Force Field Development in Protein Design

Protein design requires the rapid evaluation of very large numbers of equations during the course of a calculation. These equations must represent the important contributors to protein stability in simple and accurate terms. Some physical phenomena are relatively easy to model such as van der Waals forces. Electrostatics and solvation in a protein environment are forces that are more difficult to adequately capture. Additionally, the balance of the terms used must be determined in order to design sequences that fold to stable, specific folds. The electrostatic interactions within the protein and between the protein and solvent are important in both the stability and function of the protein. The effects of the protein-solvent interactions are evaluated using implicit models that consider the solvent as a bulk. These interactions are quantified using the Poisson-Boltzmann equation that must be solved using discrete numerical methods. We sought to avoid this performance hit by scaling a simpler model of electrostatics, Coulomb's law, to reproduce one aspect of the protein-solvent interaction: solvent screening. By dividing the Coulombic dielectric into two parts and scaling to correlate with the Poisson-Boltzmann results we significantly increased the strength of electrostatics in our force field that led to the design of a more stable engrailed homeodomain. The second part of this work describes attempts to reparameterize our protein design force field. Many protein mutants have been expressed and biophysically characterized in the literature. We sought to use the measured stabilities of protein mutants in the literature to balance the terms in the force field. While we were able to produce a force field that could reproduce experimental energies, this force field led to unsatisfactory designed sequences. To more fully satisfy the unique conditions of a protein design force field we explored other optimization techniques and found that the balance of the terms in the existing force field is nearly optimal.</p

Clinical Application of a Modular Genomics Technique in Systemic Lupus Erythematosus: Progress towards Precision Medicine

Monitoring disease activity in a complex, heterogeneous disease such as lupus is difficult. Both over- and undertreatment lead to damage. Current standard of care serologies are unreliable. Better measures of disease activity are necessary as we move into the era of precision medicine. We show here the use of a data-driven, modular approach to genomic biomarker development within lupus—specifically lupus nephritis

Repacking the Core of T4 lysozyme by automated design

Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8 Å relative to both the designed structure and wild-type. Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone

Simple electrostatic model improves designed protein sequences

Electrostatic interactions are important for both protein stability and function, including binding and catalysis. As protein design moves into these areas, an accurate description of electrostatic energy becomes necessary. Here, we show that a simple distance-dependent Coulombic function parameterized by a comparison to Poisson-Boltzmann calculations is able to capture some of these electrostatic interactions. Specifically, all three helix N-capping interactions in the engrailed homeodomain fold are recovered using the newly parameterized model. The stability of this designed protein is similar to a protein forced by sequence restriction to have beneficial electrostatic interactions