The ability to design thermostable proteins offers enormous potential for the development of novel protein bioreagents. In this work, a combined computational and experimental method was developed to increase the T m of the flavin mononucleotide based fluorescent protein Bacillus Subtilis YtvA LOV domain by 31 Celsius, thus extending its applicability in thermophilic systems. Briefly, the method includes five steps, the single mutant computer screening to identify thermostable mutant candidates, the experimental evaluation to confirm the positive selections, the computational redesign around the thermostable mutation regions, the experimental reevaluation and finally the multiple mutations combination. The adopted method is simple and effective, can be applied to other important proteins where other methods have difficulties, and therefore provides a new tool to improve protein thermostability.
Song, X., Wang, Y., Shu, Z., Hong, J., Li, T., & Yao, L. (2013). Engineering a More Thermostable Blue Light Photo Receptor Bacillus subtilis YtvA LOV Domain by a Computer Aided Rational Design Method. PLoS Computational Biology, 9(7). https://doi.org/10.1371/journal.pcbi.1003129