Constraints on the structure of (CUG)97 RNA from magic-angle-spinning solid-state NMR spectroscopy.
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3' Untranslated Regions↗
Biomedical subjects
Oliver Ohlenschläger
Publications and source records attributed to Oliver Ohlenschläger.
Tailoring broadband inversion pulses for MAS solid state NMR.
A simple approach is demonstrated for designing optimised broadband inversion pulses for MAS solid state NMR studies of biological systems. The method involves a two step numerical optimisation procedure and takes into account experimental requirements such as the pulse length, resonance offset range and extent of H(1) inhomogeneity compensation needed. A simulated annealing protocol is used initially to find appropriate values for the parameters that define the well known tanh/tan adiabatic pulse such that a satisfactory spin inversion is achieved with minimum RF field strength. This information is then used in the subsequent stage of refinement where the RF pulse characteristics are further tailored via a local optimisation procedure without imposing any restrictions on the amplitude and frequency modulation profiles. We demonstrate that this approach constitutes a generally applicable tool for obtaining pulses with good inversion characteristics. At moderate MAS frequencies the efficacy of the method is experimentally demonstrated for generating double-quantum NMR spectra via the zero-quantum dipolar recoupling scheme RFDR.
Carbon Isotopes↗
Solid state NMR at high magic angle spinning frequencies: dipolar chemical shift correlation with adiabatic inversion pulse based RF pulse schemes.
The efficacy of hetero- and homonuclear dipolar recoupling employing tanh/tan adiabatic inversion pulse based RF pulse schemes has been examined at high magic angle spinning (MAS) frequencies via numerical simulations and experimental measurements. An approach for minimising the recoupling RF power level is presented, taking into consideration the spinning speed, the range of resonance offsets and H(1) inhomogeneities and the available RF field strength. This involves the tailoring of the frequency and amplitude modulation profiles of the inversion pulses. The applicability of tanh/tan pulse based dipolar recoupling schemes to spinning speed regimes where the performance with conventional rectangular pulses may not be satisfactory is demonstrated.
Carbon Isotopes↗
Monolaterol, the first configurationally assigned phenylphenalenone derivative with a stereogenic center at C-9, from Monochoria elata.
Phytochemical analysis of the roots of Monochoria elata resulted in the structure elucidation of monolaterol, the first configurationally assigned phenylphenalenone-type natural product with a stereogenic center at the phenyl-bearing carbon, C-9, and four known phenylphenalenones by MS and NMR methods. The absolute configuration of the new phenyldihydrophenalenediol was assigned by comparing the results of quantum chemical CD calculations and experimental CD spectra, and the crystal structure was determined by X-ray diffraction analysis.
Crystallography, X-Ray↗
A novel cGUUAg tetraloop structure with a conserved yYNMGg-type backbone conformation from cloverleaf 1 of bovine enterovirus 1 RNA.
The 5'-terminal cloverleaf (CL)-like RNA structures are essential for the initiation of positive- and negative-strand RNA synthesis of entero- and rhinoviruses. SLD is the cognate RNA ligand of the viral proteinase 3C (3C(pro)), which is an indispensable component of the viral replication initiation complex. The structure of an 18mer RNA representing the apical stem and the cGUUAg D-loop of SLD from the first 5'-CL of BEV1 was determined in solution to a root-mean-square deviation (r.m.s.d.) (all heavy atoms) of 0.59 A (PDB 1Z30). The first (antiG) and last (synA) nucleotide of the D-loop forms a novel 'pseudo base pair' without direct hydrogen bonds. The backbone conformation and the base-stacking pattern of the cGUUAg-loop, however, are highly similar to that of the coxsackieviral uCACGg D-loop (PDB 1RFR) and of the stable cUUCGg tetraloop (PDB 1F7Y) but surprisingly dissimilar to the structure of a cGUAAg stable tetraloop (PDB 1MSY), even though the cGUUAg BEV D-loop and the cGUAAg tetraloop differ by 1 nt only. Together with the presented binding data, these findings provide independent experimental evidence for our model [O. Ohlenschlager, J. Wohnert, E. Bucci, S. Seitz, S. Hafner, R. Ramachandran, R. Zell and M. Gorlach (2004) Structure, 12, 237-248] that the proteinase 3C(pro) recognizes structure rather than sequence.
