The conjugation of unprotected peptide segments via thioester - cysteine ligation (native chemical ligation) is one of the most robust techniques to gain routine access to larger polypeptides and proteins. Over the last few years we have applied NCL to assemble a number of complex disulfide-rich peptides (50-83 AA, 3-5 disulfides) that modulate the activity of specific classes of membrane proteins. This approach has facilitated unique structural and functional insights into their mode of action. In this presentation, I will cover some of our recent highlights including the synthesis of the human proteins C3a and SLURP1, the scorpion toxins OD1 and Cn2 as well as the analgesic snake ‘toxin’ mambalgin-2.
Due to their remarkable reactivity, thioesters are also key intermediates in many enzyme-mediated processes, including proteolysis and peptide transpeptidation/ligation (e.g. inteins, sortase, etc.). Our own work recently has focussed on a class of cysteine proteases known as asparaginyl-endoproteases that preferentially function as peptide cyclases by coupling C-terminal propeptide cleavage with backbone cyclization. The role of this enzyme in cyclotide biosynthesis and its potential as a tool for peptide engineering will be discussed.