Backbone Dynamics of Cyclotide MCoTI‐I Free and Complexed with Trypsin

Cyclotides are a new emerging family of large plant-derived backbone-cyclized polypeptides ( 28–37 amino acids long) that share a disulfide-stabilized core (3 disulfide bonds) characterized by an unusual knotted.[1] Cyclotides contrast with other circular polypeptides in that they have a well-defined three-dimensional structure, and despite their small size, can be considered as micro-proteins. Their unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal and enzymatic degradation (Scheme 1). Furthermore, their well-defined structures have been associated with wide range of biological functions.[2,3] Cyclotides MCoTI-I/II are powerful trypsin inhibitors (Ki 20 – 30 pM) which have been recently isolated from the dormant seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family.[4] Although MCoTI cyclotides do not share significant sequence homology with other cyclotides beyond the presence of the three cystine bridges, structural analysis by NMR has shown that they adopt a similar backbonecyclic cystine-knot topology.[5,6] MCoTI cyclotides, however, show high sequence homology with related cystine-knot squash trypsin inhibitors,[4] and therefore represent interesting molecular scaffolds for drug design.[7–10] Determination of the backbone dynamics of these fascinating micro-proteins is key for understanding their physical and biological properties. Internal motions of a protein on different time scales, extending from picoseconds to second, have been suggested to play an important role in its biological function.[11] A better understanding of the backbone dynamics of the cyclotide scaffold will be extremely helpful for evaluating its utility as a scaffold for peptide-based drug discovery. Such insight will help in the design of optimal focused libraries than can be used for the discovery of new cyclotides sequences with novel biological activities. [12,13] In the current study we report for the first time the determination of internal dynamics of the cyclotide MCoTI-I in free state and complexed with trypsin. Uniformly 15 N-labeled natively folded cyclotide MCoTI-I was recombinantly produced in E. coli growing in minimal M9 medium containing 15 NH4Cl as only source of nitrogen. Concomitant backbone cyclization