3C Viral Proteases↗
Domain reorientation and induced fit upon RNA binding: solution structure and dynamics of ribosomal protein L11 from Thermotoga maritima.
L11, a protein of the large ribosomal subunit, binds to a highly conserved domain of 23S rRNA and mediates ribosomal GTPase activity. Its C-terminal domain is the main determinant for rRNA binding, whereas its N-terminal domain plays only a limited role in RNA binding. The N-terminal domain is thought to be involved in interactions with elongation and release factors as well as with the antibiotics thiostrepton and micrococcin. This report presents the NMR solution structure of the full-length L11 protein from the thermophilic eubacterium Thermotoga maritima in its free form. The structure is based on a large number of orientational restraints derived from residual dipolar couplings in addition to conventional NOE-based restraints. The solution structure of L11 demonstrates that, in contrast to many other multidomain RNA-binding proteins, the relative orientation of the two domains is well defined. This is shown both by heteronuclear 15N-relaxation and residual dipolar-coupling data. Comparison of this NMR structure with the X-ray structure of RNA-bound L11, reveals that binding not only induces a rigidification of a flexible loop in the C-terminal domain, but also a sizeable reorientation of the N-terminal domain. The domain orientation in free L11 shows limited similarity to that of ribosome-bound L11 in complex with elongation factor, EF-G.
Models, Molecular↗
TEDOR with adiabatic inversion pulses: Resonance assignments of 13C/15N labelled RNAs.
We have examined via numerical simulations the performance characteristics of different 15N RF pulse schemes employed in the transferred echo double resonance (TEDOR) experimental protocol for generating 13C-15N dipolar chemical shift correlation spectra of isotopically labelled biological systems at moderate MAS frequencies (omega(r) approximately 10 kHz). With an 15N field strength of approximately 30-35 kHz that is typically available in 5 mm triple resonance MAS NMR probes, it is shown that a robust TEDOR sequence with significant tolerance to experimental imperfections sa as H1 inhomogeneity and resonance offsets can be effectively implemented using adiabatic heteronuclear dipolar recoupling pulse schemes. TEDOR-based 15N-13C and 15N-13C-13C chemical shift correlation experiments were carried out for obtaining 13C and 15N resonance assignments of an RNA composed of 97 (CUG) repeats which has been implicated in the neuromuscular disease myotonic dystrophy.
Carbon Isotopes↗
Characterisation of hydrogen bonding networks in RNAs via magic angle spinning solid state NMR spectroscopy.
It is demonstrated that the spatial proximity of (1)H nuclei in hydrogen bonded base-pairs in RNAs can be conveniently mapped via magic angle spinning solid state NMR experiments involving proton spin diffusion driven chemical shift correlation of low gamma nuclei such as the imino and amino nitrogens of nucleic acid bases. As different canonical and non-canonical base-pairing schemes encountered in nucleic acids are characterised by topologically different networks of proton dipolar couplings, different base-pairing schemes lead to characteristic cross-peak intensity patterns in such correlation spectra. The method was employed in a study of a 100 kDa RNA composed of 97 CUG repeats, or (CUG)(97) that has been implicated in the neuromuscular disease myotonic dystrophy. (15)N-(15)N chemical shift correlation studies confirm the presence of Watson-Crick GC base pairs in (CUG)(97).
Base Pairing↗
Structural studies on LNA quadruplexes.
