Assessing the predictive capabilities of design heuristics and coarse-grain simulation toward understanding and optimizing site-specific covalent immobilization of β-lactamase.

For industrial applications, covalent immobilization of enzymes provides minimum leakage, recoverability, reusability, and high stability. Yet, the suitability of a given site on the enzyme for immobilization remains a trial-and-error procedure. Here, we investigate the reliability of design heuristics and a coarse-grain molecular simulation in predicting the optimum sites for covalent immobilization of TEM-1 β-lactamase.

Assessing site-specific PEGylation of TEM-1 β-lactamase with cell-free protein synthesis and coarse-grained simulation.

PEGylation is a broadly used strategy to enhance the pharmacokinetic properties of therapeutic proteins. It is well established that the location and extent of PEGylation have a significant impact on protein properties. However, conventional PEGylation techniques have limited control over PEGylation sites.

Coarse-grained simulation of PEGylated and tethered protein devices at all experimentally accessible surface residues on β-lactamase for stability analysis and comparison.

PEGylated and surface-tethered proteins are used in a variety of biotechnological applications, but traditional methods offer little control over the placement of the functionalization sites on the protein. Fortunately, recent experimental methods functionalize the protein at any location on the amino acid sequence, so the question becomes one of selecting the site that will result in the best protein function. This work shows how molecular simulation can be used to screen potential attachment sites for surface tethering or PEGylation.

The Effects of -Azidophenylalanine Incorporation on Protein Structure and Stability.

Functionalization is often needed to harness the power of proteins for beneficial use but can cause losses to stability and/or activity. State of the art methods to limit these deleterious effects accomplish this by substituting an amino acid in the wild-type molecule into an unnatural amino acid, such as -azidophenylalanine (pAz), but selecting the residue for substitution a priori remains an elusive goal of protein engineering. The results of this work indicate that all-atom molecular dynamics simulation can be used to determine whether substituting pAz for a natural amino acid will be detrimental to experimentally determined protein stability.

Parameterization of Unnatural Amino Acids with Azido and Alkynyl R-Groups for Use in Molecular Simulations.

Recent new methods to functionalize proteins at specific amino acid locations use unnatural amino acids that contain azido and alkynyl groups. This capability is unprecedented and enables the creation of site-specific protein devices. Because of the high specificity of these devices, many protein configurations are possible and in silico screens have shown promise in predicting optimal attachment site locations.

The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain Simulation.

Although polyethylene glycol (PEG) is commonly used to improve protein stability and therapeutic efficacy, the optimal location for attaching PEG onto proteins is not well understood. Here, we present a cell-free protein synthesis-based screening platform that facilitates site-specific PEGylation and efficient evaluation of PEG attachment efficiency, thermal stability, and activity for different variants of PEGylated T4 lysozyme, including a di-PEGylated variant. We also report developing a computationally efficient coarse-grain simulation model as a potential tool to narrow experimental screening candidates.