Assignment of the 13 C‐nmr spectra of virgin and reactive‐site modified turkey ovomucoid third domain

The virgin (reactive‐site Leu 18 ‐Glu 19 peptide bond intact) and modified (reactive‐site Leu 18 ‐Glu 19 peptide bond hydrolyzed) forms of turkey ovomucoid third domain (OMTKY3 and OMTKY3*, respectively) have been analyzed by proton‐detected 1 H{ 13 C} two‐dimensional single‐bond correlation ( 1 H{ 13 C}SBC) spectroscopy. Previous 1 H‐nmr assignments of these proteins [A. D. Robertson, W. M. Westler, and J. L. Markley (1988) Biochemistry , 27 , 2519–2529; G. I. Rhyu and J. L. Markley (1988) Biochemistry , 27 , 2529–2539] have been extended to directly bonded 13 C atoms. Assignments have been made to 52 of the 56 backbone 13 C α ‐ 1 H units and numerous side‐chain 13 C‐ 1 H groups in both OMTKY3 and OMTKY3*. The largest changes in the 13 C chemical shift upon conversion of OMTKY3 to OMTKY3* occur at or near the reactive site, and tend toward values observed in small peptides. Moreover, the side‐chain prochiral methylene protons attached to the C γ of Glu 19 and C δ of Arg 21 show nonequivalent chemical shifts in OMTKY3 but more equivalent chemical shift in OMTKY3*. These results suggest that the reactive site region becomes less ordered upon hydrolysis of the Leu 18 ‐Glu 19 peptide bond. Comparison of 13 C α chemical shifts of OMTKY3 and bovine pancreatic trypsin inhibitor [D. Brühuiler and G. Wagner (1986) Biochemistry 25 , 5839–5843; N. R. Nirmala and G. Wagner (1988) Journal of the American Chemical Society , 110 , 7557–7558] with small peptide values [R. Richarz and K. Wüthrich (1978) Biopolymers , 17 , 2133–2141] suggests that 13 C α chemical shifts of residues residing in helices are generally about 2 ppm downfield of resonances from nonhelical residues.