SOLUTION STRUCTURE AND BACKBONE DYNAMICS OF RECOMBINANT CUCURBITA-MAXIMA TRYPSIN INHIBITOR-V DETERMINED BY NMR-SPECTROSCOPY

The solution structure of recombinant Cucurbita maxima trypsin inhibit or-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distance s and 123 torsion angle constraints were utilized to generate 18 struc tures. The root mean squared deviation (RMSD) of the mean structure is 0.53 Angstrom for main-chain atoms and 0.95 Angstrom for all the non- hydrogen atoms of residues 3-40 and 49-67. The average structure of rC MTI-V is found to be almost the same as that of the native protein [Ca i, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly N-15-labeled rCMTI- V were characterized by 2D H-1-N-15 NMR methods. N-15 spin-lattice and spin-spin relaxation rate constants (R(1) and R(2), respectively) and {H-1}-N-15 steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free forma lism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Sec. I04, 4546-4559, 4559-4570], the following parameters were determined: overall tumblin g correlation time for the protein molecule (tau(m)), generalized orde r parameters for the individual N-H vectors (S-2), effective correlati on times for their internal motions (tau(e)), and terms to account for motions on a slower time scale (second) due to chemical exchange and/ or conformational averaging (R(rx)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ([S-2] = 0.83) with the ex ception of those in the binding loop (residues 41-48, (S-2) = 0.71) an d the N-terminal region ([S-2] = 0.73). Main-chain atoms in these regi ons show large RMSD values in the average NMR structure. Residues invo lved in turns also appear to have more mobility ((S-2) = 0.80). Dynami cal properties of rCMTI-V were compared with those of two other inhibi tors of the potato I family-eglin c [Peng, J. W., & Wagner, G. (1992) Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemist ry 34, 2225-2233]. The Cys(3)-Cys(48) linkage found only in rCMTI-V ap pears to somewhat reduce the N-terminal flexibility; likewise, the C-t erminal of rCMTI-V, being part of a beta-sheet, appears to be more rig id.