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

[摘要]

¹⁵N NMR assignments were made to the backbone amide nitrogen atoms at natural isotopic abundance of intact and reactive-site (Arg⁵—Ile⁶) 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 Cys³, Arg⁵, Leu⁷, Met⁸, Cys¹⁰, Cys¹⁶, Glu¹⁹, His²⁵, Tyr²⁷, Cys²⁸ and Gly²⁸ 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.

[学科分类] 生物化学/生物物理

[关键词] Inhibitor;Serine protease;Cucurbita maxima;Pumpkin;Trypsin;Activated Hageman factor;Blood coagulation;1H—15N chemical shift correlation;CMTI;Cucurbita maxima trypsin inhibitor;HPLC;high-performance liquid chromatography;NMR;nuclear magnetic resonance;HSBC;heteronuclear single bond chemical shift correlation spectroscopy;ppm;parts per million