LNAs (locked nucleic acids) are new DNA analogues with higher binding affinities toward nucleic acids than the canonical counterparts mainly due to the characteristic conformational restriction arising from the 2'-O, 4'-C methylene bridge. In light of the promising therapeutic applications and considering the advantageous characteristics of LNAs, such as their high water solubility, easy handling, and synthetic accessibility through the conventional phosphoramidite chemistry, we undertook a study concerning the capability of these nucleic acid analogues to form quadruplex structures. Particularly, we have been investigating the LNA/DNA chimeras corresponding to the well-known DNA sequences 5-GGTTGGTGTGGTTGG-3', capable of forming an unimolecular quadruplex. This article deals with the study of the sequence 5'-ggTTggTGTggTTgg-3' (upper and lower case letters represent DNA and LNA residues, respectively), which, according to CD spectroscopy, is able to fold into a quadruplex structure.
Circular Dichroism↗
NMR solution structure of a parallel LNA quadruplex.
The solution structure of a locked nucleic acid (LNA) quadruplex, formed by the oligomer d(TGGGT), containing only conformationally restricted LNA residues is reported. NMR and CD spectroscopy, as well as molecular dynamics and mechanic calculations, has been used to characterize the complex. The molecule adopts a parallel stranded conformation with a 4-fold rotational symmetry, showing a right-handed helicity and the guanine residues in an almost planar conformation with three well-defined G-tetrads. The thermal stability of Q-LNA has been found to be comparable with that of [r(UGGGU)]4, while a T(m) increment of 20 degrees C with respect to the corresponding DNA quadruplex structure [d(TGGGT)]4 has been observed. The structural features of the LNA quadruplex reported here may open new perspectives for the biological application of LNAs as novel versatile tools to design aptamer or catalyst oligonucleotides.
Base Sequence↗
Solution structure and backbone dynamics of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin mutant: a molecular analysis of its reduced thermal stability.
No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature.
Bacterial Proteins↗
Determination of the glycosidic bond angle chi in RNA from cross-correlated relaxation of CH dipolar coupling and N chemical shift anisotropy.
A new heteronuclear NMR pulse sequence, the quantitative Gamma(HCN) experiment, for the determination of the glycosidic torsion angle chi in (13)C,(15)N-labeled oligonucleotides is described. The Gamma(HCN) experiment allows measurement of CH dipole-dipole, N chemical shift anisotropy cross-correlated relaxation rates (Gamma(C1'H1',N1)(DD,CSA) and Gamma(C2'H2',N9)(DD,CSA) for pyrimidines Gamma(C1'H1'N9)(DD,CSA) and Gamma(C2'H2',N9)(DD,CSA) for purines). A nucleotide-specific parametrization for the dependence of these Gamma-rates on chi based on (15)N chemical shift tensors determined by solid-state NMR experiments on mononucleosides (Stueber, D.; Grant, D. M. J. Am. Chem. Soc. 2002, 124, 10539-10551) is presented. For a 14-mer and a 30-mer RNA of known structures, it is found that the Gamma(HCN) experiment offers a very sensitive parameter for changes in the angle chi and allows restraining of chi with an accuracy of around 10 degrees for residues which do not undergo conformational averaging. Therefore, the Gamma(HCN) experiment can be used for the determination of chi in addition to data derived from (3)J(C,H)-coupling constants. As shown for the 30-mer RNA, the derived torsion angle information can be incorporated as additional restraint, improving RNA structure calculations.
Anisotropy↗
Identification of NH...N hydrogen bonds by magic angle spinning solid state NMR in a double-stranded RNA associated with myotonic dystrophy.
RNA plays a central role in biological processes and exhibits a variety of secondary and tertiary structural features that are often stabilized via hydrogen bonds. The distance between the donor and acceptor nitrogen nuclei involved in NH...N hydrogen bonds in nucleic acid base pairs is typically in the range of 2.6-2.9 A. Here, we show for the first time that such spatial proximity between 15N nitrogen nuclei can be conveniently monitored via magic angle spinning solid state NMR on a uniformly 15N-labelled RNA. The presence of NH.N hydrogen bonds is reflected as cross-peaks between the donor and acceptor nitrogen nuclei in 2D 15N dipolar chemical shift correlation spectra. The RNA selected for this experimental study was a CUG repeat expansion implicated in the neuromuscular disease myotonic dystrophy. The results presented provide direct evidence that the CUG repeat expansion adopts a double-stranded conformation.
