Accurate Stabilities of Laccase Mutants Predicted with a Modified FoldX Protocol

作者：Christensen Niels J; Kepp Kasper P^*

来源：Journal of Chemical Information and Modeling, 2012, 52(11): 3028-3042.

DOI：10.1021/ci300398z

Fungal laccases are multicopper enzymes of Industrial importance due to their high stability, Multifunctionality, and oxidizing power. This paper reports computational protocols that quantify the relative stability (Delta Delta G of folding) of mutants of higli-redox-potential laccases (TvLIIIb and PmiL) with up to 11 simultaneously imitated sites with good correlation,against experimental stability trends. Molecular dynamics. simulations of the two laccases show that FoldX is very structure sensitive, since all :mutants and the wild type must share structural configuration to avoid artifacts of local sampling. However, using the average of 50 MD snapshots of the equilibrated trajectories restores correlation (r similar to 0.7-0.9, r(2) similar to 049-0.81) and provides a root-mean-square accuracy of similar to 1.2 kcal/inol for Delta Delta G or 3.5 degrees C for T-50, suggesting that the time average of the crystal structure is recovered. MD averaged input also reduces the spread in Delta Delta G, suggesting that local FoldX sampling overestimates free energy changes because of neglected protein relaxation: FoldX can be viewed as a simple %26quot;linear interaction energy%26quot; Method using sampling of the wild type and mutant and a parametrized relative free energy function : Thus, we show. in this work that a,Substantial %26quot;hysteresis%26quot; of similar to 1 kcal/mol applies to FoldX,%26apos; and that an improved protocol that reverses calculations and uses the average obtained Delta Delta G enhances correlation with the experimental data.%26quot; As glycosylation is ignored in FoldX, its effect: on Delta Delta G must be additive to the amino acid mutations: Quantitative structure property relationships of the FoldX energy components produced a substantially improved laccase stability predictor with error%26apos;s of similar to 1 degrees C for T-50, vs 3-5 degrees C for.a standard FOldX protocol The developed model provides Insight into the phySical.fOrCeS governing the high stability of fungal laCcaSes most notably the hydrophobic and van der Waals. interactions in the folded state, Which provide most of the predictive power.

• 出版日期2012-11