Incorporation of Noncanonical Amino Acids into Rosetta and Use in Computational Protein-Peptide Interface Design

Abstract

Noncanonical amino acids (NCAAs) can be used in a variety of protein design contexts. For example, they can be used in place of the canonical amino acids (CAAs) to improve the biophysical properties of peptides that target protein interfaces. We describe the incorporation of 114 NCAAs into the protein-modeling suite Rosetta. We describe our methods for building backbone dependent rotamer libraries and the parameterization and construction of a scoring function that can be used to score NCAA containing peptides and proteins. We validate these additions to Rosetta and our NCAA-rotamer libraries by showing that we can improve the binding of a calpastatin derived peptides to calpain-1 by substituting NCAAs for native amino acids using Rosetta. Rosetta (executables and source), auxiliary scripts and code, and documentation can be found at (http://www.rosettacommons.org/).

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

“…The main issue was to identify an appropriate incorporation site. In our previous study to re-design the mDHFR active site using a non-natural amino acid [33], we utilized a protein structure viewer along with non-natural amino acid rotamer libraries [34]. Despite the successful identification of a suitable pair of incorporation site and non-natural amino acid, the approach was based on a very qualitative visualization technique and cannot easily be applied to other systems [33].…”

“…Alternatively, considering that human DHFR is highly homologous to mDHFR with greater than 95% of amino acid sequence similarity, the crystal structure of human DHFR (PDB ID: 2W3M), ligand structures, and the rotamer library of 2Nal was utilized to construct the structural models of mDHFR variants containing 2Nal [34,36,37,[51][52][53]. Docking of mDHFR variants with DHF or FOL was performed using RosettaLigand.…”

“…We used two molecular modeling programs, RosettaLigand and RosettaDesign, version 3.4 for the computational design [34][35][36][37]. RosettaLigand takes a receptor structure and a small molecule ligand, and calculates the Rosetta score to identify a conformation and relative orientation of the receptor and ligand.…”

“…2Nal has a hydrophobic, bulkier side chain compared to other natural amino acids and was successfully used to control mDHFR inhibition [33]. The rotamer library of 2Nal was already developed for computational protein design using Rosetta programs [34]. Since the introduction of a polar amino acid into inside region of a protein likely perturbs the folded structure, a non-natural amino acid with a non-polar side chain, such as 2Nal was chosen in this study.…”

“…However, generating and testing multiple mDHFR variants containing 2Nal at different sites are not trivial. Therefore, RosettaLigand and RosettaDesign [34][35][36][37], two computational protein design tools calculating ligand docking and conformational stability scores respectively, were utilized to identify an optimal 2Nal incorporation site in the active site.…”