Engineering thermostability
Protein engineering and site-directed mutagenesis are used routinely to establish biological function and role of amino-acid residues in proteins, like the stability. The protein stability can be changed significantly by single or multiple mutations of specific amino acids, sometimes resulting in a beneficial effect. This type of work is however expensive and time-consuming so rapid prediction of how mutations might affect the stability of a protein is desirable. An important parameter in determining the protein stability, including thermostability, is the determination of the pKa value of all the ionizable residues in a protein, which however, experimentally, is also challenging and time consuming. Computational pKa prediction programs have therefore been developed [16]. One of these programs is PROPKA [17], that utilize a very fast empirical approach of pKa prediction. PROPKA is today one of the most widely used programs due to its ease of use and speed, at the same time yielding accurate results as compared to other programs [18]. Details of the method and implementation in PROPKA 3.0, the version used in this study, can be found elsewhere [19–21].