A suite of kinetically superior AEP ligases can cyclise an intrinsically disordered protein.

Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These "ligase-type" AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products.

Selective removal of individual disulfide bonds within a potato type II serine proteinase inhibitor from Nicotiana alata reveals differential stabilization of the reactive-site loop.

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues.

Biosynthesis of circular proteins in plants.

Plant cyclotides are a large family of naturally occurring circular proteins that are produced from linear precursors containing one, two or three cyclotide domains. The mechanism of excision of the cyclotide domains and ligation of the free N- and C-termini to produce the circular peptides has not been elucidated. Here, we investigate production of the prototypic cyclotide kalata B1 from the precursor Oak1 from the African plant Oldenlandia affinis.