NMR studies of RNA dynamics and structural plasticity using NMR residual dipolar couplings

An increasing number of RNAs are being discovered that perform their functions by undergoing large changes in conformation in response to a variety of cellular signals, including recognition of proteins and small molecular targets, changes in temperature, and RNA synthesis itself. The measurement of NMR residual dipolar couplings (RDCs) in partially aligned systems is providing new insights into the structural plasticity of RNA through combined characterization of large-amplitude collective helix motions and local flexibility in noncanonical regions over a wide window of biologically relevant timescales (相似文献   

Transverse relaxation optimised spin-state selective NMR experiments for measurement of residual dipolar couplings

Three transverse relaxation optimised NMR experiments (TROSY) for the measurement of scalar and dipolar couplings suitable for proteins dissolved in aqueous iso- and anisotropic solutions are described. The triple-spin-state-selective experiments yield couplings between ¹H^N-¹³C, ¹⁵N-¹³C, ¹H^N-¹³C _i–1, ¹⁵N-¹³C _i–1, ¹H^N-¹³C_i–1, ¹⁵N-¹³C_i–1, and ¹³C_i–1-¹³C _i–1 without introducing nonessential spectral crowding compared with an ordinary two-dimensional ¹⁵N-¹H correlation spectrum and without requiring explicit knowledge of carbon assignments. This set of /-J-TROSY experiments is most useful for perdeuterated proteins in studies of structure–activity relationships by NMR to observe, in addition to epitopes for ligands, also conformational changes induced by binding of ligands.     

Mg2+-induced variations in the conformation and dynamics of HIV-1 TAR RNA probed using NMR residual dipolar couplings

The effects of divalent Mg(2+) on the conformation and dynamics of the stem-loop transactivation response element (TAR) RNA from HIV-1 have been characterized using NMR residual dipolar couplings (RDCs). Order matrix analysis of one bond 13C-1H RDCs measured in TAR at [Mg(2+)]:[TAR] stoichiometric ratios of approximately 3:1 (TAR(3.0Mg)) and approximately 4.5:1 (TAR(4.5Mg)) revealed that Mg(2+) reduces the average inter-helical angle from 47(+/-5) degrees in TAR(free) to 5(+/-7) degrees in TAR(4.5Mg). In contrast to the TAR(free) state, the generalized degree of order for the two stems in TAR(4.5Mg) is found to be identical within experimental uncertainty, indicating that binding of Mg(2+) leads to an arrest of inter-helical motions in TAR(free). Results demonstrate that RDC-NMR methodology can provide new insight into the effects of Mg(2+) on both the conformation and dynamics of RNA.     

NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings

A modified version of the JHH-TOCSY experiment, `signed COSY', is presented that allows the determination of the sign of residual dipolar ¹H-¹H coupling constants with respect to the sign of one-bond ¹H-X coupling constants in linear three-spin systems X-¹H-¹H, where X = ¹³C or ¹⁵N. In contrast to the original JHH-TOCSY experiments, the signs of J _HH couplings may be determined for CH₂-CH₂ moieties and for uniformly ¹³C/¹⁵N-labelled samples. In addition, sensitivity is enhanced, diagonal peaks are suppressed and cross peaks are observed only between directly coupled protons, as in a COSY spectrum.     

Sensitivity of NMR residual dipolar couplings to perturbations in folded and denatured staphylococcal nuclease

The invariance of NMR residual dipolar couplings (RDCs) in denatured forms of staphylococcal nuclease to changes in denaturant concentration or amino acid sequence has previously been attributed to the robustness of long-range structure in the denatured state. Here we compare RDCs of the wild-type nuclease with those of a fragment that retains a folded OB-fold subdomain structure despite missing the last 47 of 149 residues. The RDCs of the intact protein and of the truncation fragment are substantially different under conditions that favor folded structure. By contrast, there is a strong correlation between the RDCs of the full-length protein and the fragment under denaturing conditions (6 M urea). The RDCs of the folded and unfolded forms of the proteins are uncorrelated. Our results suggest that RDCs are more sensitive to structural changes in folded than unfolded proteins. We propose that the greater susceptibility of RDCs in folded states is a consequence of the close packing of the polypeptide chain under native conditions. By contrast, the invariance of RDCs in denatured states is more consistent with a disruption of cooperative structure than with the retention of a unique long-range folding topology.     

