title: Chemical synthesis and biotinylation of the thrombospondin domain TSR2.
abstract: The type 1 repeat domain from thrombospondin has potent antiangiogenic activity and a structurally interesting fold, making it an attractive target for protein engineering. Chemical synthesis is an attractive approach for studying protein domains because it enables the use of unnatural amino acids for site-specific labeling and detailed structure-function analysis. Here, we demonstrate the first total chemical synthesis of the thrombospondin type 1 repeat domain by native chemical ligation. In addition to the natural domain, five sites for side chain modification were evaluated and two were found to be compatible with oxidative folding. Several challenges were encountered during peptide synthesis due to the functional complexity of the domain. These challenges were overcome by the use of new solid supports, scavengers, and the testing of multiple ligation sites. We also describe an unusual sequence-specific protecting group migration observed during cleavage resulting in +90 Da and +194 Da adducts. Synthetic access to this domain enables the synthesis of a number of variants that can be used to further our understanding of the biochemical interaction network of thrombospondin and provide insight into the structure and function of this important antitumorogenic protein domain.
authors.affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Theresa K
authors.initials: TK
authors.lastname: Tiefenbrunn
authors.forename: Philip E
authors.initials: PE
authors.lastname: Dawson
mesh_terms: D000595:Amino Acid Sequence; D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D019700:Thrombospondin 1 / Q000138:chemical synthesis* / Q000235:genetics
chemical_list: D010455:Peptides; D019700:Thrombospondin 1