and Biochemistry

Reported here is the direct synthesis and application of unnatural amino acids for the development of exploratory tools for protein studies. This work takes advantage of an expanded genetic code to extract a more precise chemical understanding of protein function with novel additions to the unnatural amino acid catalogue, as well as the expansion of techniques with previously developed compounds. The photochemical crosslinker, [D11]-p-benzoylphenylalanine (pBpa), is synthesized for isotopic labeling in proteins. When [D11]-pBpa is co-incorporated into protein with [D0]-pBpa it is a mass spectral tool for rapid and conclusive identification of crosslinked fragments. Following enzymatic digestion the fingerprint of M, M+ 11 is readily identified allowing for rapid peak identification and the determined site of crosslink formation with single amino acid accuracy. In a means to extract a level of spatiotemporal control over fluorescent labeling of protein, the photo-protected unnatural amino acid, o-nitrobenzyl cysteine (ONBC), is introduced to a small amino acid tag sequence CCPGCC. This tag is required and specifically binds the pro-fluorescent compound 5-bis(1,3,2-dithiasolan-2