title: Posttranslational incorporation of noncanonical amino acids in the RNase S system by semisynthetic protein assembly.
abstract: The unique ribonuclease S (RNase S) system, derived from proteolytic cleavage of bovine ribonuclease A (RNase A), consists of a tight complex formed by a peptide (amino acids 1-20) and a protein (21-124) part. These fragments, designated as S-peptide and S-protein, can be separated by two purification steps. By addition of synthetic S-peptide derivatives to the S-protein, semisynthetic RNase S is reassembled with high efficiency. Based on this peptide-protein complementation noncanonical amino acids can be easily introduced into a protein host. Here we describe the preparation of the S-protein from RNase A as well as the characterization of the reassembled semisynthetic RNase S complex. Complex formation can be monitored by RNase activity, circular dichroism, or fluorescence polarization. Structure-based enzyme design of the RNase S scaffold is possible based on high-resolution crystal structures of RNase S and its semisynthetic variants.
authors.affiliation: Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, University of Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany, maika.genz@gmx.de.
authors.forename: Maika
authors.initials: M
authors.lastname: Genz
authors.forename: Norbert
authors.initials: N
authors.lastname: Sträter
mesh_terms: D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D010455:Peptides; D011506:Proteins; D012260:Ribonucleases; C022542:ribonuclease S