Hydration dependent dynamics in RNA

The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms–ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in ²H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state ¹³C relaxation measurements, we establish that ns–μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.     

Structural plasticity and Mg2+ binding properties of RNase P P4 from combined analysis of NMR residual dipolar couplings and motionally decoupled spin relaxation

The P4 helix is an essential element of ribonuclease P (RNase P) that is believed to bind catalytically important metals. Here, we applied a combination of NMR residual dipolar couplings (RDCs) and a recently introduced domain-elongation strategy for measuring "motionally decoupled" relaxation data to characterize the structural dynamics of the P4 helix from Bacillus subtilis RNase P. In the absence of divalent ions, the two P4 helical domains undergo small amplitude (approximately 13 degrees) collective motions about an average interhelical angle of 10 degrees. The highly conserved U7 bulge and helical residue C8, which are proposed to be important for substrate recognition and metal binding, are locally mobile at pico- to nanosecond timescales and together form the pivot point for the collective domain motions. Chemical shift mapping reveals significant association of Mg2+ ions at the P4 major groove near the flexible pivot point at residues (A5, G22, G23) previously identified to bind catalytically important metals. The Mg2+ ions do not, however, significantly alter the structure or dynamics of P4. Analysis of results in the context of available X-ray structures of the RNA component of RNase P and structural models that include the pre-tRNA substrate suggest that the internal motions observed in P4 likely facilitate adaptive changes in conformation that take place during folding and substrate recognition, possibly aided by interactions with Mg2+ ions. Our results add to a growing view supporting the existence of functionally important internal motions in RNA occurring at nanosecond timescales.     

Isotope labeling strategies for NMR studies of RNA

The known biological functions of RNA have expanded in recent years and now include gene regulation, maintenance of sub-cellular structure, and catalysis, in addition to propagation of genetic information. As for proteins, RNA function is tightly correlated with structure. Unlike proteins, structural information for larger, biologically functional RNAs is relatively limited. NMR signal degeneracy, relaxation problems, and a paucity of long-range ¹H–¹H dipolar contacts have limited the utility of traditional NMR approaches. Selective isotope labeling, including nucleotide-specific and segmental labeling strategies, may provide the best opportunities for obtaining structural information by NMR. Here we review methods that have been developed for preparing and purifying isotopically labeled RNAs, as well as NMR strategies that have been employed for signal assignment and structure determination.     

13C NMR relaxation studies of RNA base and ribose nuclei reveal a complex pattern of motions in the RNA binding site for human U1A protein

The widespread importance of induced fit and order-disorder transition in RNA recognition by proteins and small molecules makes it imperative that RNA motional properties are characterized quantitatively. Until now, however, very few studies have been dedicated to the systematic characterization of RNA motion and to their changes upon protein or small-molecule binding. The U1A protein-RNA complexes provide some of the best-studied examples of the role of RNA motional changes upon protein binding. Here, we report (13)C NMR relaxation studies of base and ribose dynamics for the RNA internal loop target of human U1A protein located within the 3'-untranslated region (3'-UTR) of the mRNA coding for U1A itself. We also report the semi-quantitative analysis of both fast (nano- to picosecond) and intermediate (micro- to millisecond) motions for this paradigmatic RNA system. We measure (13)C T(1), T(1rho) and heteronuclear nuclear Overhauser effects (NOEs) for sugar and base nuclei, as well as the power dependence of T(1rho) at 500 MHz and 750 MHz, and analyze these results using the model-free formalism. The results provide a much clearer picture of the type of motions experienced by this RNA in the absence of the protein than was provided by the analysis of the structure based solely on NOEs and scalar couplings. They define a model where the RNA internal loop region "breathes" on a micro- to millisecond timescale with respect to the double-helical regions. Superimposed on this slower motion, the residues at the very tip of the loop undergo faster (nano- to picosecond) motions. We hypothesize that these motions allow the RNA to sample multiple conformations so that the protein can select a structure within the ensemble that optimizes intermolecular contacts.     

