Application of Hadamard spectroscopy to automated structure verification in high-throughput NMR.

Combined verification using 1-D proton and HSQC has been proved to be quite successful; the acquisition time of HSQC spectra, however, can be limiting in its high-throughput applications. The replacement with Hadamard HSQC can significantly enhance the throughput. We hereby propose a protocol to optimize the grouping of the predicted carbon chemical shifts from the proposed structure and the associated Hadamard frequencies and bandwidths. The resulting Hadamard HSQC spectra compare favorably with their Fourier-transformed counterparts, and have demonstrated to perform equivalently in terms of combined verification, but with several fold enhancement in throughput, as illustrated for 21 commercial available molecules and 16 prototypical drug compounds. Further improvement of the verification accuracy can be achieved by the cross validation from Hadamard TOCSY, which can be acquired without much sacrifice in throughput.

Protein-free ligand screening: simplification of chiral chromatographic development via novel adaptation of NMR screening methodologies.

We demonstrate here a promising NMR method that provides evidence for chiral compound selector interaction as a first-pass screening method. A novel adaptation of commonly used protein-based screening technologies, this approach relies upon ligand-to-stationary phase interaction wherein the stationary phase is tethered to sepharose beads. At only minutes per experiment, this methodology significantly reduces the time required for chiral separation methodology development and complements currently available chromatographic purity technologies.

Solution structure and backbone dynamics of the catalytic domain of matrix metalloproteinase-2 complexed with a hydroxamic acid inhibitor.

MMP-2 is a member of the matrix metalloproteinase family that has been implicated in tumor cell metastasis and angiogenesis. Here, we describe the solution structure of a catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020), determined by three-dimensional heteronuclear NMR spectroscopy. The catalytic domain, designated MMP-2C, has a short peptide linker replacing the internal fibronectin-domain insertion and is enzymatically active. Distance geometry-simulated annealing calculations yielded 14 converged structures with atomic root-mean-square deviations (r.m.s.d.) of 1.02 and 1.62 A from the mean coordinate positions for the backbone and for all heavy atoms, respectively, when 11 residues at the N-terminus are excluded. The structure has the same global fold as observed for other MMP catalytic domains and is similar to previously solved crystal structures of MMP-2. Differences observed between the solution and the crystal structures, near the bottom of the S1' specificity loop, appear to be induced by the large inhibitor present in the solution structure. The MMP-2C solution structure is compared with MMP-8 crystal structure bound to the same inhibitor to highlight the differences especially in the S1' specificity loop. The finding provides a structural explanation for the selectivity between MMP-2 and MMP-8 that is achieved by large inhibitors.

15N nuclear magnetic resonance relaxation studies on rat beta-parvalbumin and the pentacarboxylate variants, S55D and G98D.

15N relaxation data for Ca(2+)-bound rat beta-parvalbumin (a.k.a. oncomodulin) were analyzed using the Lipari-Szabo formalism and compared with existing data for rat alpha-parvalbumin. Although the average S(2) values for the two proteins are very similar (0.85 for alpha, 0.84 for beta), residue-by-residue inspection reveals systematic differences. alpha tends to have the lower S(2) value in helical regions; beta tends to have the lower value in the loop regions. Rat beta was also examined in the Ca(2+)-free state. The 59 assigned residues displayed an average order parameter (0.90) significantly greater than the corresponding residues in the Ca(2+)-loaded form. The pentacarboxylate variants of rat beta-S55D and G98D-also were examined in the Ca(2+)-bound state. Although both mutations significantly heighten Ca(2+) affinity, they utilize distinct energetic strategies. S55D improves the Ca(2+)-binding enthalpy; G98D improves the binding entropy. They also show disparate peptide backbone dynamics. Whereas beta G98D displays an average order parameter (0.87) slightly greater than that of the wild-type protein, beta S55D displays an average order parameter (0.82) slightly lower than wild-type beta. Furthermore, whereas just two backbone N-H bonds in beta G98D show internal motion on the 20-200-psec timescale, fully 52 of the 93 residues analyzed in beta S55D show this behavior. These findings suggest that the increased electrostatic repulsion attendant to introduction of an additional carboxylate into the CD site ligand array impedes backbone vibrational motion throughout the molecule.