Engineering a More Thermostable Blue Light Photo Receptor <i>Bacillus subtilis</i> YtvA LOV Domain by a Computer Aided Rational Design Method

<div><p>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 <i>T</i>_m of the flavin mononucleotide based fluorescent protein <i>Bacillus Subtilis</i> 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.</p></div

Thermal denaturation of the WT FbFP, the single point mutant N124Y, and the triple mutant N107Y-N124Y-M111F.

<p>The fluorescence intensity of the bound FMN is used to monitor the protein denaturation. As can be seen, the mutants have higher percentages of fluorescence at elevated temperature than WT suggesting mutations increase FbFP thermostability.</p

Flowchart of designing thermostable FbFP mutants.

<p>Briefly, FoldX followed by FEC (free energy calculation) are used to search for potential thermostable single mutants, from which a dozen are selected for experimental tests. The distribution of thermostable mutants is analyzed to identify the “hot spot”. Then more mutants in the “hot spot” are calculated by FEC and those predicted to be more stable are tested by experiments. Finally all stabilizing mutants are pooled together and multiple mutants are combined to further improve the protein's stability.</p

Residues in close contact with I120 (A, B), F107 (C, D), F124 (E, F) and F111 (G, H) are labeled where (A, C, E, G) are from subunit 1 and (B, D, F, H) are from subunit 2.

<p>Residues in close contact with I120 (A, B), F107 (C, D), F124 (E, F) and F111 (G, H) are labeled where (A, C, E, G) are from subunit 1 and (B, D, F, H) are from subunit 2.</p

Melting temperatures of WT and mutant FbFP.

a<p><i>T</i>_m was not determined due to the weak fluorescence of the sample at room temperature.</p

Improving Trichoderma reesei Cel7B Thermostability by Targeting the Weak Spots

For proteins that denature irreversibly, the denaturation is typically triggered by a partial unfolding, followed by a permanent change (e.g., aggregation). The regions that initiate the partial unfolding are named “weak spots”. In this work, a molecular dynamics (MD) simulation and data analysis protocol is developed to identify the weak spots of Trichoderma reesei Cel7B, an important endoglucanase in cellulose hydrolysis, through assigning the local melting temperature (<i>T</i>_mp) to individual residue pairs. To test the predicted weak spots, a total of eight disulfide bonds were designed in these regions and all enhanced the enzyme thermostability. The increased stability, quantified by Δ<i>T</i>₅₀ (which is the <i>T</i>₅₀ difference between the mutant and the wild type enzyme), is negatively correlated with the MD-predicted <i>T</i>_mp, demonstrating the effectiveness of the protocol and highlighting the importance of the weak spots. Strengthening interactions in these regions proves to be a useful strategy in improving the thermostability of <i>Tr.</i> Cel7B

Locations of mutated sites exhibiting improved thermostability.

<p>WT residues of the mutated sites are highlighted in yellow and labeled in red. The two subunits are drawn in grey and dark cyan respectively. Residues H22, V25, N107, D109, M111, V120 and N124 are from the dimer interface. The figure was drawn based on FbFP x-ray structure 2PR5 by using Discovery Studio Visualizer program.</p