Solution Structure and Backbone Dynamics of Recombinant Cucurbita maxima Trypsin Inhibitor-V Determined by NMR Spectroscopy

• Published in: Biochemistry (Easton) Vol. 35; no. 5; pp. 1516 - 1524
• Main Authors: Liu, Jianhua, Prakash, Om, Cai, Mengli, Gong, YuXi, Huang, Ying, Wen, Lisa, Wen, Joanna J, Huang, Jeng-Kuen, Krishnamoorthi, Ramaswamy
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.02.1996
• Subjects: CUCURBITA MAXIMA; ESPECTROSCOPIA RMN; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; Magnetic Resonance Spectroscopy; Models, Chemical; Models, Molecular; Nitrogen Isotopes; Plant Proteins - chemistry; Plant Proteins - genetics; Protein Structure, Secondary; Recombinant Proteins - chemistry; Solutions; SPECTROSCOPIE RMN; Trypsin Inhibitors - chemistry; Trypsin Inhibitors - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi952466d

The solution structure of recombinant Cucurbita maxima trypsin inhibitor-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 distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 Å for main-chain atoms and 0.95 Å for all the non-hydrogen atoms of residues 3−40 and 49−67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201−5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H−15N NMR methods. 15N spin−lattice and spin−spin relaxation rate constants (R 1 and R 2, respectively) and {1H}−15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546−4559, 4559−4570], the following parameters were determined:  overall tumbling correlation time for the protein molecule (τm), generalized order parameters for the individual N−H vectors (S 2), effective correlation times for their internal motions (τe), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R ex). Most of the backbone NH groups of rCMTI-V are found to be highly constrained (〈S 2〉 = 0.83) with the exception of those in the binding loop (residues 41−48, 〈S 2〉 = 0.71) and the N-terminal region (〈S 2〉 = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility (〈S 2〉 = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors 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) Biochemistry 34, 2225−2233]. The Cys3−Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a β-sheet, appears to be more rigid.