Systematic Application of Two‐Dimensional 1 H Nuclear‐Magnetic‐Resonance Techniques for Studies of Proteins

This paper describes a new nuclear magnetic resonance approach for the determination of secondary structure in globular proteins. To illustrate the practical application of the new procedure, two‐dimensional correlated spectroscopy and two‐dimensional nuclear Overhauser enhancement spectroscopy were used to obtain individual assignments for all the backbone protons of the β‐sheet secondary structures in the basic pancreatic trypsin inhibitor. First, combined connectivity diagrams of these two methods recorded in both 2 H 2 O solution and H 2 O solution of the inhibitor were employed to obtain sequential, individual resonance assignments for the separate strands in the β sheet. Second, a 2D nuclear Overhauser enhancement spectrum recorded with a long mixing time was used to determine how the separate, extended polypeptide strands are linked by hydrogen bonds in the sheet structures. By combination of these results with the identifications of the amino acid side‐chain resonances described in the preceding paper, the β‐sheet structures can, without reference to data on the spatial structure obtained with other techniques, be localized in the amino acid sequence. This investigation confirms results on limited regions of the β sheet in the inhibitor obtained previously with one‐dimensional nuclear magnetic resonance experiments and demonstrates that the entire β‐sheet structure seen in single crystals of the inhibitor is preserved in aqueous solution.