Hydration dependence of backbone and side chain polylysine dynamics: a 13C solid-state NMR and IR spectroscopy study

The molecular dynamics of solid poly-L-lysine has been studied by the following natural abundance (13)C-NMR relaxation methods: measurements of the relaxation times T(1) at two resonance frequencies, off-resonance T(1rho) at two spin-lock frequencies, and proton-decoupled T(1rho). Experiments were performed at different temperatures and hydration levels (up to 17% H(2)O by weight). The natural abundance (13)C-CPMAS spectrum of polylysine provides spectral resolution of all types of backbone and side chain carbons and thus, dynamic parameters could be determined separately for each of them. At the same time, the conformational properties of polylysine were investigated by Fourier transform infrared spectroscopy. The data obtained from the different NMR experiments were simultaneously analyzed using the correlation function formalism and model-free approach. The results indicate that in dry polylysine both backbone and side chains take part in two low amplitude motions with correlation times of the order of 10(-4) s and 10(-9) s. Upon hydration, the dynamic parameters of the backbone remain almost constant except for the amplitude of the slower process that increases moderately. The side chain dynamics reveals a much stronger hydration response: the amplitudes of both slow and fast motions increase significantly and the correlation time of the slow motion shortens by about five orders of magnitude, and at hydration levels of more than 10% H(2)O fast and slow side chain motions are experimentally indistinguishable. These changes in the molecular dynamics cannot be ascribed to any hydration-dependent conformational transitions of polylysine because IR spectra reveal almost no hydration dependence in either backbone or side chain absorption domains. The physical nature of the fast and slow motions, their correlation time distributions, and hydration dependence of microdynamic parameters are discussed.     

NMR analysis of carbohydrates with model-free spectral densities: the dispersion range revisited

Over the past decade molecular mechanics and molecular dynamics studies have demonstrated considerable flexibility for carbohydrates. In order to interpret the corresponding NMR parameters, which correspond to a time-averaged or 'virtual' conformer, it is necessary to simulate the experimental data using the averaged geometrical representation obtained with molecular modelling methods. This structural information can be transformed into theoretical NMR data using empirical Karplus-type equations for the scalar coupling constants and the appropriate formalism for the relaxation parameters. In the case of relaxation data, the 'model-free' spectral densities have been widely used in order to account for the internal motions in sugars. Several studies have been conducted with truncated model-free spectral densities based on the assumption that internal motion is very fast with respect to overall tumbling. In this report we present experimental and theoretical evidence that suggests that this approach is not justified. Indeed, recent results show that even in the case of moderate-sized carbohydrates internal motions are occurring on the same timescale as molecular reorientation. Simulations of relaxation parameters (NOESY volumes, proton cross-relaxation rates, carbon T1 and nOe values) in the dispersion range (0.1<Tc<5 ns) show that rates of internal motion can be fairly precisely defined with respect to overall tumbling. Experimental data for a variety of oligosaccharides clearly indicate similar timescales for internal and overall motion. This revised version was published online in August 2006 with corrections to the Cover Date.     

A study of protein-water exchange through the off-resonance ROESY experiment: Application to the DNA-binding domain of AlcR

Summary In this communication a new NMR experiment for the safe observation and quantification of water-protein exchange phenomena is presented. It combines a water-selective pulse, offering chemical shift-based separation, and the off-resonance ROESY dynamic filter, which permits the elimination of the unwanted intramolecular dipolar cross relaxation of protein protons. Moreover, pulsed field gradients are used for the suppression of radiation damping and the solvent signal. The straightforward incorporation of this sequence in heteronuclear experiments is demonstrated for the case of the DNA-binding domain of the alcohol regulator protein.     

Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA

In site-directed spin labeling, a nitroxide-containing side chain is introduced at selected sites in a protein. The EPR spectrum of the labeled protein encodes information about the motion of the nitroxide on the nanosecond time scale, which has contributions from the rotary diffusion of the protein, from internal motions in the side chain, and from backbone fluctuations. In the simplest model for the motion of noninteracting (surface) side chains, the contribution from the internal motion is sequence independent, as is that from protein rotary diffusion. Hence, differences in backbone motions should be revealed by comparing the sequence-dependent motions of nitroxides at structurally homologous sites. To examine this model, nitroxide side chains were introduced, one at a time, along the GCN4-58 bZip sequence, for which NMR (15)N relaxation experiments have identified a striking gradient of backbone mobility along the DNA-binding region [Bracken et al. (1999) J. Mol. Biol. 285, 2133]. Spectral simulation techniques and a simple line width measure were used to extract dynamical parameters from the EPR spectra, and the results reveal a mobility gradient similar to that observed in NMR relaxation, indicating that side chain motions mirror backbone motions. In addition, the sequence-dependent side chain dynamics were analyzed in the DNA/protein complex, which has not been previously investigated by NMR relaxation methods. As anticipated, the backbone motions are damped in the DNA-bound state, although a gradient of motion persists with residues at the DNA-binding site being the most highly ordered, similar to those of helices on globular proteins.     

Refinement of the NMR solution structure of a protein to remove distortions arising from neglect of internal motion.

