[Show abstract][Hide abstract] ABSTRACT: The sterile alpha motif (SAM) domain of VTS1p, a posttranscriptional gene regulator, belongs to a family of SAM domains conserved from yeast to humans. Even though SAM domains were originally classified as protein-protein interaction domains, recently, it was shown that the yeast VTS1p-SAM and the SAM domain of its Drosophila homolog Smaug can specifically recognize RNA hairpins termed Smaug recognition element (SRE). Structural studies of the SRE-RNA complex of VTS1p-SAM revealed that the SAM domain primarily recognizes the shape of the RNA fold induced by the Watson-Crick base-pairing in the RNA pentaloop. Only the central G nucleotide is specifically recognized. The VTS1p-SAM domain recognizes SRE-RNAs with a CNGGN pentaloop where N is any nucleotide. The C1-G4 base pair in the wild type can be replaced by any pair of nucleotides that can form base pairs even though the binding affinity is greatest with a pyrimidine in position 1 and a purine in position 4. The interaction thus combines elements of sequence-specific and non-sequence-specific recognitions. The lack of structural rearrangements in either partner following binding is rather intriguing, suggesting that molecular dynamics may play an important role in imparting relaxed specificity with respect to the exact combination of nucleotides in the loop, except for the central nucleotide. In this work, we extend our previous studies of SRE-RNA interaction with VTS1p, by comparing the dynamics of the VTS1p-SAM domain both in its free form and when bound to SRE-RNA. The 15N relaxation studies of backbone dynamics suggest the presence of a dynamic interaction interface, with residues associated with specific G3 recognition becoming more rigid on RNA binding while other regions attain increased flexibility. The results parallel the observations from our studies of dynamics changes in SRE-RNA upon binding to VTS1p-SAM and shows that molecular dynamics could play a crucial role in modulating binding affinity and possibly contribute to the free energy of the interaction through an entropy-driven mechanism.
[Show abstract][Hide abstract] ABSTRACT: RNA recognition by proteins is often accompanied by significant changes in RNA dynamics in addition to conformational changes. However, there are very few studies which characterize the changes in molecular motions in RNA on protein binding. We present a quantitative (13)C NMR relaxation study of the changes in RNA dynamics in the pico-nanosecond time scale and micro-millisecond time scale resulting from interaction of the stem-loop SRE-RNA with the VTS1p-SAM domain. (13)C relaxation rates of the protonated carbons of the nucleotide base and anomeric carbons have been analyzed by employing the model-free formalism, for a fully (13)C/(15)N-labeled sample of the SRE-RNA in the free and protein-bound forms. In the free RNA, the nature of molecular motions are found to be distinctly different in the stem and the loop region. On binding to the protein, the nature of motions becomes more homogeneous throughout the RNA, with many residues showing increased flexibility at the aromatic carbon sites, while the anomeric carbon sites become more rigid. Surprisingly, we also observe indications of a slow collective motion of the RNA in the binding pocket of the protein. The observation of increased motions on binding is interesting in the context of growing evidence that binding does not always lead to motional restrictions and the resulting entropy gain could favor the free energy of association.
Journal of the American Chemical Society 10/2008; 130(36):12007-20. DOI:10.1021/ja8023115 · 11.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chemical shift anisotropy (CSA) tensor parameters have been determined for the protonated carbons of the purine bases in an RNA kissing complex in solution by extending the model-independent approach [Fushman, D., Cowburn, D. (1998) J. Am. Chem. Soc. 120, 7109-7110]. A strategy for determining CSA tensor parameters of heteronuclei in isolated X-H two-spin systems (X = 13C or 15N) in molecules undergoing anisotropic rotational diffusion is presented. The original method relies on the fact that the ratio kappa2=R2 auto/R2 cross of the transverse auto- and cross-correlated relaxation rates involving the X CSA and the X-H dipolar interaction is independent of parameters related to molecular motion, provided rotational diffusion is isotropic. However, if the overall motion is anisotropic kappa2 depends on the anisotropy D(parallel)/D (perpendicular) of rotational diffusion. In this paper, the field dependence of both kappa2 and its longitudinal counterpart kappa1=R1 auto/R1 cross are determined. For anisotropic rotational diffusion, our calculations show that the average kappa(av) = 1/2 (kappa1+kappa2), of the ratios is largely independent of the anisotropy parameter D(parallel)/D (perpendicular). The field dependence of the average ratio kappa(av) may thus be utilized to determine CSA tensor parameters by a generalized model-independent approach in the case of molecules with an overall motion described by an axially symmetric rotational diffusion tensor.
