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. We use this simulation method as a novel tool to evaluate the locational impact of PEGylation. Using this screen, we also evaluated the predictive impact of PEGylation site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings indicate that PEGylation efficiency, protein stability, and protein activity varied considerably with PEGylation site, variations that were not well predicted by common PEGylation guidelines. Overall our results suggest current guidelines are insufficiently predictive, highlighting the need for experimental and simulation screening systems such as the one presented here.

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. Previous simulation work has shown promise in this regard for a model protein, but these studies are limited to screening only a few of the surface-accessible sites or only considered surface tethering or PEGylation separately rather than their combined effects. This work is done to overcome these limitations by screening all surface-accessible functionalization sites on a protein of industrial and therapeutic importance (TEM-1) and to evaluate the effects of tethering and PEGylation simultaneously in an effort to create a more accurate screen. The results show that functionalization site effectiveness appears to be a function of super-secondary and tertiary structures rather than the primary structure, as is often currently assumed. Moreover, sites in the middle of secondary structure elements, and not only those in loops regions, are shown to be good options for functionalization—a fact not appreciated in current practice. Taken as a whole, the results show how rigorous molecular simulation can be done to identify candidate amino acids for functionalization on a protein to facilitate the rational design of protein devices.

The literature is divided on the proper parameterization of linear-angle containing dihedrals (LACD). Some works include LACD parameters while others, including the GAFF and CGenFF databases, exclude LACD parameters. To more fully understand both methods, A 4-bodied model, depicted in Figure S1, is used to explain the cause of ramping in potential energy for LACDs. The purpose of this model is to focus attention on the angle and dihedral by eliminating translational movement of the system and only allowing significant movements for one atom (A) in the 4-bodied system. This was done by increasing the relative mass of sites B, C and D to be magnitudes greater than A and by fixing B, C and D to their initial positions using a strong harmonic spring potential. As a result, sites B-D only experience movements ± 0.001Å in any direction during simulation. The entire model still uses the CHARMM FF and these changes are only used to help visualize the relevant motion. Specifically, using this setup allowed the BC bond to be fixed along the z-axis and the BCD plane to be restricted to the yz-plane.

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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 wildtype molecule into an unnatural amino acid, such as pazidophenylalanine (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. These results offer significant hope that local deviations from wild-type structure caused by pAz incorporation observed in simulations can be a predictive metric used to reduce the number of costly experiments that must be done to find active proteins upon substitution with pAz and subsequent functionalization.