posted on 2024-02-28, 06:13authored byAnna Charalampidou, Thomas Nehls, Christian Meyners, Satish Gandhesiri, Sebastian Pomplun, Bradley L. Pentelute, Frederik Lermyte, Felix Hausch
Engineering at the amino acid level is key to enhancing
the properties
of existing proteins in a desired manner. So far, protein engineering
has been dominated by genetic approaches, which have been extremely
powerful but only allow for minimal variations beyond the canonical
amino acids. Chemical peptide synthesis allows the unrestricted incorporation
of a vast set of unnatural amino acids with much broader functionalities,
including the incorporation of post-translational modifications or
labels. Here we demonstrate the potential of chemical synthesis to
generate proteins in a specific conformation, which would have been
unattainable by recombinant protein expression. We use recently established
rapid automated flow peptide synthesis combined with solid-phase late-stage
modifications to rapidly generate a set of FK506-binding protein 51
constructs bearing defined intramolecular lactam bridges. This trapped
an otherwise rarely populated transient pocketas confirmed
by crystal structureswhich led to an up to 39-fold improved
binding affinity for conformation-selective ligands and represents
a unique system for the development of ligands for this rare conformation.
Overall, our results show how rapid automated flow peptide synthesis
can be applied to precision protein engineering.