NMR analysis of bovine tRNATrp: conformation dependence of Mg2+ binding

NMR was used to study the solution structure of bovine tRNA(Trp) hyperexpressed in Escherichia coli. With the use of (15)N labeling and site-directed mutagenesis to assign overlapping resonances through the base pair replacement of U(71)A(2) by G(2)C(71), U(27)A(43) by G(27)C(43), and G(12)C(23) by U(12)A(23), the resonances of all 26 observable imino protons in the helical regions and in the tertiary interactions were assigned unambiguously by means of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear single quantum coherence methods. When the discriminator base A(73) and the G(12)C(23) base pair on the D stem, two identity elements on bovine tRNA(Trp) that are important for effective recognition by tryptophanyl-tRNA synthetase, were mutated to the ineffective forms of G(73) and U(12)A(23), respectively, NMR analysis revealed an important conformational change in the U(12)A(23) mutant but not in the G(73) mutant molecule. Thus A(73) appears to be directly recognized by tryptophanyl-tRNA synthetase, and G(12)C(23) represents an important structural determinant. Mg(2+) effects on the assigned resonances of imino protons allowed the identification of strong, medium, and weak Mg(2+) binding sites in tRNA(Trp). Strong Mg(2+) binding modes were associated with the residues G(7), s(4)U(8) (where s(4)U is 4-thiouridine), G(12), and U(52). The observations that G(42) was associated with strong Mg(2+) binding in only the U(12)A(23) mutant tRNA(Trp) but not the wild type or G(73) mutant tRNA(Trp) and that the G(7), s(4)U(8), G(24), and G(22) imino protons are associated with a two-site Mg(2+) binding mode in wild type and G(73) mutant but only a one-site mode in the U(12)A(23) mutant established the occurrence of conformational change in the U(12)A(23) mutant tRNA(Trp). These observations also established the dependence of Mg(2+) binding on tRNA conformation and the usefulness of Mg(2+) binding sites as conformational probes. The thermal titration of tRNA(Trp) in the presence and absence of 10 mm Mg(2+) indicated that overall tRNA(Trp) structure stability was increased by more than 15 degrees C by the presence of Mg(2+).     

Conformational studies of Lewis X and Lewis A trisaccharides using NMR residual dipolar couplings.

The conformations of the histo-blood group carbohydrate antigens Lewis X (Le(x)) and Lewis A (Le(a)) were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. A strategy for rapid calculation of the difference between theoretical and experimental dipolar couplings of a large number of model structures generated by computer simulations was developed, resulting in an accurate model structure for the compounds. Monte Carlo simulations were used to generate models for the trisaccharides, and orientations of each model were sought that could reproduce the experimental residual dipolar coupling values. For both, Le(a) and Le(x), single low energy models giving excellent agreement with experiment were found, implying a compact rigidly folded conformation for both trisaccharides. The new approach was also applied to the pentasaccharides lacto-N-fucopentaose 2 (LNF-2) and lacto-N-fucopentaose 3 (LNF-3) proving its consistency and robustness. For describing the conformation of tightly folded oligosaccharides, a definition for characterization of ring planes in pyranoside chairs is proposed and applied to the analysis of the relation between the fucose and galactose residues in the epitopes, revealing the structural similarity between them.     

Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

We present a protocol for determining the relative orientation and dynamics of A-form helices in 13C/15N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal Watson-Crick base pairs in helical stems are experimentally identified using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic field. The order tensors are translated into Euler angles defining the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.     

NMR experiments for the measurement of proton-proton and carbon-carbon residual dipolar couplings in uniformly labelled oligosaccharides

A 2D-HSQC-carbon selective/proton selective-constant time COSY, 2D-HSQC-(sel C, sel H)-CT COSY experiment, which is applicable to uniformly ¹³C isotopically enriched samples (U-¹³C) of oligosaccharides or oligonucleotides is proposed for the measurement of proton–proton RDC in crowded regions of 2D-spectra. In addition, a heteronuclear constant time-COSY experiment, ¹³C-¹³C CT-COSY, is proposed for the measurement of one bond carbon–carbon RDC in these molecules. These two methods provide an extension, to U-¹³C molecules, of the original homonuclear constant time-COSY experiment proposed by Tian et al. (1999) for saccharides. The combination of a number of these RDC with NOE data may provide the method of choice to study oligosaccharide conformation in the free and receptor-bound state.     

