Base-type-selective high-resolution 13C edited NOESY for sequential assignment of large RNAs

Extensive spectral overlap presents a major problem for the NMR study of large RNAs. Here we present NMR techniques for resolution enhancement and spectral simplification of fully ¹³C labelled RNA. High-resolution ¹H-¹³C correlation spectra are obtained by combining TROSY-type experiments with multiple-band-selective homonuclear ¹³C decoupling. An additional C-C filter sequence performs base-type-selective spectral editing. Signal loss during the filter is significantly reduced because of TROSY-type spin evolution. These tools can be inserted in any ¹³C-edited multidimensional NMR experiment. As an example we have chosen the ¹³C-edited NOESY which is a crucial experiment for sequential resonance assignment of RNA. Application to a 33-nucleotide RNA aptamer and a 76-nucleotide tRNA illustrates the potential of this new methodology.     

Biphasic folding kinetics of RNA pseudoknots and telomerase RNA activity

Using a combined master equation and kinetic cluster approach, we investigate RNA pseudoknot folding and unfolding kinetics. The energetic parameters are computed from a recently developed Vfold model for RNA secondary structure and pseudoknot folding thermodynamics. The folding kinetics theory is based on the complete conformational ensemble, including all the native-like and non-native states. The predicted folding and unfolding pathways, activation barriers, Arrhenius plots, and rate-limiting steps lead to several findings. First, for the PK5 pseudoknot, a misfolded 5' hairpin emerges as a stable kinetic trap in the folding process, and the detrapping from this misfolded state is the rate-limiting step for the overall folding process. The calculated rate constant and activation barrier agree well with the experimental data. Second, as an application of the model, we investigate the kinetic folding pathways for human telomerase RNA (hTR) pseudoknot. The predicted folding and unfolding pathways not only support the proposed role of conformational switch between hairpin and pseudoknot in hTR activity, but also reveal molecular mechanism for the conformational switch. Furthermore, for an experimentally studied hTR mutation, whose hairpin intermediate is destabilized, the model predicts a long-lived transient hairpin structure, and the switch between the transient hairpin intermediate and the native pseudoknot may be responsible for the observed hTR activity. Such finding would help resolve the apparent contradiction between the observed hTR activity and the absence of a stable hairpin.     

Automated assignment and 3D structure calculations using combinations of 2D homonuclear and 3D heteronuclear NOESY spectra

The NOAH/DIAMOD suite uses feedback filtering and self-correcting distance geometry to generate 3D structures from unassigned NOESY spectra. In this study we determined the minimum set of experiments needed to generate a high quality structure bundle. Different combinations of 3D ¹⁵N-edited, ¹³C-edited HSQC-NOESY and 2D homonuclear ¹H-¹H NOESY spectra of the 77 amino acid protein, myeloid progenitor inhibitory factor-1 (MPIF-1) were used as input for NOAH/DIAMOD calculations. The quality of the assignments of NOESY cross peaks and the accuracy of the automatically generated 3D structures were compared to those obtained with a conventional manual procedure. Combining data from two types of experiments synergistically increased the number of peaks assigned unambiguously in both individual spectra. As a general trend for the accuracy of the structures we observed structural variations in the backbone fold of the final structures of about 2 Å for single spectral data, of 1 Å to 1.5 Å for double spectral data, and of 0.6 Å for triple spectral data sets. The quality of the assignments and 3D structures from the optimal data using all three spectra were similar to those obtained from traditional assignment methods with structural variations within the bundle of 0.6 Å and 1.3 Å for backbone and heavy atoms, respectively. Almost all constraints (97%) of the automatic NOESY cross peak assignments were cross compatible with the structures from the conventional manual assignment procedure, and an even larger proportion (99%) of the manually derived constraints were compatible with the automatically determined 3D structures. The two mean structures determined by both methods differed only by 1.3 Å rmsd for the backbone atoms in the well-defined regions of the protein. Thus NOAD/DIAMOD analysis of spectra from labeled proteins provides a reliable method for high throughput analysis of genomic targets.     

