Disulfide Bond Mutagenesis and the Structure and Function of the Head-to-Tail Macrocyclic Trypsin Inhibitor SFTI-1,

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman−Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA3,11]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by 1H NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA3,11]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 ± 0.09 and 1.10 ± 0.22 Å, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone Hα protons of two disulfide-deficient linear analogues of SFTI-1, [ABA3,11]SFTI-1[6,5] and [ABA3,11]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.