Rational stabilization of enzymes by computational redesign of surface charge–charge interactions

Abstract

Here, we report the application of a computational approach that allows the rational design of enzymes with enhanced thermostability while retaining full enzymatic activity. The approach is based on the optimization of the energy of charge-charge interactions on the protein surface. We experimentally tested the validity of the approach on 2 human enzymes, acylphosphatase (AcPh) and Cdc42 GTPase, that differ in size (98 vs. 198-aa residues, respectively) and tertiary structure. We show that the designed proteins are significantly more stable than the corresponding WT proteins. The increase in stability is not accompanied by significant changes in structure, oligomerization state, or, most importantly, activity of the designed AcPh or Cdc42. This success of the design methodology suggests that it can be universally applied to other enzymes, on its own or in combination with the other strategies based on redesign of the interactions in the protein core.Until man duplicates a blade of grass, nature will laugh at his so-called scientific knowledge.Thomas Edison computational design ͉ protein engineering ͉ protein stability R ational engineering of proteins to enhance stability and yet retain their enzymatic activity is well motivated (1). One motivation is the practical significance of expanding the use of enzymes in many areas of the modern world, including protein therapeutics, enzymes for food industry, diagnostics, and other areas of industrial biotechnology. Another motivation is validation of the existing scientific knowledge. In this case, predictions made by the existing models for protein stability are subjected to thorough experiments, testing their applicability to protein design. In this paper, we present the results of rational design of enzymes with enhanced stability and unchanged enzymatic activity. This approach has 2 major differences from previously described successful protein design methods (2-5): (i) it concentrates only on the residues on the protein surface, and (ii) it optimizes just one type of interactions, namely, charge-charge interactions on the protein surface (6-15).One of the most important aspects of engineering proteins with enhanced stability, retaining the enzymatic activity, is often forgotten. However, for all of these design efforts to be practically useful, it is important that the engineered proteins retain their biological and enzymatic activity. This issue is particularly important when enhanced protein stability is achieved by redesigning the charge-charge interactions on the protein surface. Such redesign can lead to several potentially detrimental effects on the activity: (i) it can affect the electrostatic potential in the active center, thus reducing or even abolishing the activity; (ii) it can affect substrate/product binding and again reduce or abolish the enzymatic activity; or (iii) it can have effects on the kinetics of substrate binding and, thus, lower the activity via reduced rates of electrostatic steering (3,(16)(17)(18)(19)(20)(21)(22).To this end, it is imp...

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

“…We choose to obtain each binding free energy by combining data from three independent, local simulations (starting from different configurations). Each simulation is considered to sample one representative region of phase space, hence, their results are combined as if merging samples within the exponential average of FEP: (9) We assume additivity, meaning that the global free energy of internal hydration is estimated as the sum of local free energies weighted by the occupancy n i of each site: (10) In turn, the mean occupancy of each site is obtained from the binding free energies as: (11) C is a factor common to all sites in one protein, and is fitted so that the total predicted occupancy matches the average number of bound water molecules measured in long simulations, in other words: (12) We find the optimal value of C to be 0.6 for both the mesophile and thermophile.…”

Linking computation and experiments to study the role of charge–charge interactions in protein folding and stability

“…To experimentally demonstrate this, the design algorithm was applied to seven different proteins: ubiquitin, the activation domain of human procarboxypeptidase A2, the fibronectin type III domain of human tenascin, the N-terminal RNAbinding domain of human U1A protein, Fyn SH3 domain, human acylphosphatase, and CDC42 GTPase [62,[83][84][85]. These proteins differ in size (from 59 to 198 amino acid residues) and in tertiary fold topology.…”

“…Importantly, the designed proteins remain enzymatically active. In the case of CDC42 in addition to unperturbed GTPase activity, the interactions with cellular activator protein CDC42GAP also remained unchanged [85]. The substitutions in ACP and CDC42 were analyzed in terms of evolutionary conservation.…”

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

“…Larger errors were detected for mutations that introduced changes in the electrostatics of buried residues or large structure fluctuation: mutations to glycine, involving bulky and/or well packed residues, etc.. Due to the impossibility of scoring the entire sequence (mutation) space, several strategies have been developed to focus the search in smaller regions. These include: i) the identification of flexible backbone sites which can be rigidified [129,130] introducing salt bridges [131] and/or disulphide bonds [132]; ii) the optimization of surface charge-charge interactions [133][134][135]; iii) the optimization of core packing [136]; iv) the removal of unsatisfied buried polar groups [137]; v) the localization of critical residues in the active site entry tunnels, especially for cosolute tolerance, with MD [138] or other algorithms like our in-house software PELE [113].…”