Automated combined assignment of NOESY spectra and three-dimensional protein structure determination

A procedure for automated protein structure determination is presented that is based on an iterative procedure during which the NOESY peak list assignment and the structure calculation are performed simultaneously. The input consists of a list of NOESY peak positions and a list of chemical shifts as obtained from sequence-specific resonance assignment. For the present applications of this approach the previously introduced NOAH routine was implemented in the distance geometry program DIANA. As an illustration, experimental 2D and 3D NOESY cross-peak lists of six proteins have been analyzed, for which complete sequence-specific ¹H assignments are available for the polypeptide backbone and the amino acid side chains. The automated method assigned 70–90% of all NOESY cross peaks, which is on average 10% less than with the interactive approach, and only between 0.8% and 2.4% of the automatically assigned peaks had a different assignment than in the corresponding manually assigned peak lists. The structures obtained with NOAH/DIANA are in close agreement with those from manually assigned peak lists, and with both approaches the residual constraint violations correspond to high-quality NMR structure determinations. Systematic comparisons of the bundles of conformers that represent corresponding automatically and interactively determined structures document the absence of significant bias in either approach, indicating that an important step has been made towards automation of structure determination from NMR spectra.     

Stereochemical structure determination of p-cymene Ru(II) complexes containing the PPh2Py ligand with 2-D NOESY and HMQC NMR experiments

Complexes [Ru(p-cymene)Cl₂(PPh₂Py)] (1) and [Ru(p-cymene)Cl(PPh₂Py)]BF₄ (2) were studied by means of ¹H, ¹³C{¹H} 2-D NOESY and HMQC NMR spectral methods. NMR data agree with C₁ and C_s symmetries for complexes 1 and 2, respectively. NOESY cross-peaks allowed the assignment of signals to CH arene protons and in the case of complex 2 the determination of the molecular stereochemistry. These results are in agreement with the X-ray molecular structures of both complexes.     

Optimization of resolution and sensitivity of 4D NOESY using Multi-dimensional Decomposition

Highly resolved multi-dimensional NOE data are essential for rapid and accurate determination of spatial protein structures such as in structural genomics projects. Four-dimensional spectra contain almost no spectral overlap inherently present in lower dimensionality spectra and are highly amenable to application of automated routines for spectral resonance location and assignment. However, a high resolution 4D data set using conventional uniform sampling usually requires unacceptably long measurement time. Recently we have reported that the use of non-uniform sampling and multi-dimensional decomposition (MDD) can remedy this problem. Here we validate accuracy and robustness of the method, and demonstrate its usefulness for fully protonated protein samples. The method was applied to 11 kDa protein PA1123 from structural genomics pipeline. A systematic evaluation of spectral reconstructions obtained using 15–100% subsets of the complete reference 4D ¹H–¹³C–¹³C–¹H NOESY spectrum has been performed. With the experimental time saving of up to six times, the resolution and the sensitivity per unit time is shown to be similar to that of the fully recorded spectrum. For the 30% data subset we demonstrate that the intensities in the reconstructed and reference 4D spectra correspond with a correlation coefficient of 0.997 in the full range of spectral amplitudes. Intensities of the strong, middle and weak cross-peaks correlate with coefficients 0.9997, 0.9965, and 0.83. The method does not produce false peaks. 2% of weak peaks lost in the 30% reconstruction is in line with theoretically expected noise increase for the shorter measurement time. Together with good accuracy in the relative line-widths these translate to reliable distance constrains derived from sparsely sampled, high resolution 4D NOESY data.     

Variability in automated assignment of NOESY spectra and three-dimensional structure determination: A test case on three small disulfide-bonded proteins

Three independent runs of automatic assignment and structure calculations were performed on three small proteins, calcicludine from the venom of the green mamba Dendroaspis angusticeps, -conotoxin PVIIA from the purple cone Conus purpurascens and HsTX1, a short scorpion toxin from the venom of Heterometrus spinnifer. At the end of all the runs, the number of cross peaks which remained unassigned (0.6%, 1.4% and 2% for calcicludine, -conotoxin and HsTX1, respectively), as well as the number of constraints which were rejected as producing systematic violations (2.7%, 1.0%, and 1.4% for calcicludine, -conotoxin and HsTX1, respectively) were low. The conformation of the initial model used in the procedure (linear model or constructed by homology) has no influence on the final structures. Mainly two parameters control the procedure: the chemical shift tolerance and the cut-off distance. Independent runs of structure calculations, using the same parameters, yield structures for which the rmsd between averaged structures and the rmsd around each averaged structure were of the same order of magnitude. A different cut-off distance and a different chemical shift tolerance yield rmsd values on final average structures which did not differ more than 0.5 Å compared to the rmsd obtained around the averaged structure for each calculation. These results show that the procedure is robust when applied to such a small disulfide-bonded protein.     

