Synergistic stabilization of a double mutant in CI2 from anin-celllibrary screen

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 not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ/mol 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 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

“…Our analysis of the crystal structures of the four CI2 variants showed that the structures are very similar and only subtle structural changes are observed throughout the protein. 32 This picture is mirrored by our current NMR analysis. The very small changes in dynamics between the variants are thus scattered throughout the protein.…”

“…We expressed wild-type CI2 (UniProt: P01053, residues 22-84 with an additional N-terminal Met) and the three variants L49I, I57V, and L49I/I57V using previously described DNA constructs. 32 E. coli BL21(DE3) carrying the expression plasmid was grown in M9 minimal medium at 37 ºC to OD 600 =0.6 before induction with 0.4 mM isopropyl b-D-1-thiogalactopyranoside (IPTG). Four different isotope compositions of the M9 media were used.…”

“…50 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. 32 The proteins were placed in a cubic box of 30.66 nm 3 (resulting in 0.5 nm distance from the protein to all box edges) and solvated with 6728 to 6836 water molecules. The net charge of CI2 is zero, so no ions were needed to neutralize the system.…”

“…31 We recently showed that L49I destabilizes the folded state of CI2 (DDG f = 3.4±0.1 kJ/mol), while I57V stabilizes it slightly (DDG f = -2.2±0.1 kJ/mol). 32 Notably, the double mutant (L49I/I57V) is significantly more stable than either of the single mutants (DDG f = -3.8±0.1 kJ/mol; Figure 1). This synergistic stabilizing effect of the double mutant is unexpected and could not be predicted using commonly applied methods such as FoldX 33 and Rosetta.…”

A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern

“…We recently described a workflow employing second-site saturation-suppressor mutagenesis (SSSM) coupled to yeast surface display to rapidly identify stabilizing mutations (33). Recent work describes related approaches to identify stabilizing point mutants of GFP and CI2 from multi-mutant libraries in E. coli (47)(48)(49). In the present study, we employ this to identify stabilizing mutations of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2.…”