COMBINED USE OF C-13 CHEMICAL-SHIFT AND H-1-ALPHA-C-13-ALPHA HETERONUCLEAR NOE DATA IN MONITORING A PROTEIN NMR STRUCTURE REFINEMENT

A large portion of the C-13 resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28 degrees C has been determined a t natural isotope abundance. Sequence-specific C-13 assignments are re ported for 100% of the assignable C alpha, 96% of the C beta, 86% of t he aromatic and 70% of the remaining peripheral aliphatic resonances o f mEGF. A good correlation was observed between experimental and back- calculated C alpha chemical shifts for regions of regular P-sheet stru cture. These assignments also provide the basis for interpreting H-1 a lpha-C-13 alpha heteronuclear NOE (HNOE) values in mEGF at natural iso tope abundance. Some of the backbone polypeptide segments with high in ternal mobility, indicated by these H-1 alpha-C-13 alpha HNOE measurem ents, correlate with locations of residues involved in the putative mE GF-receptor binding site. Using four families of mEGF structures obtai ned over the last few years, we demonstrate that standard deviations b etween experimental and back-calculated Delta delta C alpha values can be used to monitor the refinement of this protein's structure, partic ularly for P-sheet regions. Improved agreement between calculated and observed values of Delta delta C alpha is correlated with other measur es of structure quality, including lowered values of residual constrai nt violations and more negative values of conformational energy These results support the view that experimental conformation-dependent chem ical shifts, Delta delta C alpha, can provide a reliable source of inf ormation for monitoring the process of protein structure refinement an d are potentially useful restraints for driving the refinement.