An efficient strategy for assignment of cross-peaks in 3D heteronuclear NOESY experiments

The question is addressed of how maximal structural NOE data on double labelled proteins can be acquired with a minimal set of NOESY experiments. Two 3D-NOESY spectra are reported which, in concert with other commonly used spectra, provide a convenient strategy for NOE assignment. The 3D CNH-NOESY and 3D NCH-NOESY provide NOE connectivities between amide protons and carbon-bound protons and constitute orthogonal heteronuclear filters which eliminate diagonal signals, considerably improving spectral quality. Two different heteronuclear chemical shift dimensions are recorded in the spectra, thus exploiting the extra dispersion of the heteronucleus and considerably simplifying assignment.     

2D and 3D TROSY-enhanced NOESY of 15N labeled proteins

Recently, several TROSY-based experiments have been designed for backbone chemical shift assignment and measurement of the NOEs of ²H, ¹³C and ¹⁵N labeled proteins. Here, we present TROSY-enhanced NOESY experiments, namely the 2D S3E-NOESY-S3E, 3D TROSY-NOESY-S3E and S3E-NOESY-TROSY experiments. These experiments use the spin-state selective excitation method (S3E), and have the TROSY effect in all the indirectly and directly detected dimensions, and so provide optimal resolution for amide protons. The first two experiments provide an additional useful feature in that the diagonal peaks of the amide proton region are cancelled or greatly reduced, allowing clear identification of NOE cross peaks that are close to diagonal peaks.     

RNA2D3D: A program for Generating,Viewing, and Comparing 3-Dimensional Models of RNA

Abstract

Using primary and secondary structure information of an RNA molecule, the program RNA2D3D automatically and rapidly produces a first-order approximation of a 3-dimensional conformation consistent with this information. Applicable to structures of arbitrary branching complexity and pseudoknot content, it features efficient interactive graphical editing for the removal of any overlaps introduced by the initial generating procedure and for making conformational changes favorable to targeted features and subsequent refinement. With emphasis on fast exploration of alternative 3D conformations, one may interactively add or delete base-pairs, adjacent stems can be coaxially stacked or unstacked, single strands can be shaped to accommodate special constraints, and arbitrary subsets can be defined and manipulated as rigid bodies. Compaction, whereby base stacking within stems is optimally extended into connecting single strands, is also available as a means of strategically making the structures more compact and revealing folding motifs. Subsequent refinement of the first-order approximation, of modifications, and for the imposing of tertiary constraints is assisted with standard energy refinement techniques. Previously determined coordinates for any part of the molecule are readily incorporated, and any part of the modeled structure can be output as a PDB or XYZ file. Illustrative applications in the areas of ribozymes, viral kissing loops, viral internal ribosome entry sites, and nanobiology are presented.     

RNA structure and RNA-protein interactions in purified yeast U6 snRNPs

The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2p-8p proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal function. However, the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. Here, we have investigated the secondary structure of the U6 snRNA in purified U6 snRNPs and compared it with its naked form. Using RNA structure-probing techniques, we demonstrate that within the U6 snRNP a large internal region of the U6 snRNA is unpaired and protected from chemical modification by bound Prp24p. Several of these U6 nucleotides are available for base-pairing interaction, as only their sugar backbone is contacted by Prp24p. Thus, Prp24p can present them to the U4 snRNA and facilitate formation of U4/U6 stem I. We show that the 3' stem-loop is not bound strongly by U6 proteins in native particles. However, when compared to the 3' stem-loop in the naked U6 snRNA, it has a more open conformation, which would facilitate formation of stem II with the U4 snRNA. Our data suggest that the combined association of Prp24p and the LSm complex confers upon U6 nucleotides a conformation favourable for U4/U6 base-pairing. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs.     

Free energy landscapes of RNA/RNA complexes: with applications to snRNA complexes in spliceosomes