Conformational studies of blood group A and blood group B oligosaccharides using NMR residual dipolar couplings

The conformations of two synthetic trisaccharides of blood group A and B (alpha-L-Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-alpha-D-Galp and alpha-L-Fucp-(1-->2)-[alpha-D-Galp-(1-->3)]-alpha-D-Galp, respectively) and of a type A tetrasaccharide alditol, Fucp-(1-->2)-[alpha-D-GalpNAc-(1-->3)]-beta-D-Galp-(1-->3)-GalNAc-ol, were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. The conformations of the three oligosaccharides were analyzed by generating thousands of structures using a Monte-Carlo method. Two different strategies were applied to calculate theoretical dipolar couplings for these structures. In the first method, the orientation of the molecule was calculated from the optimal fit of the molecular model to the experimental data, while in the second method the orientation tensor was calculated directly from the moment of inertia of the molecular model. Both methods of analysis give similar results but with slightly better agreement with experiment for the former one. The analysis of the results implies a single unique conformation for both blood group epitopes in solution in disagreement with theoretical models suggesting the existence of two conformers in solution.     

NMR analysis of the Mg2+-binding properties of aequorin, a Ca2+-binding photoprotein

Aequorin, which is a calcium-sensitive photoprotein and a member of the EF-hand superfamily, binds to Mg2+ under physiological conditions, which modulates its light emission. The Mg2+ binding site and its stabilizing influence were examined by NMR spectroscopy. The binding of Mg2+ to aequorin prevented the molecule from aggregating and stabilized it in the monomeric form. To determine the structural differences between Mg2+-bound and free aequorin, we have performed backbone NMR assignments of aequorin in the Mg2+-free state. Mg2+ binding induces conformational changes that are localized in the EF-hand loops. The chemical shift difference data indicated that there are two Mg2+-binding sites, EF-hands I and III. The Mg2+ titration experiment revealed that EF-hand III binds to Mg2+ with higher affinity than EF-hand I, and that only EF-hand III seems to be occupied by Mg2+ under physiological conditions.     

Solution structure and metal-ion binding of the P4 element from bacterial RNase P RNA

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)3+(6)) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)3+(6) is a useful spectroscopic probe for Mg(H2O)2+(6)-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)3+(6). We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C.G base pair is replaced by U.G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3' direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)3+(6) and RNA protons were used to locate the site of Co(NH3)3+(6) binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5' by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.     

G350 of Escherichia coli RNase P RNA contributes to Mg2+ binding near the active site of the enzyme

G350 of Escherichia coli RNase P RNA is a highly conserved residue among all bacteria and lies near the known magnesium binding site for the RNase P ribozyme, helix P4. Mutations at G350 have a dramatic effect on substrate cleavage activity for both RNA alone and holoenzyme; the G350C mutation has the most severe phenotype. The G350C mutation also inhibits growth of cells that express the mutant RNA in vivo under conditions of magnesium starvation. The results suggest that G350 contributes to Mg(2+) binding at helix P4 of RNase P RNA.     

Study of phospholipid structure by 1H, 13C, and 31P dipolar couplings from two-dimensional NMR.

Various motionally averaged 31P-1H, 13C-1H, 1H-1H, and 31P-13C dipolar couplings were measured for natural-abundance and unoriented phosphocholine in the L alpha phase. The couplings were obtained and assigned by a variety of advanced and partly novel two-dimensional solid-state NMR experiments. Whereas 31P-1H and 31P-13C dipolar couplings provide long-range structural constraints, geminal 1H-1H couplings and the signs of 13C-1H couplings are important new elements in a segmental order-tensor analysis of the lipid headgroup and glycerol backbone. The implications of these measured dipolar couplings for the conformational exchange of the lipid headgroup and the bending of the headgroup from the glycerol backbone are discussed. These dipolar couplings are also analyzed semiquantitatively in terms of the segmental order tensor.     

