Helena Kovacs

Optimization of 13C direct detection NMR methods.

Abstract13C-detected experiments are still limited by their inherently lower sensitivity, as compared to the equivalent 1H-detected experiments. Improving the sensitivity of 13C detection methods remains a significant area of NMR research that may provide better means for studying large macromolecular systems by NMR. In this communication, we show that 13C-detected experiments are less sensitive to the salt concentration of the sample solution than 1H-detected experiments. In addition, acquisition can be started with anti-phase coherence, resulting in higher sensitivity due to the elimination of the final INEPT transfer step.

13C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides

We present here a set of 13C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4′ nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1′,H1′ ribose signals. The experiments were applied to two RNA hairpin structures. The current set of 13C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, 13C-direct detected NMR methods constitute useful complements to the conventional 1H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.

Solution structure of the coiled-coil trimerization domain from lung surfactant protein D

Surfactant protein D (SP-D) is one of four known protein components of the pulmonary surfactant lining the lung alveoli. It is involved in immune and allergic responses. SP-D occurs as a tetramer of trimers. Trimerization is thought to be initiated by a coiled coil domain. We have determined the solution structure of a 64-residue peptide encompassing the coiled coil domain of human SP-D. As predicted, the domain forms a triple-helical parallel coiled coil. As with all symmetric oligomers, the structure calculation was complicated by the symmetry degeneracy in the NMR spectra. We used the symmetry-ADR (ambiguous distance restraint) structure calculation method to solve the structure. The results demonstrate that the leucine zipper region of SP-D is an autonomously folded domain. The structure is very similar to the independently determined X-ray crystal structure, differing mainly at a single residue, Tyr248. This residue is completely symmetric in the solution structure, and markedly asymmetric in the crystalline phase. This difference may be functionally important, as it affects the orientation of the antigenic surface presented by SP-D.

Fast experiments for structure elucidation of small molecules: Hadamard NMR with multiple receivers

We propose several significant improvements to the PANSY (Parallel NMR SpectroscopY) experiments—PANSY COSY and PANSY‐TOCSY. The improved versions of these experiments provide sufficient spectral information for structure elucidation of small organic molecules from just two 2D experiments. The PANSY‐TOCSY‐Q experiment has been modified to allow for simultaneous acquisition of three different types of NMR spectra—1D C‐13 of non‐protonated carbon sites, 2D TOCSY and multiplicity edited 2D HETCOR. In addition the J‐filtered 2D PANSY‐gCOSY experiment records a 2D HH gCOSY spectrum in parallel with a 1J‐filtered HC long‐range HETCOR spectrum as well as offers a simplified data processing. In addition to parallel acquisition, further time savings are feasible because of significantly smaller F1 spectral windows as compared to the indirect detection experiments. Use of cryoprobes and multiple receivers can significantly alleviate the sensitivity issues that are usually associated with the so called direct detection experiments. In cases where experiments are sampling limited rather than sensitivity limited further reduction of experiment time is achieved by using Hadamard encoding. In favorable cases the total recording time for the two PANSY experiments can be reduced to just 40 s. The proposed PANSY experiments provide sufficient information to allow the CMCse software package (Bruker) to solve structures of small organic molecules. Copyright

Parallel NMR spectroscopy with simultaneous detection of (1) H and (19) F nuclei.

Recording NMR signals of several nuclear species simultaneously by using parallel receivers provides more information from a single measurement and at the same time increases the measurement sensitivity per unit time. Here we present a comprehensive series of the most frequently used NMR experiments modified for simultaneous direct detection of two of the most sensitive NMR nuclei - (1) H and (19) F. We hope that the presented material will stimulate interest in and further development of this technique.

Direct 13C-detected NMR experiments for mapping and characterization of hydrogen bonds in RNA

In RNA secondary structure determination, it is essential to determine whether a nucleotide is base-paired and not. Base-pairing of nucleotides is mediated by hydrogen bonds. The NMR characterization of hydrogen bonds relies on experiments correlating the NMR resonances of exchangeable protons and can be best performed for structured parts of the RNA, where labile hydrogen atoms are protected from solvent exchange. Functionally important regions in RNA, however, frequently reveal increased dynamic disorder which often leads to NMR signals of exchangeable protons that are broadened beyond 1H detection. Here, we develop 13C direct detected experiments to observe all nucleotides in RNA irrespective of whether they are involved in hydrogen bonds or not. Exploiting the self-decoupling of scalar couplings due to the exchange process, the hydrogen bonding behavior of the hydrogen bond donor of each individual nucleotide can be determined. Furthermore, the adaption of HNN-COSY experiments for 13C direct detection allows correlations of donor–acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed 13C direct detected experiments therefore provide information about molecular sites not amenable by conventional proton-detected methods. Such information makes the RNA secondary structure determination by NMR more accurate and helps to validate secondary structure predictions based on bioinformatics.

Probing water-protein contacts in a MMP-12/CGS27023A complex by nuclear magnetic resonance spectroscopy

Using the case of the catalytic domain of MMP-12 in complex with the known inhibitor CGS27023A, a recently assembled 3D 15N-edited/14N,12C-filtered ROESY experiment is used to monitor and distinguish protein amide protons in fast exchange with bulk water from amide protons close to water molecules with longer residence times, the latter possibly reflecting water molecules of structural or functional importance. The 15N-edited/14N,12C-filtered ROESY spectra were compared to the original 15N-edited/14N,12C-filtered NOESY and the conventional amide-water exchange experiment, CLEANEX. Three protein backbone amide protons experiencing direct dipolar cross relaxation with water in the 15N-edited/14N,12C-filtered ROESY spectrum were assigned. In an ensemble of six crystal structures, two conserved water molecules within 3 Å of the three amide protons were identified. These two water molecules are buried into cavities in the protein surface and thus sufficiently slowed down by the protein topology to account for the observed dipolar interaction. Structural analysis of an ensemble of six crystal structures ruled out any exchange-relayed contributions for the amide-water interactions of interest.