Chemical chaperones assist intracellular folding to buffer mutational variations

Abstract

Hidden genetic variations have the potential to lead to the evolution of new traits. Molecular chaperones, which assist protein folding, may conceal genetic variations in protein-coding regions. Here we investigate whether the chemical milieu of cells has the potential to alleviate intracellular protein folding, a possibility that could implicate osmolytes in concealing genetic variations. We found that the model osmolyte trimethylamine N-oxide (TMAO) can buffer mutations that impose kinetic traps in the folding pathways of two model proteins. Using this information, we rationally designed TMAO-dependent mutants in vivo, starting from a TMAO-independent protein. We show that different osmolytes buffer a unique spectrum of mutations. Consequently, the chemical milieu of cells may alter the folding pathways of unique mutant variants in polymorphic populations and lead to unanticipated spectra of genetic buffering.

Figure 1

Chemical chaperone assists rapid attainment of native structure. (a) Single amino acid substitution renders MBP TMAO dependent. Fold change in refolding rates of DM-MBP, SM-MBP and Wt-MBP, in presence of TMAO as compared to spontaneous refolding. The rates were obtained by fitting the curves to single exponential equations. (b) Kinetic simulation output showing steady state level of unfolded protein increasing with decreasing folding rates. Accumulation of non-native protein with varying folding rates is shown for different rates of degradation of the non-native protein. Degradation rate was varied from 0.001 to 0.02 s⁻¹. (c) Induction of UPR as observed by increase in GFP fluorescence on Galactose induction of DM-MBP as compared to SM-MBP. GFP fluorescence was recorded using FACS. (d) UPR Induction observed by increase in GFP fluorescence on inducing DM-MBP expression in presence of different concentrations (50mM, 100mM, 200mM and 500mM) of TMAO. (e) Refolding rates of DM-MBP as a function of TMAO concentration. Effect of TMAO was measured by carrying out refolding of DM-MBP in buffer containing 100mM to 500mM TMAO. Goodness of fit is provided in Supplementary Figure 8. (f) In vitro refolding rate obtained at different concentrations of TMAO are plotted against the median UPR-induced GFP fluorescence obtained in S. cerevisiae, at the same concentrations of TMAO. All averages shown in panel a, e and f are averages of 3 independent experiments. (See also Supplementary Figure 1-10)

Figure 2

Rationally designing TMAO-dependent conditional activity. (a) Schematic of Glycine-duplet substitution library showing representative mutations and the pattern of mutations. Two consecutive residues were substituted by Glycine-Glycine residues to obtain a comprehensive library of Glycine-duplet substitutions. (b) The mutants were grown with gentamicin selection in absence of TMAO at 37°C (LB), in presence of TMAO at 37°C (TMAO), and in absence of TMAO at 30°C (30D). Growth normalized with respect to cells transformed with wtGmr, that grew equally well in these three conditions. Mutants are sorted according to their growth in LB and growth in the other two conditions is plotted as bars for each mutant. (c) Gentamicin resistance as obtained by growing each mutant in 5 different concentrations of gentamicin (2 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml and 32μg/ml) in presence and absence of TMAO. Growth is shown as a colormap with increasing color density representing increase in growth. Details of GmR Glycine-duplet mutants used for this analysis are mentioned at the bottom of the panel. (See also supplemental Figure 11-17)

Figure3

Analysis of TMAO assisted buffering. (a) Structural features of osmolyte and temperature sensitive mutants. First row: fractional Accessible Surface Area (ASA) of the main chain atoms of the residues calculated from the crystal structure 1bo4. Second row: secondary structure cartoon of 1bo4. Third to Seventh row: Color scale showing growth of different mutants at different conditions. Growth at 37°C(third row), in presence of TMAO at 37°C(fourth row), at 37°C in 200mM Proline (fifth row), at 37°C in Glycerol (sixth row), at 30°C (seventh row). Mutants exhibiting differential growth are enclosed in colored boxes. (b). Residues in GmR structure (1bo4) colored in orange, which when mutated to glycine-duplets lead to TMAO-dependent activity (left panel) or temperature-dependent activity (right panel). (c). Box plot showing side chain fractional ASA (top panel) and main chain fractional ASA (bottom panel) of all the residues that were mutated to obtain the library, mutants that are more active in presence of TMAO and mutants that are more active at 30°C. The significance is based on two tailed Students-t test. (d). Box plot for predicted ΔΔG (kcal⁻¹mol⁻¹ FU ) of all the residues mutated to obtain the library, mutants that are more active in presence of TMAO and -mutants that are more active at 30°C. Since ΔΔG_FU values do not follow a normal distribution, significance was calculated using non-parametric Mann-Whitney test (see also Supplementary Figure 18,19).

Figure4

Chemical chaperones have specific spectrum of mutational buffering. (a). Gentamicin resistance of 12 random mutants in presence of osmolytes is shown as colormap. WtGmR transformed cells were taken as control. (b) Properties of mutated residues are plotted. Conservation (top panel), aggregation propensity (middle panel) and main chain fractional accessible surface area (bottom panel) are shown along with the residues that were buffered by the osmolytes. Line in the middle panel at aggregation propensity value of 1 denotes the generally accepted cutoff value; peptide segments above this value are considered to be aggregation prone. The line in the lower panel signifies the ASA of 0.1. Residues lying below this line are considered to be completely buried. (c) Residues buffered by osmolytes are mapped on the crystal structure of GmR, 1bo4. Color codes are listed in the legend. (d) Residues involved in mutational buffering is mapped to the crystal structure of Ccdb, 3vub (e) Properties of CcdB mutants were analyzed as described in (b) (see also Supplementary Figure 20-27).

Figure5

Stress response pathways do not mediate osmolyte mediated buffering. (a) Expression levels of GroEL, DnaK and Trigger Factor in presence of osmolytes like TMAO, Proline and glycerol, measured using western-blotting (left panel)quantified and is denoted as bar graph (right panel). (b). color map showing growth of four mutants of glycine-duplet substitution library, transformed in ΔdnaK and ΔotsA strains, at different gentamicin concentrations.(c). Intracellular trehalose concentrations estimated in cells grown at different conditions. Triplicate measurements were made for each growth conditions and the experiments were repeated twice to average the values. (See also Supplementary Figure 28-34).