Interestingly, in the evolved KE07 variant some mutated residues destabilized the transition state (Figure 2). Residue contributions to the reorganization energy were used to screen for mutations that facilitate evolution of the

original KE07 design. Residues, which did not compromise the reorganization energy were selected [28•]. The predicted mutations were in agreement with libraries of active variants from different rounds of directed evolution [37]. This indicates that screening should also allow those residues, which are not involved in catalysis directly, but enable structural changes required along the pathway. KE07 analysis also demonstrates that reorganization energy can be optimized during evolution via small rotamer changes and smaller scale rearrangements in the electrostatic interaction pattern. Besides KE07 Kemp eliminase, further examples selective HDAC inhibitors indicate that electrostatic preorganization could be tuned in directed evolution [6••, 31 and 32••]. This implies that variants, where the preorganization

effect was maximized, could serve as promising starting points for further laboratory optimization. As reorganization energy is invested upon protein folding [44], so evaluating it could affect scaffold ranking and selection. The proposed flowchart of the computer aided design complemented by reorganization energy calculations is shown in Figure 3. First, ab initio calculations are employed to determine the reaction mechanism, the TS geometry and the parameters for the energy functionals for the reactant and the product state. Second, a high-throughput scaffold search is performed based on shape complementarity this website from and TS binding energy. Third, global reorganization energy is computed for top-ranked scaffolds, and will serve as a basis of filtering. Selected variants will be further optimized based on comparing individual reorganization energy contributions of the original and mutated residues. Successful enzyme designs provide insights

into how catalysis can be evoked. The performance of artificial enzymes varies in a wide range, but even with the assistance of directed evolution remains inferior to natural enzymes. Moderate efficiency of man-made constructs indicates the absence of a major catalytic factor, which can also be optimized in laboratory. Electrostatic preorganization has dominant contribution to the catalytic effect and it can also be significantly improved by directed evolution. On the basis of the reactant and product energy functions, reorganization energy can be computed in an economical manner and individual contributions can be determined. We propose to utilize global reorganization energy for refinement and final evaluation of top-ranked scaffolds. Screening based on individual contributions can result in variants similar to evolved libraries, which also include stabilizing or compensatory mutations in addition to those, which have direct impact on catalysis.