Combined use of 13C chemical shift and 1H alpha-13C alpha heteronuclear NOE data in monitoring a protein NMR structure refinement.

A large portion of the 13C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28 degrees C has been determined at natural isotope abundance. Sequence-specific 13C assignments are reported for 100% of the assignable C alpha, 96% of the C beta, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C alpha chemical shifts for regions of regular beta-sheet structure. These assignments also provide the basis for interpreting 1H alpha-13C alpha heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these 1H alpha-13C alpha HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated delta delta C alpha values can be used to monitor the refinement of this protein's structure, particularly for beta-sheet regions. Improved agreement between calculated and observed values of delta delta C alpha is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, delta delta C alpha, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.

The role of asparagine-32 in forming the receptor-binding epitope of human epidermal growth factor.

The highly conserved asparagine residue at position 32 (Asn32) in the 'hinge' region of epidermal growth factor (EGF) separates the N- and C-terminal structural motifs of the EGF molecule and is therefore an appropriate target for structure-function studies. Analogs of human EGF (hEGF) were generated in which Asn32 was substituted with aspartate, glycine, isoleucine, lysine, proline and tryptophan. The relative affinity of the EGF receptor for mutant hEGF analogs was determined by radioreceptor competition assay. A wide range of receptor affinities was observed depending on the amino acid substitution. N32K and N32W hEGF analogs had relatively high receptor affinity, while the N32G and N32D analogs showed decreased affinity, 35% and 25% respectively, relative to wild type hEGF. However, no binding of the N32P analog was detected by radioreceptor competition assay. The N32P mutant displayed an NMR spectrum significantly different from that of native wild type hEGF, indicating gross structural perturbation. In contrast, the N32K and N32D analogs exhibited spectra similar to that of native wild type hEGF. Genetically combining the N32D hEGF with an hEGF species having either the mutation L26G in the N-terminal region or L47A in the C-terminal region, generated double-mutant hEGF species which had relative affinities essentially equal to the product of the relative affinities of the parent hEGF mutants, indicating functionally independent changes in ligand-receptor interaction. These studies indicate the requirement for H-bond donor functionality in the side chain of residue number 32 in forming a fully competent receptor-binding epitope.