[Show abstract][Hide abstract] ABSTRACT: DNA A-tracts have been defined as four or more consecutive A.T base pairs without a TpA step. When inserted in phase with the DNA helical repeat, bending is manifested macroscopically as anomalous migration on polyacrylamide gels, first observed >20 years ago. An unsolved conundrum is why DNA containing in-phase A-tract repeats of A(4)T(4) are bent, whereas T(4)A(4) is straight. We have determined the solution structures of the DNA duplexes formed by d(GCAAAATTTTGC) [A4T4] and d(CGTTTTAAAACG) [T4A4] with NH(4)(+) counterions by using NMR spectroscopy, including refinement with residual dipolar couplings. Analysis of the structures shows that the ApT step has a large negative roll, resulting in a local bend toward the minor groove, whereas the TpA step has a positive roll and locally bends toward the major groove. For A4T4, this bend is nearly in phase with bends at the two A-tract junctions, resulting in an overall bend toward the minor groove of the A-tract, whereas for T4A4, the bends oppose each other, resulting in a relatively straight helix. NMR-based structural modeling of d(CAAAATTTTG)(15) and d(GTTTTAAAAC)(15) reveals that the former forms a left-handed superhelix with a diameter of approximately 110 A and pitch of 80 A, similar to DNA in the nucleosome, whereas the latter has a gentle writhe with a pitch of >250 A and diameter of approximately 50 A. Results of gel electrophoretic mobility studies are consistent with the higher-order structure of the DNA and furthermore depend on the nature of the monovalent cation present in the running buffer.
Proceedings of the National Academy of Sciences 02/2004; 101(5):1177-82. DOI:10.1073/pnas.0308143100 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Two sets of cross-correlated relaxation rates involving chemical shift anisotropy and dipolar interactions have been measured in an RNA kissing complex. In one case, both the CSA and dipolar interaction tensors are located on the same nucleotide base and are rigidly fixed with respect to each other. In the other case, the CSA tensor is located on the nucleotide base whereas the dipolar interaction is located on the adjoining ribose unit. Analysis of the measured rates in terms of isotropic or anisotropic rotational diffusion has been carried out for both cases. A marked difference between the two models is observed for the cross-correlation rates involving rigidly fixed spin interactions. The influence of internal motions about the glycosidic linkage between the nucleotide base and the ribose unit on cross-correlated relaxation rates has been estimated by applying a model of restricted rotational diffusion. Local motions seem to have a more pronounced effect on cross-correlated relaxation rates when the two spin interactions are not rigidly fixed with respect to each other.
[Show abstract][Hide abstract] ABSTRACT: Exchange transferred effects in solution-state NMR experiments allow one to determine the conformation of ligands that are weakly bound to macromolecules. Exchange-transferred nuclear Overhauser effect spectroscopy ('TR-NOESY') provides information about internuclear distances in a ligand in the bound state. Recently the possibility of obtaining dihedral angle information from a ligand in the bound state by exchange-transferred cross-correlation spectroscopy ('TR-CCSY') has been reported. In both cases the analysis of the signal amplitudes is usually based on the assumption that rapid exchange occurs between the free and bound forms of the ligand. In this paper we show that the fast exchange condition is not easily attained for observing exchange-transferred cross-correlation effects even in systems where exchange-transferred NOE can be observed. Extensive simulations based on analytical expressions for signal intensities corresponding to fast, intermediate, and slow chemical exchange have been carried out on a test system to determine the exchange regimes in which the fast exchange condition can be fulfilled for successfully implementing TR-NOESY and TR-CCSY.
Journal of Magnetic Resonance 09/2003; 163(2):199-207. DOI:10.1016/S1090-7807(03)00156-3 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: New nuclear magnetic resonance (NMR) methods are described for the measurement of cross-correlation rates of zero- and double-quantum coherences involving two nitrogen nuclei belonging to successive amino acids in proteins and peptides. Rates due to the concerted fluctuations of two NH(N) dipole-dipole interactions and to the correlated modulations of two nitrogen chemical shift anisotropies have been obtained in a sample of doubly labeled Ubiquitin. Ambiguities in the determination of dihedral angles can be lifted by comparison of different rates. By defining a heuristic order parameter, experimental rates can be compared with those expected for a rigid molecule. The cross-correlation order parameter that can be derived from a model-free approach can be separated into structural and dynamic contributions.
[Show abstract][Hide abstract] ABSTRACT: Double-quantum heteronuclear local field NMR is performed on a sample of a 13C2-labeled disaccharide, in which the two 13C spins are located on opposite sides of the glycosidic linkage. The evolution of the double-quantum coherences is found to be consistent with the solid-state conformation of the molecule, as previously determined by X-ray diffraction. The dependence of the double-quantum evolution on the glycosidic torsional angles is examined by using a graphical molecular manipulation program interfaced to a numerical spin simulation module.
Journal of Magnetic Resonance 08/2001; 151(1):136-41. DOI:10.1006/jmre.2001.2357 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein enzymes often use ionizable side chains, such as histidine, for general acid-base catalysis because the imidazole pK(a) is near neutral pH. RNA enzymes, on the other hand, are comprised of nucleotides which do not have apparent pK(a) values near neutral pH. Nevertheless, it has been recently shown that cytidine and adenine protonation can play an important role in both nucleic acid structure and catalysis. We have employed heteronuclear NMR methods to determine the pK(a) values and time scales of chemical exchanges associated with adenine protonation within the catalytically essential B domain of the hairpin ribozyme. The large, adenine-rich internal loop of the B domain allows us to determine adenine pK(a) values for a variety of non-Watson-Crick base pairs. We find that adenines within the internal loop have pK(a) values ranging from 4.8 to 5.8, significantly higher than the free mononucleotide pK(a) of 3. 5. Adenine protonation results in potential charge stabilization, hydrogen bond formation, and stacking interactions that are expected to stabilize the internal loop structure at low pH. Fast proton exchange times of 10-50 micros were determined for the well-resolved adenines. These results suggest that shifted pK(a) values may be a common feature of adenines in non-Watson-Crick base pairs, and identify two adenines which may participate in hairpin ribozyme active site chemistry.
