Access to Synthetic Fold-switching Proteins Using Automated Flow Chemistry

This paper introduces a robust pipeline for preparing and studying synthetic fold-switching proteins using automated flow protein synthesis. Fold-switching proteins are an expanding class of proteins that remarkably switch from one stable fold to another in response to subtle changes in the sequence or chemical environment, without populating unfolded or aggregated states. The polymorphism of these proteins permits important regulatory functions across biological processes and diseases, but also hinders their preparation and characterization using recombinant methods. To provide reliable access to fold-switching proteins, we used automated flow chemistry to establish the synthesis and to provide a robust platform for answering fundamental questions of protein folding with unnatural amino acids. The synthetic methods are demonstrated with 56-amino acid homologs of GA and GB, which were successfully synthesized and purified as sequences that are 95% and 98% identical. Biophysical studies show that the synthetic proteins adopt a folded conformation consistent with the 3-α and 4β+α conformations observed in prior studies. The success of these protein syntheses shows that automated flow chemistry continues to push the boundaries of high-fidelity protein synthesis, by enabling the preparation of polymorphic proteins that are sensitive to mutations. These synthetic proteins will be used to study noncovalent interactions at the interface of two protein folds that cannot be resolved with biosynthetic proteins.