WP2 will develop the necessary tools to estimate the effect of specific point mutations on the free energy. Alchemical transformation of one amino acid into another will be accomplished using the free energy perturbation approach combined with soft-core potentials. While we previously used the linear interaction energy approach to probe the effect of point mutations at protein-protein interfaces, the theoretically exact machinery of free energy perturbation represents a better and more accurate alternative. Based on comparative bioinformatics analysis of sequences from organisms adapted to different working temperatures key mutations will be identified. The temperature dependence characteristic for the mutations will subsequently be examined by performing calculations at different temperatures to yield the enthalpy and entropy of the mutations.
Using systems for which experimental data is available will serve to assess and validate the performance of the methods and protocols. The Norwegian Structural Biology Centre and ArcticZymes will serve as our experimental collaborations to construct new mutants and provide detailed characterizations of their kinetic and thermodynamic properties.