Automated Stereospecific ¹H NMR Assignments and Their Impact on the Precision of Protein Structure Determinations in Solution

Two sets of constraints on proton-proton distances and dihedral angles, which mimic data that can be obtained from nuclear magnetic resonance experiments in solution, were derived from the crystal structure of the protein basic pancreatic trypsin inhibitor (BPTI). In one of these data sets, all prochiral groups of protons were replaced by pseudoatoms. In the second set, stereospecific assignments were used for all 0-methylene groups, all protons of glycine and proline, the methyl groups of valine and leucine, and the ring protons of phenylalanine and tyrosine. Comparison of the BPTI structures calculated from these data with the distance geometry program Disman Showed that, with otherwise identical distance constraints, the use of stereospecific assignments results in significantly improved precision of the structure determination for the polypeptide backbone as well as the amino acid side chains. The paper further describes the program Habas, Which determines stereospecific assignments by a systematic analysis of the proton-proton scalar couplings and the intraresidual and sequential proton-proton nuclear Overhauser effects. To investigate to what extent stereospecific assignments could be obtained for a predetermined completeness and precision of the input data set, Habas Was used for test calculations with a standard dipeptide unit and a database derived from a group of high-resolution protein crystal structures. From these data we estimate that with the precision presently achieved for NMR measurements with proteins, stereospecific assignments can be obtained for approximately half of the β-methylene protons. Quite generally, this ratio can be expected to be higher for 0-proteins than for those that contain predominantly a-helical secondary structure.