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Chemical synthesis and biotinylation of the thrombospondin domain TSR2
Author(s) -
Tiefenbrunn Theresa K.,
Dawson Philip E.
Publication year - 2009
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.107
Subject(s) - native chemical ligation , thrombospondin , chemistry , computational biology , egf like domain , chemical biology , function (biology) , biotinylation , chemical synthesis , protein domain , protein engineering , biochemistry , combinatorial chemistry , biology , genetics , gene , enzyme , metalloproteinase , in vitro
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.