Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo. This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations.
Submitter: Marie Ary
Submission Date: March 13, 2017, 4:37 p.m.
|Number of data points||5707|
|Assays/Quantities/Protocols||Experimental Assay: CcdA binding to refolded mutants (TSA) ; Experimental Assay: fast phase refolding rate constant (k1) ; Experimental Assay: MSseq (BWpTig) ; Experimental Assay: unfolding rate constant (K1) ; Experimental Assay: amplitude for unfolding (A1) ; Experimental Assay: amplitude for burst phase for refolding (a0) ; Experimental Assay: amplitude for burst phase for unfolding (A0) ; Experimental Assay: amplitude for fast phase of refolding (a1) ; Experimental Assay: MSseq (BWΔTig) ; Experimental Assay: MSseq (BWΔlon) ; Experimental Assay: MSseq (BWΔclpP) ; Experimental Assay: Fractional increase in solubility (Tig) ; Experimental Assay: Fractional increase in solubility (SecB) ; Experimental Assay: MSeq (BWΔSecB) ; Experimental Assay: Gyrase binding to refolded mutants (BLI) ; Experimental Assay: [GdmCl]m ; Experimental Assay: Tm (apparent) ; Experimental Assay: slow phase refolding rate constant (k2) ; Experimental Assay: Tm increase upon CcdA binding ; Experimental Assay: amplitude for slow phase of refolding (a2) ; Experimental Assay: MSseq (BWΔhchA) ; Experimental Assay: MSseq ; Experimental Assay: Soluble fraction ; Experimental Assay: Relative in vivo activity (compared to WT) ; Experimental Assay: MSseq (BW25113) ; Experimental Assay: MSseq (BWpSecB) ; Experimental Assay: Insoluble fraction ; Experimental Assay: Relative RU change upon Gyrase binding (compared to WT) ; Derived Quantity: ΔTm (WT−mutant) ; Computational Protocol: Residue depth from surface ; Computational Protocol: Side chain solvent accessibility|
|Libraries||In vivo activity and solubility in presence and absence of ATP-independent chaperones (Table 4) ; Correlating in vivo solubility and activity with gyrase binding (Fig. S4C) ; Kinetic parameters for refolding and unfolding; CcdA and gyrase binding (Table 3) ; Mutational sensitivity, side-chain accessibility, and depth for all the sequenced point mutants of CcdB (1664 mutants) (S1 Appendix) ; In vivo solubilities, apparent Tms, etc. of selected mutants (Table S5) ; GdmCl denaturation of selected mutants (to compare with apparent Tms) (Table S8) ; In vivo activity in protease deletion strains (Table S9)|