The effect of internal motion on the quality of a protein structure derived from nuclear magnetic resonance (NMR) cross relaxation has been investigated experimentally. Internal rotation of the tyrosine-31 ring of turkey ovomucoid third domain was found to mediate magnetization transfer; the effect led to underestimation of proton-proton distances in its immediate neighborhood. Experimental methods that distinguish pure cross relaxation from chemical exchange mediated cross relaxation were used to separate true distances from distorted ones. Uncorrected and corrected sets of distances, where the corrections took internal motion into account, each were used as input to a distance geometry program for structural modeling. Each set of distances yielded a family of similar (converged) structures. The two families of structures differed considerably (2 A) in the region of tyrosine-31. In addition, differences as large as 1 A were observed at other positions throughout the structure. These results emphasize the importance of analyzing the effects of internal motions in order to obtain more accurate NMR solution structures.     

Errors in RNA NOESY distance measurements in chimeric and hybrid duplexes: differences in RNA and DNA proton relaxation.

Nuclear magnetic resonance experiments reveal that the base H8/H6 protons of oligoribonucleotides (RNA) have T1 relaxation times that are distinctly longer than those of oligodeoxyribonucleotides (DNA). Similarly, the T1 values for the RNA H1' protons are approximately twice those of the corresponding DNA H1' protons. These relaxation differences persist in single duplexes containing covalently linked RNA and DNA segments and cause serious overestimation of distances involving RNA protons in typical NOESY spectra collected with a duty cycle of 2-3 s. NMR and circular dichroism experiments indicate that the segments of RNA maintain their A-form geometry even in the interior of DNA-RNA-DNA chimeric duplexes, suggesting that the relaxation times are correlated with the type of helix topology. The difference in local proton density is the major cause of the longer nonselective T1s of RNA compared to DNA, although small differences in internal motion cannot be completely ruled out. Fortunately, any internal motion differences that might exist are shown to be too small to affect cross-relaxation rates, and therefore reliable distance data can be obtained from time-dependent NOESY data sets provided an adequately long relaxation delay is used. In hybrid or chimeric RNA-DNA duplexes, if the longer RNA relaxation times are not taken into account in the recycle delay of NOESY pulse sequences, serious errors in measuring RNA proton distances are introduced.     

The UAA/GAN internal loop motif: a new RNA structural element that forms a cross-strand AAA stack and long-range tertiary interactions

Analysis of aligned RNA sequences and high-resolution crystal structures has revealed a new RNA structural element, termed the UAA/GAN motif. Found in internal loops of the 23 S rRNA, as well as in RNase P RNA and group I and II introns, this six-nucleotide motif adopts a distinctive local structure that includes two base-pairs with non-canonical conformations and three conserved adenine bases, which form a cross-strand AAA stack in the minor groove. Most importantly, the motif invariably forms long-range tertiary contacts, as the AAA stack typically forms A-minor interactions and the flipped-out N nucleotide forms additional contacts that are specific to the structural context of each loop. The widespread presence of this motif and its propensity to form long-range contacts suggest that it plays a critical role in defining the architectures of structured RNAs.     

Backbone motions in a crystalline protein from field-dependent 2H-NMR relaxation and line-shape analysis

We have used 2H-nmr to study backbone dynamics of the 2H-labeled, slowly exchanging amide sites of fully hydrated, crystalline hen egg white lysozyme. Order parameters are determined from the residual quadrupole coupling and values increase from S2 = 0.85 at 290 K to S2 = 0.94 at 200 K. Dynamical rates are determined from spin-lattice relaxation at three nmr frequencies (38.8, 61.5, and 76.7 MHz). The approach used here is thus distinct from solution nmr studies where dynamical amplitudes and rates are both determined from relaxation measurements. At temperatures below 250 K, relaxation is independent of the nmr frequency indicating that backbone motions are fast compared to the nmr frequencies. However, as the temperature is increased above 250 K, relaxation is significantly more efficient at the lowest frequency, which shows, in addition, the presence of motions that are slow compared to the nmr frequencies. Using the values of S2 determined from the residual quadrupole coupling and a model-free relaxation formalism that allows for fast and slow internal motions, we conclude that these slow motions have correlation times in the range of 0.1 to 1.0 microsecond and are effectively frozen out at 250 K where fast motions of the amide planes with approximately 15 ps effective correlation times and 9 degrees rms amplitudes dominate relaxation. The fast internal motions increase slightly in amplitude as the temperature rises toward 290 K, but the correlation time, as is also observed in solution nmr studies of RNase H, is approximately constant. These findings are consistent with hypotheses of dynamic glass transitions in hydrated proteins arising from temperature-dependent damping of harmonic modes of motion above the transition point.     

