Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli

Abstract

Most single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is often not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay in E. coli to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ mol−1 more stable than the wild-type. In one case, the increased stability is the effect of the single substitution D55G. The other case is a double mutant, L49I/I57V, which is 5.1 kJ mol−1 more stable than the sum of the effects of the individual mutations. In addition to demonstrating the strength of our selection system for finding stabilizing mutations, our work also demonstrate how subtle conformational effects may modulate stability.

Double Mutant of Chymotrypsin Inhibitor 2 Stabilized through Increased Conformational Entropy

“…We performed the simulations using the Amber a99SB- disp force field with the TIP4P-D-based a99SB- disp water model . Crystal structures of the CI2 WT (PDB: 7A1H) and its mutated variants L49I (PDB: 7AOK), I57V (PDB: 7A3M), and L49I/I57V (PDB: 7AON) were used as the starting conformations for the simulations . The proteins were placed in a cubic box with a volume of 30.66 nm 3 (resulting in a 0.5 nm distance from the protein to all box edges) and solvated with 6728 to 6836 water molecules.…”

“…We recently showed that L49I destabilizes the folded state of CI2 (ΔΔ G f = 3.4 ± 0.1 kJ/mol), while I57V stabilizes it slightly (ΔΔ G f = −2.2 ± 0.1 kJ/mol) . Notably, the double mutant (L49I/I57V) is significantly more stable than either of the single mutants (ΔΔ G f = −3.8 ± 0.1 kJ/mol; Figure ).…”

Increasing protein stability by inferring substitution effects from high-throughput experiments

“…We therefore asked whether it would have been possible to point them out as likely candidates beforehand. We calculated variant effects using two methods; structure based stabilities calculations using Rosetta and a direct coupling analysis (lbsDCA) of a multiple sequence alignment (MSA) of natural thioredoxins as such analyses have previously been shown to predict stability effects 22,30,31 (Figure 4). While both methods gave rise to reasonable overall Pearson correlations of 0.54 and 0.59 respectively, they failed to identify many of the substitutions which were inferred to be stabilizing (negative ΔΔG-values) in our GMMA.…”

“…More quantitative analyses have been enabled by a thermodynamic model that considers doubly substituted protein variants to infer the effect of single amino acid substitutions on both protein-protein interaction and folding free energies 18,19 , which was later shown to match chemical unfolding stabilities well 20 . We have recently used a folding sensor based on a bacterial heat shock response 21 to select for variants with improved thermodynamic stability of an already stable protein 22 . In line with this, we have shown that single substitution effects may be obtained by analysing the results of a DMS experiment with many diverse multiple-substituted protein variants, and suggested a global multi-mutant analysis (GMMA) for this task 23 .…”

A dual-reporter system for investigating and optimizing protein translation and folding in E. coli

“…Even for systems with a lower dynamic range, it has previously been shown possible to identify stabilized protein variants 23 , 49 . In a recent study, the present reporter system has been used to successfully identify stabilized variants of the CI2 protein 50 .…”