We develop a statistical mechanical model for RNA/RNA complexes with both intramolecular and intermolecular interactions. As an application of the model, we compute the free energy landscapes, which give the full distribution for all the possible conformations, for U4/U6 and U2/U6 in major spliceosome and U4atac/U6atac and U12/U6atac in minor spliceosome. Different snRNA experiments found contrasting structures, our free energy landscape theory shows why these structures emerge and how they compete with each other. For yeast U2/U6, the model predicts that the two distinct experimental structures, the four-helix junction structure and the helix Ib-containing structure, can actually coexist and specifically compete with each other. In addition, the energy landscapes suggest possible mechanisms for the conformational switches in splicing. For instance, our calculation shows that coaxial stacking is essential for stabilizing the four-helix junction in yeast U2/U6. Therefore, inhibition of the coaxial stacking possibly by protein-binding may activate the conformational switch from the four-helix junction to the helix Ib-containing structure. Moreover, the change of the energy landscape shape gives information about the conformational changes. We find multiple (native-like and misfolded) intermediates formed through base-pairing rearrangements in snRNA complexes. For example, the unfolding of the U2/U6 undergoes a transition to a misfolded state which is functional, while in the unfolding of U12/U6atac, the functional helix Ib is found to be the last one to unfold and is thus the most stable structural component. Furthermore, the energy landscape gives the stabilities of all the possible (functional) intermediates and such information is directly related to splicing efficiency.     

NMR structure of 2′-O-(2-methoxyethyl) modified and C5-methylated RNA dodecamer duplex

The solution-state structure of 2′-O-(2-methoxyethly) substituted dodecamer r(*CG*CGAA*U*U*CG*C)d(G), 2′-MOE RNA, with all cytosines and uracils methylated at the C5-position has been determined by NMR spectroscopy. The chemical modifications were used to improve the oligonucleotide's drug-like properties. The 2′-MOE group drives pseudorotational equilibrium of the ribofuranose moiety to the N-type conformation and supposedly results in structural preorganization leading to high affinity of a modified oligonucleotide towards its complementary biological target, improved pharmacokinetic and toxicological properties. The high melting temperature of the antiparallel duplex structure adopted by 2′-MOE RNA was explained through the formation of a stable A-form RNA consistent with effective base-pairing and stacking interactions. The comparison of the solution-state structure with the crystal structure of a non-methylated analogue shows an increase in the stacking at the base pair steps for the C5-methylated 2′-MOE RNA duplex. The MOE substituents adopt a well-defined structure in the minor groove with the predominant gauche conformations around the ethylene bond.     

Improved simulation of NOESY spectra by RELAX-JT2 including effects of J-coupling, transverse relaxation and chemical shift anisotropy

RELAX-JT2 is an extension of RELAX, a program for the simulation of 1H 2D NOESY spectra and (15)N or (13)C edited 3D NOESY-HSQC spectra of biological macromolecules. In addition to the already existing NOE-simulation it allows the proper simulation of line shapes by the integrated calculation of T(2) times and multiplet structures caused by J-couplings. Additionally the effects of relaxation mediated by chemical shift anisotropy are taken into account. The new routines have been implemented in the program AUREMOL, which aims at the automated NMR structure determination of proteins in solution. For a manual or automatic assignment of experimental spectra that is based on the comparison with the corresponding simulated spectra, the additional line shape information now available is a valuable aid. The new features have been successfully tested with the histidine-containing phosphocarrier protein HPr from Staphylococcus carnosus.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

NMR structure of a 4 x 4 nucleotide RNA internal loop from an R2 retrotransposon: identification of a three purine-purine sheared pair motif and comparison to MC-SYM predictions

The NMR solution structure is reported of a duplex, 5'GUGAAGCCCGU/3'UCACAGGAGGC, containing a 4 × 4 nucleotide internal loop from an R2 retrotransposon RNA. The loop contains three sheared purine-purine pairs and reveals a structural element found in other RNAs, which we refer to as the 3RRs motif. Optical melting measurements of the thermodynamics of the duplex indicate that the internal loop is 1.6 kcal/mol more stable at 37°C than predicted. The results identify the 3RRs motif as a common structural element that can facilitate prediction of 3D structure. Known examples include internal loops having the pairings: 5'GAA/3'AGG, 5'GAG/3'AGG, 5'GAA/3'AAG, and 5'AAG/3'AGG. The structural information is compared with predictions made with the MC-Sym program.     

Refinement of the structure of protein–RNA complexes by residual dipolar coupling analysis

The main limitation in NMR-determined structures of nucleic acids and their complexes with proteins derives from the elongated, non-globular nature of physiologically important DNA and RNA molecules. Since it is generally not possible to obtain long-range distance constraints between distinct regions of the structure, long-range properties such as bending or kinking at sites of protein recognition cannot be determined accurately nor precisely. Here we show that use of residual dipolar couplings in the refinement of the structure of a protein–RNA complex improves the definition of the long-range properties of the RNA. These features are often an important aspect of molecular recognition and biological function; therefore, their improved definition is of significant value in RNA structural biology.