Internal bulge and tetraloop of the catalytic domain 5 of a group II intron ribozyme are flexible: implications for catalysis

RNA molecules have an inherent flexibility that enables recognition of other interacting partners through potential disorder-order transitions, yet studies to quantify such motional dynamics remain few. With an increasing database of three-dimensional structures of biologically important RNA molecules, quantifying such motions becomes important to link structural deformations with function. One such system studied intensely is domain 5 (D5) from the self-splicing group II introns, which is at the heart of its catalytic machinery. We report the dynamics of a 36 nucleotide D5 from the Pylaiella littoralis group II intron in the presence and absence of magnesium ions, and at a range of temperatures (298K-318 K). Using high-resolution NMR experiments of heteronuclear nuclear Overhauser enhancement (NOE), spin-lattice (R(1)), and spin-spin (R(2)) (13)C relaxation rates, we determined the rotational diffusion tensor of D5 using the ROTDIF program modified for RNA dynamic analysis (ROTDIF_RNA). The D5 rotational diffusion tensor has an axial symmetric ratio (D(||)/D(perpendicular)) of 1.7+/-0.3, consistent with an estimated overall rotational correlation time of tau(m)=(2D(||)+4D(perpendicular))(-1) of 6.1(+/-0.3) ns at 298 K and 4.1(+/-0.2) ns at 318 K. The measured relaxation data were analyzed with the reduced spectral density mapping formalism using assumed values of the chemical shift anisotropy of the (13)C spins. Both the relaxation data and the values of the spectral density function reveal that the functional groups in D5 implicated in magnesium ion binding and catalysis (catalytic triad, internal bulge, and tetraloop regions) exhibit thermally induced motion on a wide variety of timescales. Because these motions parallel those observed in the intramolecular stem-loop of the U6 element within the spliceosome, we hypothesize that such extensive dynamic disorder likely facilitates D5 engaging both binding and catalytic regions of the ribozyme, and these may be a conserved feature of the catalytic machinery essential for catalysis.     

Preparation, resonance assignment, and preliminary dynamics characterization of residue specific 13C/15N-labeled elongated DNA for the study of sequence-directed dynamics by NMR

DNA is a highly flexible molecule that undergoes functionally important structural transitions in response to external cellular stimuli. Atomic level spin relaxation NMR studies of DNA dynamics have been limited to short duplexes in which sensitivity to biologically relevant fluctuations occurring at nanosecond timescales is often inadequate. Here, we introduce a method for preparing residue-specific ¹³C/¹⁵N-labeled elongated DNA along with a strategy for establishing resonance assignments and apply the approach to probe fast inter-helical bending motions induced by an adenine tract. Preliminary results suggest the presence of elevated A-tract independent end-fraying internal motions occurring at nanosecond timescales, which evade detection in short DNA constructs and that penetrate deep (7 bp) within the DNA helix and gradually fade away towards the helix interior.     

Flexibility of ras lipid modifications studied by 2H solid-state NMR and molecular dynamics simulations

Human posttranslationally modified N-ras oncogenes are known to be implicated in numerous human cancers. Here, we applied a combination of experimental and computational techniques to determine structural and dynamical details of the lipid chain modifications of an N-ras heptapeptide in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes. Experimentally, 2H NMR spectroscopy was used to study oriented membranes that incorporated ras heptapeptides with two covalently attached perdeuterated hexadecyl chains. Atomistic molecular dynamics simulations of the same system were carried out over 100 ns including 60 DMPC and 4 ras molecules. Several structural and dynamical experimental parameters could be directly compared to the simulation. Experimental and simulated 2H NMR order parameters for the methylene groups of the ras lipid chains exhibited a systematic difference attributable to the absence of collective motions in the simulation and to geometrical effects. In contrast, experimental 2H NMR spin-lattice relaxation rates for Zeeman order were well reproduced in the simulation. The lack of slower collective motions in the simulation did not appreciably influence the relaxation rates at a Larmor frequency of 115.1 MHz. The experimental angular dependence of the 2H NMR relaxation rates with respect to the external magnetic field was also relatively well simulated. These relaxation rates showed a weak angular dependence, suggesting that the lipid modifications of ras are very flexible and highly mobile in agreement with the low order parameters. To quantify these results, the angular dependence of the 2H relaxation rates was calculated by an analytical model considering both molecular and collective motions. Peptide dynamics in the membrane could be modeled by an anisotropic diffusion tensor with principal values of Dparallel=2.1x10(9) s(-1) and Dperpendicular=4.5x10(5) s(-1). A viscoelastic fitting parameter describing the membrane elasticity, viscosity, and temperature was found to be relatively similar for the ras peptide and the DMPC host matrix. Large motional amplitudes and relatively short correlation times facilitate mixing and dispersal with the lipid bilayer matrix, with implications for the role of the full-length ras protein in signal transduction and oncogenesis.