Structural consequences of the natural substitution, E9K, on reactive-site-hydrolyzed squash (Cucurbita maxima) trypsin inhibitor (CMTI), as studied by two-dimensional NMR.
Sequence-specific hydrogen-1 NMR assignments were made to all of the 29 amino acid residues of reactive-site-hydrolyzed Cucurbita maxima trypsin inhibitor I (CMTI-I*) by the application of two-dimensional NMR (2D NMR) techniques, and its secondary structural elements (two tight turns, a 3(10)-helix, and a triple-stranded beta-sheet) were identified on the basis of short-range NOESY cross peaks and deuterium-exchange kinetics. These secondary structural elements are present in the intact inhibitor [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648] and are unaffected by the hydrolysis of the reactive-site peptide bond between Arg5 and Ile6, in accordance with the earlier conclusion reached for CMTI-III* [Krishnamoorthi, R., Gong, Y.-X., Lin, C. S., & VanderVelde, D. (1992) Biochemistry 31, 898-904]. Chemical shifts of backbone hydrogen atoms, peptide NH's, and C alpha H's, of CMTI-I* were compared with those of the intact inhibitor, CMTI-I, and of the reactive-site-hydrolyzed, natural, E9K variant, CMTI-III*. Cleavage of the Arg5-Ile6 peptide bond resulted in changes of chemical shifts of most of the backbone atoms of CMTI-I, in agreement with the earlier results obtained for CMTI-III. Comparison of chemical shifts of backbone hydrogen atoms of CMTI-I* and CMTI-III* revealed no changes, except for residues Glu9 and His25. However, the intact forms of the same two proteins, CMTI-I and CMTI-III, showed small but significant perturbations of chemical shifts of residues that made up the secondary structural elements of the inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)
Krishnamoorthi
R
R
Department of Biochemistry, Kansas State University, Manhattan 66506.
Lin
C L
CL
VanderVelde
D
D
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014361:Trypsin Inhibitors; D014675:Vegetables
C081870:CMTI protein, Cucurbita maxima; D010940:Plant Proteins; D014361:Trypsin Inhibitors; C031809:trypsin inhibitor, Cucurbita sp.