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.
Highlights
Most single point mutations destabilize folded proteins
The algorithms are better at predicting deletion mutations in the hydrophobic core, than mutations that increase the size of the side chain, mutations on the protein surface and mutations where electrostatic interactions contribute to the stabilization
To select variants of chymotrypsin inhibitor 2 (CI2) that are more stable than the starting point we opted to use a destabilized background as starting point
Summary
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. The algorithms are better at predicting deletion mutations in the hydrophobic core, than mutations that increase the size of the side chain, mutations on the protein surface and mutations where electrostatic interactions contribute to the stabilization This is partly a result of the data available for training the algorithms that mainly consist of deletion mutations in the hydrophobic core[26], A particular challenge in predicting stabilizing protein variants is that among the few single substitutions that are stabilizing the effects are often small, so that multiple substitutions may be needed to create a substantial stabilizing effect[17]. CI2 has been extensively studied by mutagenesis, only few variants that stabilize the protein relative to the wild-type are reported and all of these are substitutions of R48 with a hydrophobic residue[35] Extending this set of stabilized CI2 variants could, in the context of the extensive amount of data available for CI2, provide additional insight into how multiple mutations may interact to stabilize a protein
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