Engineering proteins to withstand a broad range of conditions continues to be a coveted objective, holding the potential to advance biomedicine, industry, and our understanding of disease. One way of achieving this goal lies in elucidating the underlying interactions that define protein stability. It has been shown that the hydrophobic effect, hydrogen bonding, and packing interactions between residues in the protein interior are dominant factors that define protein stability. The role of surface residues in protein stability has received much less attention. It has been believed that surface residues are not important for protein stability particularly because their interactions with the solvent should be similar in the native and unfolded states. In the case of surface charged residues, it was sometimes argued that solvent exposure meant that the high dielectric of the solvent will further decrease the strength of the charge-charge interactions. In this paper, we challenge the notion that the surface charged residues are not important for protein stability. We computationally redesigned sequences of five different proteins to optimize the surface charge-charge interactions. All redesigned proteins exhibited a significant increase in stability relative to their parent proteins, as experimentally determined by circular dichroism spectroscopy and differential scanning calorimetry. These results suggest that surface charge-charge interactions are important for protein stability and that rational optimization of charge-charge interactions on the protein surface can be a viable strategy for enhancing protein stability.

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...

Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb2+ on the structure and membrane-binding properties of C2α. C2α is a peripheral membrane-binding domain of Protein Kinase Cα (PKCα), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2α binds Pb2+ with higher affinity than its natural cofactor, Ca2+. To gain insight into the coordination geometry of protein-bound Pb2+, we determined the crystal structures of apo and Pb2+-bound C2α at 1.9 Å and 1.5 Å resolution, respectively. A comparison of these structures revealed that the metal-binding site is not pre-organized and that rotation of the oxygen-donating sidechains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb2+ ions coexist within a single protein molecule. Using protein-to-membrane Förster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb2+ displaces Ca2+ from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb2+ associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb2+ on the PKCα activity.

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