Supercharged Phosphotriesterase for improved Paraoxon activity.

Phosphotriesterases (PTEs) represent a class of enzymes capable of efficient neutralization of organophosphates (OPs), a dangerous class of neurotoxic chemicals. PTEs suffer from low catalytic activity, particularly at higher temperatures, due to low thermostability and low solubility. Supercharging, a protein engineering approach via selective mutation of surface residues to charged residues, has been successfully employed to generate proteins with increased solubility and thermostability by promoting charge-charge repulsion between proteins. We set out to overcome the challenges in improving PTE activity against OPs by employing a computational protein supercharging algorithm in Rosetta. Here, we discover two supercharged PTE variants, one negatively supercharged (with -14 net charge) and one positively supercharged (with +12 net charge) and characterize them for their thermodynamic stability and catalytic activity. We find that positively supercharged PTE possesses slight but significant losses in thermostability, which correlates to losses in catalytic efficiency at all temperatures, whereas negatively supercharged PTE possesses increased catalytic activity across 25°C-55°C while offering similar thermostability characteristic to the parent PTE. The impact of supercharging on catalytic efficiency will inform the design of shelf-stable PTE and criteria for enzyme engineering.

Computational Design of Phosphotriesterase Improves V-Agent Degradation Efficiency.

Organophosphates (OPs) are a class of neurotoxic acetylcholinesterase inhibitors including widely used pesticides as well as nerve agents such as VX and VR. Current treatment of these toxins relies on reactivating acetylcholinesterase, which remains ineffective. Enzymatic scavengers are of interest for their ability to degrade OPs systemically before they reach their target. Here we describe a library of computationally designed variants of phosphotriesterase (PTE), an enzyme that is known to break down OPs. The mutations G208D, F104A, K77A, A80V, H254G, and I274N broadly improve catalytic efficiency of VX and VR hydrolysis without impacting the structure of the enzyme. The mutation I106 A improves catalysis of VR and L271E abolishes activity, likely due to disruptions of PTE's structure. This study elucidates the importance of these residues and contributes to the design of enzymatic OP scavengers with improved efficiency.

Biomolecularmodeling and simulation: a field coming of age.

We assess the progress in biomolecular modeling and simulation, focusing on structure prediction and dynamics, by presenting the field's history, metrics for its rise in popularity, early expressed expectations, and current significant applications. The increases in computational power combined with improvements in algorithms and force fields have led to considerable success, especially in protein folding, specificity of ligand/biomolecule interactions, and interpretation of complex experimental phenomena (e.g. NMR relaxation, protein-folding kinetics and multiple conformational states) through the generation of structural hypotheses and pathway mechanisms. Although far from a general automated tool, structure prediction is notable for proteins and RNA that preceded the experiment, especially by knowledge-based approaches. Thus, despite early unrealistic expectations and the realization that computer technology alone will not quickly bridge the gap between experimental and theoretical time frames, ongoing improvements to enhance the accuracy and scope of modeling and simulation are propelling the field onto a productive trajectory to become full partner with experiment and a field on its own right.

Impact of phenylalanines outside the dimer interface on phosphotriesterase stability and function.

We explore the significance of phenylalanine outside of the phosphotriesterase (PTE) dimer interface through mutagenesis studies and computational modeling. Previous studies have demonstrated that the residue-specific incorporation of para-fluorophenylalanine (pFF) into PTE improves stability, suggesting the importance of phenylalanines in stabilization of the dimer. However, this comes at a cost of decreased solubility due to pFF incorporation into other parts of the protein. Motivated by this, eight single solvent-exposed phenylalanine mutants are evaluated viarosetta and good correspondence between experiments and these predictions is observed. Three residues, F304, F327, and F335, appear to be important for PTE activity and stability, even though they do not reside in the dimer interface region or active site. While the remaining mutants do not significantly affect structure or activity, one variant, F306L, reveals improved activity at ambient and elevated temperatures. These studies provide further insight into role of these residues on PTE function and stability.