The N-terminal domain of ribosomal protein L9 (NTL9) contains a small three-stranded anti-parallel β-sheet sandwiched between two helices. NTL9 is one of the simplest examples of a common supersecondary structural motif, the split β-α-β motif. Additionally, the effects of electrostatic interactions in the denatured state ensemble (DSE) on the overall stability of NTL9 are known to influence stability changes caused by mutations. The EmCAST (Empirical C-Alpha Stability Tool) represents a promising computational tool for predicting the impact of mutations on protein stability (www.emcast.org). It optimizes protein structures using Cα dihedral angle preferences of four-residue sequences in the Protein Data Bank, providing a way to enhance stability through targeted mutations. Since EmCAST primarily predicts stability based on structured regions, NTL9 provides a useful system for testing how much changes in DSE stability affect the accuracy of EmCAST predictions. In this study, we applied EmCAST and GdnHCl denaturation monitored by circular dichroism (CD) spectroscopy to analyze the stability of wild-type NTL9 and various mutants. Our goal was to evaluate the predictive power of EmCAST in identifying stabilizing mutations in an α/β domain where the DSE affects stability. EmCAST predicted that K10P, A22P, G24E or G24D, E38P, and N43A mutations would enhance NTL9 stability. Thesemutations if all added to NTL9 were predicted to stabilize NTL9 by 3.27 kcal/mol. Our results demonstrate close agreement for some of these mutation sites, demonstrating the efficacy of EmCAST for predicting and improvingstability. However, our experimental results revealed discrepancies in certain cases such as E38P and N43A. These two variants demonstrate that long-range hydrogen bonds are not well-accounted for by EmCAST when it predicts stabilizing mutations. Future studies could refine EmCAST to account for these types of long-range interactions.
Esfahani et al. (Sun,) studied this question.