Fourier Transform Multiple Quantum Nuclear Magnetic Resonance

The excitation and detection of multiple quantum transitions in systems of coupled spins offers, among other advantages, an increase in resolution over single quantum n.m.r. since the number of lines decreases as the order of the transition increases. This paper reviews the motivation for detecting multiple quantum transitions by a Fourier transform experiment and describes an experimental approach to high resolution multiple quantum spectra in dipolar systems along with results on some protonated liquid crystal systems. A simple operator formalism for the essential features of the time development is presented and some applications in progress are discussed

Toward a Structure Determination Method for Biomineral-Associated Protein Using Combined Solid- State NMR and Computational Structure Prediction

SummaryProtein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution nuclear magnetic resonance (NMR). Here we report a method for determining the structure of biomineral-associated proteins. The method combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. In addition, the algorithm is able to identify lattice geometries most compatible with ssNMR constraints, representing a quantitative, novel method for investigating crystal-face binding specificity. We use this method to determine most of the structure of human salivary statherin interacting with the mineral phase of tooth enamel. Computation and experiment converge on an ensemble of related structures and identify preferential binding at three crystal surfaces. The work represents a significant advance toward determining structure of biomineral-adsorbed protein using experimentally biased structure prediction. This method is generally applicable to proteins that can be chemically synthesized

Monitoring tat peptide binding to TAR RNA by solid-state (31)P–(19)F REDOR NMR

Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 Å decrease in the separation between phosphorothioate and 2′F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR–tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, (31)P–(19)F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA–peptide complex

Dynamic Impact of Methylation at the M. HhaI Target Site: A Solid-State Deuterium NMR Study

Base methylation plays an important role in numerous biological functions of DNA, from inhibition of cleavage by endonucleases to inhibition of transcription factor binding. Studies of nucleic acid structure have shown little differences in unmethylated DNAs and the identical sequence containing methylated analogues. We have investigated changes in the local dynamics of DNA upon substitution of a methylated cytosine analogue for cytosine using solid-state deuterium NMR. In particular, we have observed changes in the local dynamics at the target site of the M. HhaI restriction system. These studies observe changes in the amplitudes of the local backbone dynamics at the actual target site of the HhaI methyltransferase. This conclusion is another indication that the significant result of base methylation is to perturb the local dynamics, and therefore the local conformational flexibility, of the DNA helix, inhibiting or restricting the protein\u27s ability to manipulate the DNA helix in order to perform its chemical alterations

A Solid-State Deuterium NMR Study of the Localized Dynamics at the C9pG10 Step in the DNA Dodecamer [d(CGCGAATTCGCG)]2

A solid-state deuterium NMR study of localized mobility at the C9pG10 step in the DNA dodecamer [d(CGCGAATTCGCG)]2 is described. In contrast to the results of earlier deuterium NMR studies of furanose ring and backbone dynamics within the d(AATT) moiety, the furanose ring and helix backbone of dC9 display large amplitudes of motion on the 0.1 ms time scale at hydration levels characteristic of the B form structure. Solid-state deuterium NMR line shape data obtained from labeled dC9 DNA are interpreted using a composite motion model, in which the DNA oligomer is treated as rotating as a whole about the helix axis, while the base, furanose ring, and phosphodiester backbone execute localized motions. Consistent with past solid-state NMR studies, the amplitude and rate of the uniform rotation of the dC9-labeled oligomer are found to be sensitive to hydration level. Amplitudes of localized reorientational motions of C−D bonds in the furanose ring and backbone of dC9 are found to be larger than the librational amplitudes for the C−D bonds in the base of dC9, indicating that the pyrimidine base sugar does not move as a rigid entity and intersects a locally flexible region of the phosphodiester backbone. At hydration levels corresponding to 10−12 waters per nucleotide, Zeeman relaxation times for the furanose ring and backbone deuterons of dC9 in B form DNA equal 0.025 and 0.03 ms, respectively, and are the shortest relaxation times observed thus far for any deuteron in the DNA dodecamer at comparable hydration levels. The results of this solid-state NMR study suggest the existence of a significant dynamic component of sequence-specific recognition in this system