Base Pairing↗
(1)H, (13)C and (15)N sequence-specific resonance assignments of the two-domain thrombin inhibitor dipetalin.
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Animals↗
Homonuclear chemical shift correlation in rotating solids via RNnu n symmetry-based adiabatic RF pulse schemes.
The efficacy of RN(nu) (n) symmetry-based adiabatic Zero-Quantum (ZQ) dipolar recoupling schemes for obtaining chemical shift correlation data at moderate magic angle spinning frequencies has been evaluated. RN(nu) (n) sequences generally employ basic inversion elements that correspond to a net 180 degrees rotation about the rotating frame x-axis. It is shown here via numerical simulations and experimental measurements that it is also possible to achieve efficient ZQ dipolar recoupling via RN(nu) (n) schemes employing adiabatic pulses. Such an approach was successfully used for obtaining (13)C chemical shift correlation spectra of a uniformly labelled sample of (CUG)(97) - a triplet repeat expansion RNA that has been implicated in the neuromuscular disease myotonic dystrophy. An analysis of the (13)C sugar carbon chemical shifts suggests, in agreement with our recent (15)N MAS-NMR studies, that this RNA adopts an Alpha-helical conformation.
Nuclear Magnetic Resonance, Biomolecular↗
The structure of the stemloop D subdomain of coxsackievirus B3 cloverleaf RNA and its interaction with the proteinase 3C.
Stemloop D (SLD) of the 5' cloverleaf RNA is the cognate ligand of the coxsackievirus B3 (CVB3) 3C proteinase (3Cpro). Both are indispensable components of the viral replication initiation complex. SLD is a structurally autonomous subunit of the 5' cloverleaf. The SLD structure was solved by NMR spectroscopy to an rms deviation of 0.66 A (all heavy atoms). SLD contains a novel triple pyrimidine mismatch motif with a central Watson-Crick type C:U pair. SLD is capped by an apical uCACGg tetraloop adopting a structure highly similar to stable cUNCGg tetraloops. Binding of CVB3 3Cpro induces changes in NMR spectra for nucleotides adjacent to the triple pyrimidine mismatch and of the tetraloop implying them as sites of specific SLD:3Cpro interaction. The binding of 3Cpro to SLD requires the integrity of those structural elements, strongly suggesting that 3Cpro recognizes a structural motif instead of a specific sequence.
3C Viral Proteases↗
RFDR with adiabatic inversion pulses: application to internuclear distance measurements.
In the context of the structural characterisation of biomolecular systems via MAS solid state NMR, the potential utility of homonuclear dipolar recoupling with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. The results obtained suggest that it is possible to obtain reliable estimates of internuclear distances via an analysis of the initial cross-peak intensity buildup curves generated from two-dimensional adiabatic inversion pulse driven longitudinal magnetisation exchange experiments.
Computer Simulation↗
Triple resonance MAS NMR with (13C, 15N) labelled molecules: reduced dimensionality data acquisition via 13C-15N heteronuclear two-spin coherence transfer pathways.
A reduced dimensionality magic angle spinning solid-state NMR experimental protocol for obtaining chemical shift correlation spectra of dipolar coupled nuclei in uniformly ((13)C, (15)N) labelled biological systems is described and demonstrated. The method involves a mapping of the evolution frequencies of heteronuclear (13)C-(15)N zero- and double-quantum coherences. In comparison to a reduced dimensionality procedure involving the simultaneous incrementation of two single-quantum chemical shift evolution periods, the approach described here could be potentially advantageous for minimising the heat dissipated in the probe by high power (1)H decoupling in experiments requiring long t (1) acquisition times.
Carbon Isotopes↗