SOLUTION CONFORMATION OF A SYNTHETIC BIS-HEADED INHIBITOR OF TRYPSIN AND CARBOXYPEPTIDASE-A - NEW STRUCTURAL ALIGNMENT BETWEEN THE SQUASH INHIBITORS AND THE POTATO CARBOXYPEPTIDASE INHIBITOR

The trypsin carboxypeptidase peptide inhibitor (TCPI) which inhibits b oth trypsin and carboxypeptidase A has been chemically engineered by m odification of the Ecballium elaterium trypsin inhibitor II (EETI-II). The solution conformation of TCPI, studied by two-dimensional nuclear magnetic resonance, was shown to be very close to those of squash inh ibitors. Only limited deviations of the trypsin binding loop compared to its location in the EETI-II/trypsin complex were detected. It was a lso shown that the position of the C-terminal tail did not significant ly change from the position observed in the complex between carboxypep tidase A and the potato carboxypeptidase inhibitor (PCI). The conforma tion of TCPI was carefully compared with the PCI one and a new structu ral alignment between the two microproteins is proposed. This alignmen t points out the very good conservation in the two inhibitors of a sub domain comprising segments 7-15, 19-22 and 25-28. Most importantly, th e 2-19 disulfide bridge of TCPI was not structurally conserved in PCI and appeared to be rather unimportant for the early folding process of these molecules. This result agrees with the recent observation that the 2-19 bridge is the last to be formed in the folding of the squash inhibitor EETI-II and suggests that this is also the case during the f olding of the potato carboxypeptidase inhibitor.