[Show abstract][Hide abstract] ABSTRACT: Double-quantum heteronuclear local field NMR was applied to two 13C2-labeled carbohydrate samples, [1,2-13C2]-glucose and methyl-α-d-[1,3-13C2]-glucose. The geometry of the H−13C−13C−H moeity was estimated using the evolution of double-quantum coherences under correlated heteronuclear dipolar interactions. For [1,2-13C2]-glucose, double-quantum techniques were used both in solution and solid phases. The measured H−C1−C2−H torsion angles in crystalline glucose were 170° ± 5° for the β-anomer and 40° ± 15° for the α-anomer, in good agreement with reported crystal structures. In the solution phase we give a full analysis of an experiment in which the cross-correlation effects are isolated by the use of a heteronuclear multiple-quantum filter. We consider the influence of anisotropic rotational diffusion, chemical shift anisotropy, and proton−proton spin diffusion on the torsion angle estimate. We show that it is possible to determine the torsion angle and the rotational correlation time independently. The measured H−C1−C2−H torsion angles in solution differ slightly from the solid-state results: 159° ± 10° for the β-anomer and 57° ± 7° for the α-anomer. For methyl-α-d-[1,3-13C2]-glucose, the solid phase double-quantum heteronuclear local field experiment was applied for the first time to a HCCH moiety in which the carbons are not directly bonded. These techniques may be applied to other structural problems such as the determination of glycosidic linkage conformations and the conformation of sugar rings in nucleotides.
Journal of the American Chemical Society 01/2000; 122(6). DOI:10.1021/ja9910863 · 11.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dynamics of poly(vinyl acetate) in toluene solution has been examined by 13C and proton relaxation. 13C spin−lattice relaxation time and nuclear Overhauser enhancement measurements were carried out as a function of temperature at 50.3 and 100.6 MHz. The spin−lattice relaxation times for backbone protons were measured at different temperatures at 200 MHz. The relaxation data have been analyzed using the Hall−Weber−Helfand (HWH) model, which describes backbone dynamics in terms of conformational transitions and the Dejean−Laupretre−Monnerie (DLM) model, which includes bond librations in addition to conformational transitions. The parameters obtained from the analysis of 13C relaxation data were utilized to predict the proton relaxation data. The DLM model was found to be more successful in reproducing the experimental results. To study the influence of libration further, proton relaxation data for poly(vinyl acetate) over a wider range of temperature reported in the literature were analyzed by these two models. The DLM model could reproduce the experimental data at all temperatures whereas the HWH model was found to be successful only in accounting for the experimental data at high temperatures. The results demonstrate the importance of including the librational mode in the description of the backbone dynamics in polymers.
The Journal of Physical Chemistry 12/1996; 100(51):19939–19944. DOI:10.1021/jp961226k · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dynamics of poly(isobutyl methacrylate) in toluene solution has been examined by 13C spin−lattice relaxation time and NOE measurements as a function of temperature. The experiments were performed at 50.3 and 100.6 MHz. The backbone carbon relaxation data have been analyzed using the Dejean−Laupretre−Monnerie (DLM) model, which describes the dynamical processes in the backbone in terms of conformational transitions and bond librations. The relaxation data of the side chain nuclei have been analyzed by assuming different motional models, namely, unrestricted rotational diffusion, three site jumps, and restricted rotational diffusion. The different models have been compared for their ability to reproduce the experimental spin−lattice relaxation times and also to predict the behavior of NOE as a function of temperature. Conformational energy calculations have been carried out on a model compound by using the semiempirical quantum chemical method, AM1, and the results confirm the validity of the motional models used to describe the side-chain motion.
[Show abstract][Hide abstract] ABSTRACT: The dynamics of poly(2-vinylpyridine) in chloroform solution has been examined by C-13 spin-lattice relaxation time and NOE measurements as a function of temperature. The experiments were performed at 50.3 and 100.6 MHz. The backbone carbon relaxation data have been analyzed in terms of six motional models. Among these models, the models which consider conformational transitions and bond librations for the backbone were found to be more successful. Pyridyl ring motion has been modeled as a restricted rotation with the rotational amplitude varying with temperature. The activation energy parameters obtained from the relaxation data of the pyridyl ring carbon have been compared with the energy barrier for ring rotation estimated from conformational energy calculations using the AM1 semiempirical quantum chemical method. The results of the conformational energy calculations support the description of pyridyl ring motion as a restricted rotation.