Glycine Substitution of Residues with Unfavored Dihedral Angles Improves Protein Thermostability

Single mutations that can substantially enhance stability are highly desirable for protein engineering. However, it is generally rare for this kind of mutant to emerge from directed evolution experiments. This study used computational approaches to identify hotspots in a diacylglycerolspecific lipase for mutagenesis with functional hotspot and sequence consensus strategies, followed by Delta Delta G calculations for all possible mutations using the Rosetta ddg_monomer protocol. Single mutants with significant Delta Delta G changes (<=-2.5 kcal/mol) were selected for expression and characterization. Three out of seven tested mutants showed a significantly enhanced thermostability, with Q282W and A292G in the catalytic pocket and D245G located on the opposite surface of the protein. Remarkably, A292G increased the T-50(15) (the temperature at which 50% of the enzyme activity was lost after a 15 min of incubation) by similar to 7 degrees C, concomitant with a twofold increase in enzymatic activity at the optimal reaction temperature. Structural analysis showed that both A292 and D245 adopted unfavored dihedral angles in the wild-type (WT) enzyme. Substitution of them by glycine might release a steric strain to increase the stability. In sum, substitution by glycine might be a promising strategy to improve protein thermostability.

Automated summary

This study used computational approaches and mutagenesis strategies to enhance the stability of a diacylglycerol-specific lipase. Several mutations were found to significantly improve the thermostability and enzymatic activity of the enzyme. Structural analysis revealed how these mutations can release steric strain and increase stability. This suggests that substitution with glycine could be a promising strategy for improving protein thermostability.