Natural abundance 15N NMR assignments delineate structural differences between intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III

• Published in: FEBS letters 1992-06, Vol.304 (2), p.149-152
• Main Authors: Krishnamoorthi, Ramaswamy, Nemmers, Sylvia, Tobias, Brian
• Format: Article
• Language: English
• Subjects: 1H— 15N chemical shift correlation; Activated Hageman factor; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Blood coagulation; CMTI; Cucurbita maxima; Cucurbita maxima trypsin inhibitor; Disaccharides; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glucans; heteronuclear single bond chemical shift correlation spectroscopy; high-performance liquid chromatography; HPLC; HSBC; Hydrolases; Hydrolysis; Inhibitor; Magnetic Resonance Spectroscopy; Molecular Sequence Data; NMR; nuclear magnetic resonance; parts per million; Peptide Fragments - chemistry; Plant Proteins - chemistry; Plants, Edible - chemistry; ppm; Pumpkin; Serine protease; Trypsin; Trypsin Inhibitors - chemistry
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(92)80607-I

15N NMR assignments were made to the backbone amide nitrogen atoms at natural isotopic abundance of intact and reactive-site (Arg 5—Ile 6) hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III and CMTI-III*, respectively) by means of 2D proton-detected heteronuclear single bond chemical shift correlation (HSBC) spectroscopy, utilizing the previously made sequence-specific 1H NMR assignments (Krishnamoorthi et al. (1992) Biochemistry 31. 898–904). Comparison of the 15N chemical shifts of the two forms of the inhibitor molecule revealed significant changes not only for residues located near the reactive-site region, but also for those distantly located. Residues Cys 3, Arg 5, Leu 7, Met 8, Cys 10, Cys 16, Glu 19, His 25, Tyr 27, Cys 28 and Gly 28 showed significant chemical shift changes ranging from 0.3 to 6.1 ppm, thus indicating structural perturbations that were transmitted throughout the molecule. These findings confirm the earlier conclusions based on 1H NMR investigations.