| Chemical methods for the synthesis of peptides and proteins are a valuable tool for the study of protein structure and function. The type 1 repeat domain from thrombospondin has potent antiangiogenic activity and a structurally interesting fold, making it an attractive target for protein engineering. After significant optimization, a robust synthetic route to this domain was identified using both Boc and Fmoc SPPS. Synthetic access to this domain has enabled the synthesis of a number of analogs containing mutations that perturb the cation-pi stack in the core of the TSR2 domain. Arginine was replaced with citrulline, an uncharged isostere, ornithine, containing a localized charge, and dimethylarginine. Finally, the middle tryptophan of the stack was replaced with benzothienylalanine, which changes the cation binding energy of the pi system. The relative stabilities of these variants, investigated via thermal denaturation followed by circular dichroism, provide insights into the stability afforded by cation-pi interactions in the context of a native protein fold. The results suggest that van der Waals forces provide the dominant stabilizing interactions in the TSR2 domain.;In a different application, a method for site-selective labeling of exosites of natural serine proteases was developed. In this method, an exosite binding peptide is used to target a chemically reactive group such as a photocrosslinker to a specific portion of a protein. The N- or C-terminus of the peptide can also contain a fluorescent group or other desired chemical moiety. Methods were developed both for the labeling of a protein with a peptide and fluorophore and for the labeling of a protein with a fluorophore using a cleavable peptide that can be removed after it is used to target the desired exosite. |
