Nuclear magnetic resonance of labile protons in the basic pancreatic trypsin inhibitor

Abstract

In the 1H nuclear magnetic resonance (n.m.r.) spectrum of the basic pancreatic trypsin inhibitor in 2H 20 at p 2H 4·6 and 36°C, resonances of 33 labile protons were identified. Additional labile protons were observed in H 2O. All the labile protons observed in 2H 20 come from amide groups of the polypeptide backbone and the side-chains of asparagine and glutamine. Individual assignments for 18 amide protons have been previously described (Dubs et al., 1979). Here, the backbone amide protons are characterized by their chemical shifts, the chemical shifts of the at-protons and, in so far as possible, of the side-chain protons, the spin-spin coupling constants with the α-protons, and the stability with respect to exchange with solvent protons. The spectral parameters are analyzed on the basis of the known locations of individual protons in the spatial structure of the protein. In H 2O, the hydrogen-bonded amide protons of the α-helix and the β-pleated sheet in the trypsin inhibitor gave well-resolved n.m.r. lines, even at extreme values of pH and temperature, showing that the conformation observed in single crystals is preserved over a pH range from at least 1·0 to 12·0 at 25°C, and at temperatures up to at least 90°C at neutral pH. The Appendix presents a Table with all the presently known individual proton resonance assignments in the trypsin inhibitor, including all the known spin systems with labile protons. This is the first in a series of three papers on n.m.r. studies of the amide protons in the basic pancreatic trypsin inhibitor.