GroEL/ES and protease Lon compete for binding to molten globule intermediate of DHFR, resulting in a peculiar symmetry in their action: overexpression of GroEL/ES and deletion of Lon both restore growth of deleterious DHFR mutants and most of the slow-growing orthologous DHFR strains. Kinetic steady-state modeling predicts and experimentation verifies that mutations affect fitness by shifting the flux balance in cellular milieu between protein production, folding, and degradation orchestrated by PQC through the interaction with folding intermediates.

Figure 1. GroEL/ES over-expression and lon deletion rescue a near-lethal phenotype of a DHFR mutant

Growth curves of wt and mutant (I91L+W133V) DHFR strains at 42°C. (A, B) Strains with (red) and without (blue) GroEL/ES over-expression. (C, D) Strains carrying lon gene (blue) or lon deletion (red). Error bars represent s.d. of 16 independent measurements.

Figure 2. The effect of GroEL/ES over-expression and lon deletion on the growth rates of DHFR mutant strains

(A) Growth rates of the DHFR mutant strains at 30 °C, 37 °C and 42°C were sorted from the lowest to the highest (black). Growth rates obtained with GroEL/ES over-expression (right) and lon deletion (left) are shown in red. (See also Table S1). Dashed line indicates the wt DHFR strain. Error bars represent s.d. of 16 independent measurements. (B – D) Change in the growth rates of DHFR mutants upon lon deletion and GroEL/ES over-expression is linearly correlated at all temperatures tested (30°C, 37°C, and 42°C). Change in growth rate is calculated by subtracting the growth rates under endogenous conditions from the growth rates upon lon deletion or GroEL/ES over-expression. Error bars for changes in the growth rate were calculated by summation of the relative s.d. of endogenous and lon deletion or GroEL/ES over-expression values. R, and P-values are calculated by ANOVA.

Figure 3. Detection of folding intermediate by bis-ANS fluorescence

Bis-ANS fluorescent probe was used in vitro to monitor the population of folding intermediates during thermal unfolding of the purified DHFR proteins (25°C –70°C) (see Experimetal Procedures). Panels A and B show representative behavior, the data for all mutants are in Figure S3. (A) Wild-type DHFR protein is characterized by a very low fluorescent signal up to 50°C and a sharp transition through a folding intermediate around 60°C (T_m, mid-transition temperature). (B) V75H+I155A DHFR mutant exhibits a very pronounced folding intermediate already at room temperature (25°C) that tends to disappear with temperature increase due to unfolding. (C) T_m^app values of the DHFR proteins derived from bis-ANS profiling during thermal unfolding (see also Figure S3) is correlated with T_m^app values derived from the thermal unfolding measured by Differential Scanning Calorimetry (Bershtein et al., 2012).

Figure 4. Dependence of fitness on protein abundance

(A) Positions of lacO binding sites introduced upstream of the folA gene to provide IPTG-controllable expression (see Experimental Procedures for a detailed description). Nucleotide sequence represents the non-coding area upstream to folA ORF. Nucleotides in red denote the sequence of the introduced lacO V and IV operators. Underlined sequences represent the −33 and −10 promoter signals. Low case nucleotides indicate the initiation of transcription (+1). The first ATG of the coding sequence is in italic. CAT upstream to the ATG codon was introduced for the cloning purposes. (B) The effect of down-regulation of the wt DHFR expression on growth rate at 30°C. Growth rates of the B3 strain with IPTG-controlled folA expression were measured in the range of IPTG concentrations (see Experimental Procedures) and presented as a function of the corresponding DHFR expression level. Solid line is a fit to Michaelis-Menten-like kinetics (see Experimental Procedures). Error bars represent s.d. of 4 independent experiments.

Figure 5. Protein abundance is correlated with population of folding intermediate (I)

(A) Abundance of the insoluble DHFR fraction detected for all lon deleted DHFR mutant strains is correlated to bis-ANS fluorescence measured in vitro at 30°C. (B) Insoluble fraction of the DHFR proteins in lon+ strains does not correlate with bis-ANS fluorescence (see also Figure S6). Insoluble abundance is normalized against V75H+I91L DHFR strain (arbitrary set to 1). (C) Intracellular protein abundance of the soluble DHFR proteins in the strains grown at 30°C inversely correlates with bis-ANS fluorescence, as predicted by Eq. 3. Abundances are normalized against wt DHFR (arbitrary set to 1). bis-ANS fluorescence is normalized against wt DHFR (arbitrary set to 1), see also Table S2. Error bars respresent s.d. of 3 independent experiments.

Figure 6. The effect of GroEL/ES over-expression and lon deletion on the growth rates of DHFR strains with orthologous replacements of the folA gene

(A) Growth rates of the strains at 30 °C, 37 °C and 42°C were sorted from the lowest to the highest (black). Growth rates obtained with GroEL/ES over-expression (right) and lon deletion (left) are shown in red (see Experimental Procedures and Table S3). Dashed line marks the strain expressing E. coli’s DHFR. Error bars represent s.d. of 16 independent measurements. (B – D) Change in the growth rates of the orthologously replaced DHFR strains upon lon deletion and GroEL/ES over-expression is linearly correlated at all temperatures tested (30°C, 37°C, and 42°C). Change in growth rate is calculated by subtracting the growth rates under endogenous conditions from the growth rates upon lon deletion or GroEL/ES over-expression. Error bars for changes in the growth rate were calculated by summation of the relative s.d. of endogenous and lon deletion or GroEL/ES over-expression values. R, and P-values are calculated by ANOVA.

Figure 7. Distribution of growth rates of DHFR strains with orthologous replacements of the folA gene

The effect of Δlon, GroEL+, and Δlon with GroEL+ on the distribution of growth rates of the orthologous DHFR strains was measured at 30°C (upper panel), 37°C (middle panel), and 42°C (lower panel). σ is standard deviation. Dashed line indicates the growth rate of the strain expressing E. coli’s DHFR.