The 46-kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP. The reaction is inhibited by N-(phosphonomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, we have used dipolar recovery at the magic angle (DRAMA) and rotational-echo double resonance (REDOR) to determine intra- and interligand internuclear distances. DRAMA was used to determine the single 31P-31P distance, while REDOR was used to determine one 31P-15N distance and five 31P-13C distances. These experimental distances were used as restraints in molecular dynamics simulations of an S3P-Glp complex to examine the geometry of the two ligands relative to one another in the ternary complex. The simulations were compared to unrestrained simulations of the EPSP synthase tetrahedral intermediate and its phosphonate analog. The results suggest that Glp is unlikely to bind in the same fashion as PEP, a conclusion that is consistent with recent studies that have questioned the role of Glp as a transition-state or intermediate analog.

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The combination of transferred-echo double resonance (TEDOR) with rotational-echo double resonance (REDOR) has been used to measure an 8-Á fluorine-carbon internuclear distance in a nine-residue fragment of the peptide antibiotic emerimicin. The fragment is 19FCH2C0-Phe-MeA-MeA-[l-13C]MeA-[15N]Val-Gly-Leu-MeA-MeA-0Bzl (MeA = a-methylalanine or aminoisobutyric acid). The TEDOR part of this magic-angle-spinning, solid-state NMR experiment selects the 13C label by its dipolar coupling to 15N and suppresses the natural-abundance carbon background. The REDOR part of the experiment measures dipolar coupling of the selected carbon to 19F. The TEDOR-REDOR combined experiment works with a variety of spin */2 nuclei and can be used to characterize intemuclear distances and geometry in macromolecular aggregates that do not crystallize.Recent advances in X-ray crystallography and solution-state NMR spectroscopy have yielded three-dimensional structures of many soluble proteins and peptides.1"3 Unfortunately, the same sort of information, which is essential in elucidating mechanisms of action, is largely unavailable for molecules which function within biological membranes. The problem lies primarily in the inability to prepare samples in a crystalline form suitable for X-ray analysis or to interpret NMR spectra displaying the broad lines of membrane-bound species with solid-like properties.We have previously demonstrated4 the utility of solid-state, rotational-echo double-resonance (REDOR) NMR spectroscopy5,6 in providing accurate structural information for biological solids. REDOR involves the dephasing of transverse, S-spin magnetization by rotor-synchronized I-spin pulses; I and S refer to different types of dipolar coupled rare spins. When the interactions with all other spins can be ignored or suppressed, the comparison of S-spin echo intensities with (S) and without (S0) I-spin dephasing pulses leads directly to the strength of the I-S dipolar coupling and hence I-S intemuclear distances.4The natural-abundance S-spin background complicates the interpretation of REDOR experiments by a contribution to S0 that is unrelated to I-S dipolar coupling. Sometimes this contribution can be measured in separate experiments on unlabeled samples.7 This approach is generally successful if the concentration of label is high or the I-S dipolar coupling is strong. When these conditions are not met, we propose selecting the dipolar coupled spins from among the background of uncoupled spins by a coherence transfer from I to S. Dephasing of the selected S-signal by rotor-synchronized ir pulses applied to a third rare spin (X) can now be used to measure S-X dipolar coupling and intemuclear distances with no background interferences.In this paper, we report the results of background-free distance measurements on a triple-labeled emerimicin 1-9. This peptide sequence is a fluorinated analogue of the N-terminal portion of the emerimicins III and IV, which are members of the peptaibol family of antibiotics known to function as ion channels in ...

Constrained systematic search was used in an exhaustive conformational analysis of a structurally diverse set of substance P (SP) antagonists to identify a unique hypothesis for their bound conformation at the neurokinin-1 receptor. In this conformation, two aromatic groups essential for high affinity adopt a perpendicular or edge-on arrangement. This pharmacophore hypothesis for the receptor-bound conformation was used in a comparative molecular field analysis (CoMFA) of an expanded set of SP antagonists, and the predictive ability of the resulting three-dimensional quantitative structure-activity relationship (3D-QSAR) was evaluated against a test set of SP antagonists different from those in the training set. This CoMFA model based on the Constrained Search alignment yielded significant cross-validated, conventional, and predictive r2 values equal to 0.70, 0.93, and 0.82, respectively. For comparison, the SP antagonists were forced into an alternative poorer alignment in which the two aromatic rings were parallel and then subjected to a CoMFA analysis. Both the parallel and perpendicular arrangements of the aromatic rings are seen in X-ray structures of SP antagonists and have been proposed as candidates for the receptor-bound conformation. The parallel (or stacked) conformation yielded a poorer correlation with a cross-validated r2 = 0.57, a conventional r2 = 0.90, and a predictive r2 = 0.78. Our results indicate that although both alignments could generate a reasonable CoMFA correlation, the stacked conformation is unlikely to be the receptor-bound conformation, as the covalent structure of the antagonists precludes a common geometry in which the aromatic rings are stacked.