Structural analysis of Mg2+ and Ca2+ binding to CaBP1, a neuron-specific regulator of calcium channels

CaBP1 (calcium-binding protein 1) is a 19.4-kDa protein of the EF-hand superfamily that modulates the activity of Ca(2+) channels in the brain and retina. Here we present data from NMR, microcalorimetry, and other biophysical studies that characterize Ca(2+) binding, Mg(2+) binding, and structural properties of recombinant CaBP1 purified from Escherichia coli. Mg(2+) binds constitutively to CaBP1 at EF-1 with an apparent dissociation constant (K(d)) of 300 microm. Mg(2+) binding to CaBP1 is enthalpic (DeltaH = -3.725 kcal/mol) and promotes NMR spectral changes, indicative of a concerted Mg(2+)-induced conformational change. Ca(2+) binding to CaBP1 induces NMR spectral changes assigned to residues in EF-3 and EF-4, indicating localized Ca(2+)-induced conformational changes at these sites. Ca(2+) binds cooperatively to CaBP1 at EF-3 and EF-4 with an apparent K(d) of 2.5 microM and a Hill coefficient of 1.3. Ca(2+) binds to EF-1 with low affinity (K(d) >100 microM), and no Ca(2+) binding was detected at EF-2. In the absence of Mg(2+) and Ca(2+), CaBP1 forms a flexible molten globule-like structure. Mg(2+) and Ca(2+) induce distinct conformational changes resulting in protein dimerization and markedly increased folding stability. The unfolding temperatures are 53, 74, and 76 degrees C for apo-, Mg(2+)-bound, and Ca(2+)-bound CaBP1, respectively. Together, our results suggest that CaBP1 switches between structurally distinct Mg(2+)-bound and Ca(2+)-bound states in response to Ca(2+) signaling. Both conformational states may serve to modulate the activity of Ca(2+) channel targets.     

NMR spectroscopic evidence for Mn(2+)(Mg(2+)) binding to a precursor-tRNA microhelix near the potential RNase P cleavage site

The binding of Mg(2+)/Mn(2+) to acceptor stem microhelices as minimal models for precursor-tRNA(Gly) is demonstrated by NMR spectroscopy. From the evaluation of COSY and NOESY spectra, binding sites for Mg(2+)/Mn(2+) can be inferred. In particular, one binding site exists near the ribose moiety of nucleotide -1 at the position of cleavage by RNase P. From comparison with a variant possessing a deoxynucleotide at this position, it is concluded that the 2'-OH group of this nucleotide is indispensable for coordinating the divalent metal ion. Hence, this catalytically important metal ion is "pre-bound" to the precursor-tRNA before complexation with RNase P.     

Impact of static and dynamic A-form heterogeneity on the determination of RNA global structural dynamics using NMR residual dipolar couplings

We examined how static and dynamic deviations from the idealized A-form helix propagate into errors in the principal order tensor parameters determined using residual dipolar couplings (rdcs). A 20-ns molecular dynamics (MD) simulation of the HIV-1 transactivation response element (TAR) RNA together with a survey of spin relaxation studies of RNA dynamics reveals that pico-to-nanosecond local motions in non-terminal Watson-Crick base-pairs will uniformly attenuate base and sugar one bond rdcs by approximately 7%. Gaussian distributions were generated for base and sugar torsion angles through statistical comparison of 40 RNA X-ray structures solved to <3.0 A resolution. For a typical number (>or=11) of one bond C-H base and sugar rdcs, these structural deviations together with rdc uncertainty (1.5 Hz) lead to average errors in the magnitude and orientation of the principal axis of order that are <9% and <4 degrees, respectively. The errors decrease to <5% and <4 degrees for >or=17 rdcs. A protocol that allows for estimation of error in A-form order tensors due to both angular deviations and rdc uncertainty (Aform-RDC) is validated using theoretical simulations and used to analyze rdcs measured previously in TAR in the free state and bound to four distinct ligands. Results confirm earlier findings that the two TAR helices undergo large changes in both their mean relative orientation and dynamics upon binding to different targets.     

Towards structural genomics of RNA: rapid NMR resonance assignment and simultaneous RNA tertiary structure determination using residual dipolar couplings

We report a new residual dipolar couplings (RDCs) based NMR procedure for rapidly determining RNA tertiary structure demonstrated on a uniformly (15)N/(13)C-labeled 27 nt variant of the trans-activation response element (TAR) RNA from HIV-I. In this procedure, the time-consuming nuclear Overhauser enhancement (NOE)-based sequential assignment step is replaced by a fully automated RDC-based assignment strategy. This approach involves examination of all allowed sequence-specific resonance assignment permutations for best-fit agreement between measured RDCs and coordinates for sub-structures in a target RNA. Using idealized A-form geometries to model Watson-Crick helices and coordinates from a previous X-ray structure to model a hairpin loop in TAR, the best-fit RDC assignment solutions are determined very rapidly (相似文献