• serine proteinase inhibitor;
  • specificity;
  • NMR structure;
  • flexibility

The solution structure of three small serine proteinase inhibitors, two natural and one engineered protein, SGCI␣(Schistocerca gregaria chymotrypsin inhibitor), SGCI[L30R, K31M] and SGTI (Schistocerca gregaria trypsin inhibitor), were determined by homonuclear NMR-spectroscopy. The molecules exhibit different specificities towards target proteinases, where SGCI is a good chymotrypsin inhibitor, its mutant is a potent trypsin inhibitor, and SGTI inhibits both proteinases weakly. Interestingly, SGTI is a much better inhibitor of insect proteinases than of the mammalian ones used in common assays. All three molecules have a similar fold composed from three antiparallel β-pleated sheets with three disulfide bridges. The proteinase binding loop has a somewhat distinct geometry in all three peptides. Moreover, the stabilization of the structure is different in SGCI and SGTI. Proton–deuterium exchange experiments are indicative of a highly rigid core in SGTI but not in SGCI. We suggest that the observed structural properties play a significant role in the specificity of these inhibitors.