title: Protein side-chain translocation mutagenesis via incorporation of peptoid residues.
abstract: For the last few decades, chemistry has played an important role in protein engineering by providing a variety of synthetic tools such as chemoselective side-chain modifications, chemical conjugation, incorporation of non-natural amino acids, and the development of protein-mimetic heteropolymers. Here we study protein backbone engineering in order to better understand the molecular mechanism of protein function and to introduce protease stable, non-natural residues into a protein structure. Using a combination of genetic engineering and chemical synthesis, we were able to introduce peptoid residues (N-substituted glycine residues) at defined positions into bovine pancreatic ribonuclease A. This results in a side-chain translocation from the Cα carbon to the neighboring backbone nitrogen atom. To generate these peptoid substitutions, we removed the N-terminal S-peptide of the protein by proteolysis and chemically conjugated synthetic peptide-peptoid hybrids to the new N-terminus. A triple peptoid mutant containing a catalytic His12 peptoid mutation was active with a k(cat)/K(m) value of 1.0 × 10(4) M(-1) s(-1). This k(cat)/K(m) value is only 10-fold lower than the control wild-type conjugate and comparable in magnitude to many other natural enzymes. The peptoid mutations increased the chain flexibility at the site of peptoid substitution and at its C-terminal neighboring residue. Our ability to translocate side chains by one atom along the proten backbone advances a synthetic mutagenesis tool and opens up a new level of protein engineering.
authors.affiliation: Biological Nanostructures Facility, The Molecular Foundry, Lawrence Berkeley National Laboratory, California 94720, USA.
authors.forename: Byoung-Chul
authors.initials: BC
authors.lastname: Lee
authors.forename: Ronald N
authors.initials: RN
authors.lastname: Zuckermann
mesh_terms: D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D034444:Peptoids / Q000138:chemical synthesis / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D034444:Peptoids; D012259:Ribonuclease, Pancreatic