A Fast and Precise Approach for Computational Saturation Mutagenesis and its Experimental Validation by Using an Artificial (beta alpha)(8)-Barrel Protein

作者：Fischer Andre; Seitz Tobias; Lochner Adriane; Sterner Reinhard; Merkl Rainer; Bocola Marco^*

来源：ChemBioChem, 2011, 12(10): 1544-1550.

DOI：10.1002/cbic.201100051

• beta-alpha-barrels
• molecular dynamics
• molecular modeling
• mutagenesis
• protein engineering

We present a computational saturation mutagenesis protocol (CoSM) that predicts the impact on stability of all possible amino acid substitutions for a given site at an internal protein interface. CoSM is an efficient algorithm that uses a combination of rotamer libraries, side-chain flips, energy minimization, and molecular dynamics equilibration. Because CoSM considers full side-chain and backbone flexibility in the local environment of the mutated position, amino acids larger than the wild-type residue are also modeled in a proper manner. To assess the performance of CoSM, the effect of point mutations on the stability of an artificial (beta alpha)(8)-barrel protein that has been designed from identical (beta alpha)(4)-half barrels, was studied. In this protein, position 234(N) is a previously identified stability hot-spot that is located at the interface of the two half barrels. By using CoSM, changes in protein stability were predicted for all possible single point mutations replacing wild-type Val234(N). In parallel, the stabilities of 14 representative mutants covering all amino acid classes were experimentally determined. A linear correlation of computationally and experimentally determined energy values yielded an R-2 value of 0.90, which is statistically significant. This degree of coherence is stronger than the ones we obtained for established computational methods of mutational analysis.

• 出版日期2011-7-4