Abstract
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.
Highlights
There is a considerable interest in proteins as therapeutics, biochemical reagents and catalysts
The majority of the stable mutants have the occurrence frequency at mutated sites smaller than that of the were from x-ray crystallography (WT). This finding strongly suggests that FoldX and free energy calculations explore very different sequence spaces from the consensus analysis method
FoldX followed by free energy calculations are performed to identify stable single mutant candidates
Summary
There is a considerable interest in proteins as therapeutics, biochemical reagents and catalysts. There are three main protein engineering methods, namely directed evolution [1], sequence consensus [2] and rational design [3]. Rational design method relies on the 3D structure of a protein. The success of rational design depends on the accurate understanding of the relationship between protein structure and stability. Many computational screening methods have been developed to pursue this goal, such as CC/ PBSA [11], EGAD [12], FoldX [13], I-mutant 2.0 [14], Rosetta [15,16,17] and etc. A more recent study by Seeliger et al [19] using an alchemical free energy method to study point mutation effects on the thermostability of a barnase demonstrated much better accuracy. In the catalytic formation of the chromophores, the requirement for molecular oxygen as a cofactor [21] hinders their applications for anaerobic microorganisms and
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
