Site-wise Diversification of Combinatorial Libraries Using Insights from Structure-guided Stability Calculations.

TitleSite-wise Diversification of Combinatorial Libraries Using Insights from Structure-guided Stability Calculations.
Publication TypeJournal Article
Year of Publication2022
AuthorsDolgikh B, Woldring D
JournalMethods Mol Biol
Date Published2022
KeywordsGene Library, Humans, Ligands, Mutation, Protein Engineering, Proteins

Many auspicious clinical and industrial accomplishments have improved the human condition by means of protein engineering. Despite these achievements, our incomplete understanding of the sequence-structure-function relationship prevents rapid innovation. To tackle this problem, we must develop and integrate new and existing technologies. To date, directed evolution and rational design have dominated as protein engineering principles. Even so, prior to screening for novel or improved functions, a large collection of variants, within a protein library, exist along an ambiguous mutational terrain. Complicating things further, the choice of where to initialize investigation along a vast sequence space becomes even more difficult given that the majority of any sequence lacks function entirely. Unfortunately, even when considering functionally relevant positions, random substitutions can prove to be destabilizing, causing a hindrance to an otherwise function-inducing, stability-reliant folding process. To enhance productivity in the field, we seek to address this issue of destabilization, and subsequent disfunction, at protein-protein and protein-ligand interacting regions. Herein, the process of choosing amenable positions - and amino acids at those positions - allows for a refined, knowledge-based approach to combinatorial library design. Using structural data, we perform computational stability prediction with FoldX's PositionScan and Rosetta's ddG_monomer in tandem, allowing for the refinement of our thermodynamic stability data through the comparison of results. In turn, we provide a process for selecting in silico predicted mutually stabilizing positions and avoiding overly destabilizing ones that guides the site-wise diversification of combinatorial libraries.

Alternate JournalMethods Mol Biol
PubMed ID35482184
PubMed Central ID2867005