Engineering proteins to enhance thermal stability is a widely utilized approach for creating industrially relevant biocatalysts. The development of new experimental datasets and computational tools to guide these engineering efforts remains an active area of research. Thus, to complement the previously reported measures of T 50 and kinetic constants, we are reporting an expansion of our previously published dataset of mutants for β-glucosidase to include both measures of T M and ΔΔG. For a set of 51 mutants, we found that T 50 and T M are moderately correlated, with a Pearson correlation coefficient and Spearman’s rank coefficient of 0.58 and 0.47, respectively, indicating that the two methods capture different physical features. The performance of predicted stability using nine computational tools was also evaluated on the dataset of 51 mutants, none of which are found to be strong predictors of the observed changes in T 50, T M, or ΔΔG. Furthermore, the ability of the nine algorithms to predict the production of isolatable soluble protein was examined, which revealed that Rosetta ΔΔG, FoldX, DeepDDG, PoPMuSiC, and SDM were capable of predicting if a mutant could be produced and isolated as a soluble protein. These results further highlight the need for new algorithms for predicting modest, yet important, changes in thermal stability as well as a new utility for current algorithms for prescreening designs for the production of mutants that maintain fold and soluble production properties.

The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

Engineering proteins to enhance thermal stability is a widely utilized approach for creating industrially relevant biocatalysts. Computational tools that guide these engineering efforts remain an active area of research with new data sets and develop algorithms. To aid in these efforts, we are reporting an expansion of our previously published data set of mutants for a -glucosidase to include both measures of TM and G, to complement the previously reported measures of T50 and kinetic constants (kcat and KM). For a set of 51 mutants, we found that T50 and TM are moderately correlated with a Pearson correlation coefficient (PCC) of 0.58, indicated the two methods capture different physical features. The performance of predicted stability using five computational tools are also evaluated on the 51 mutants dataset, none of which are found to be strong predictors of the observed changes in T50, TM, or G. Furthermore, the ability of the five algorithms to predict the production of isolatable soluble protein is examined, which revealed that Rosetta ΔΔG, ELASPIC, and DeepDDG are capable of predicting if a mutant could be produced and isolated as a soluble protein.These results further highlight the need for new algorithms for predicting modest, yet important, changes in thermal stability as well as a new utility for current algorithms for prescreening designs for the production of soluble mutants. ASSOCIATED CONTENT Supporting Information (SI)SI 1-1. SDS-PAGE images for 51 BglB mutants and WT. (PDF) SI 1-2. A distribution analysis of temperatures observed for TM and T50. SI 1-3. PPC graph between ΔTM and ΔΔG of BglB mutants. (PDF) SI 1-4. Evaluation of five computational methods on protein expression. (PDF) SI 2. Images of TM fluorescence graphs, derivative graphs, and Van't Hoff plot for 51 mutants and WT. (Zip) SI 3. Rosetta ΔΔG and FoldX PSSM correlations graphs with ΔTM and experimental ΔΔG. Excel files of all the parameters from data acquisition. Excel files of the total system energy for DeepDDG, ELASPIC, and PoPMuSiC (Zip)SI 4. Jupyter notebook for all thermal stability data acquisitions with all TM raw data files. (ipynb) SI 5. Example files for Rosetta_ddg_monomer run. (Zip)