Backbone resonance assignments of the 48 kDa dimeric putative 18S rRNA-methyltransferase Nep1 from Methanocaldococcus jannaschii.

Nep1 from Methanocaldococcus jannaschii is a 48 kDa dimeric protein belonging to the SPOUT-class of S-adenosylmethionine dependent RNA-methyltransferases and acting as a ribosome assembly factor. Mutations in the human homolog are the cause of Bowen-Conradi syndrome. We report here 1H, 15N and 13C chemical shift assignments for the backbone of the protein in its apo state.

Protein oligomerization induced by oleic acid at the solid-liquid interface--equine lysozyme cytotoxic complexes.

Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self-assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid-liquid interface within an ion-exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed similarity with both amyloid oligomers and the complexes with oleic acid and its structural homologous protein alpha-lactalbumin, known as human alpha-lactalbumin made lethal for tumour cells (HAMLET). As determined by NMR diffusion measurements, ELOA may consist of 4-30 lysozyme molecules. Each lysozyme molecule is able to bind 11-48 oleic acids in various preparations. Equine lysozyme acquired a partially unfolded conformation in ELOA, as evident from its ability to bind hydrophobic dye 8-anilinonaphthalene-1-sulfonate. CD and NMR spectra. Similar to amyloid oligomers, ELOA also interacts with thioflavin-T dye, shows a spherical morphology, assembles into ring-shaped structures, as monitored by atomic force microscopy, and exerts a toxic effect in cells. Studies of well-populated ELOA shed light on the nature of the amyloid oligomers and HAMLET complexes, suggesting that they constitute one large family of cytotoxic proteinaceous species. The hydrophobic surfaces can be used profitably to produce complexes with very distinct properties compared to their precursor proteins.

Cytosine ribose flexibility in DNA: a combined NMR 13C spin relaxation and molecular dynamics simulation study.

Using (13)C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C-H vectors in all cytosine ribose moieties within the Dickerson-Drew dodecamer (5'-CGCGAATTCGCG-3') are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase.

NMR and MD studies of the temperature-dependent dynamics of RNA YNMG-tetraloops.

In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin.

Conserved nucleotides in an RNA essential for hepatitis B virus replication show distinct mobility patterns.

The number of regulatory RNAs with identified non-canonical structures is increasing, and structural transitions often play a role in their biological function. This stimulates interest in internal motions of RNA, which can underlie structural transitions. Heteronuclear NMR relaxation measurements, which are commonly used to study internal motion, only report on local motions of few sites within the molecule. Here we have studied a 27-nt segment of the human hepatitis B virus (HBV) pregenomic RNA, which is essential for viral replication. We combined heteronuclear relaxation with the new off-resonance ROESY technique, which reports on internal motions of H,H contacts. Using off-resonance ROESY, we could for the first time detect motion of through-space H,H contacts, such as in intra-residue base-ribose contacts or inter-nucleotide contacts, both essential for NMR structure determination. Motions in non-canonical structure elements were found primarily on the sub-nanosecond timescale. Different patterns of mobility were observed among several mobile nucleotides. The most mobile nucleotides are highly conserved among different HBV strains, suggesting that their mobility patterns may be necessary for the RNA's biological function.

Quantitative gamma-HCNCH: determination of the glycosidic torsion angle chi in RNA oligonucleotides from the analysis of CH dipolar cross-correlated relaxation by solution NMR spectroscopy.

A novel NMR pulse sequence is introduced to determine the glycosidic torsion angle chi in (13)C,(15)N-labeled oligonucleotides. The quantitative Gamma-HCNCH measures the dipolar cross-correlated relaxation rates Gamma(DD,DD)(C8H8,C1'H1') (pyrimidines) and Gamma(DD,DD)(C6H6,C1'H1') (purines). Cross-correlated relaxation rates of a (13)C,(15)N-labeled RNA 14mer containing a cUUCGg tetraloop were determined and yielded chi-angles that agreed remarkably well with data derived from the X-ray structure of the tetraloop. In addition, the method was applied to the larger stemloop D (SLD) subdomain of the Coxsackievirus B3 cloverleaf 30mer RNA and the effect of anisotropic rotational motion was examined for this molecule. It could be shown that the chi-angle determination especially for nucleotides in the anti conformation was very accurate and the method was ideally suited to distinguish between the syn and the anti-conformation of all four types of nucleotides.

Structure induction of the T-cell receptor zeta-chain upon lipid binding investigated by NMR spectroscopy.

The conformation of the cytoplasmic part of the zeta-chain of the T-cell receptor (TCR) in its free form and bound to detergent micelles has been investigated by heteronuclear NMR spectroscopy. The zeta-chain is considered to be a mediator between the extracellular antigen and the intracellular signal-transduction cascade leading to T-cell activation. Earlier studies suggested a T-cell activation mechanism that involved a TCR-state-dependent lipid incorporation propensity of the zeta-chain accompanied by a helical folding transition. In order to support this proposed mechanism, standard protein NMR assignment and secondary-structure-elucidation techniques have been applied to the free TCR zeta-chain and to the zeta-chain bound to the detergent LMPG, which forms a micelle, in order to obtain the structural characteristics of this folding transition in a residue-resolved manner. We could assign the resonances of the free zeta-chain at 278 K, and this formed the basis for chemical-shift-perturbation studies to identify lipid binding sites. Our NMR results show that the free TCR zeta-chain is indeed intrinsically unstructured. Regions around the ITAM2 and ITAM3 sequences are involved in a highly dynamic binding of the free zeta-chain to a detergent micelle formed by the acidic lipid LMPG.

Residue specific ribose and nucleobase dynamics of the cUUCGg RNA tetraloop motif by MNMR 13C relaxation.

The dynamics of the nucleobase and the ribose moieties in a 14-nt RNA cUUCGg hairpin-loop uniformly labeled with 13C and 15N were studied by 13C spin relaxation experiments. R1, R1rho and the 13C-[1H] steady-state NOE of C6 and C1' in pyrimidine and C8 and C1' in purine residues were obtained at 298 K. The relaxation data were analyzed by the model-free formalism to yield dynamic information on timescales of pico-, nano- and milli-seconds. An axially symmetric diffusion tensor with an overall rotational correlation time tau(c) of 2.31 +/- 0.13 ns and an axial ratio of 1.35 +/- 0.02 were determined. Both findings are in agreement with hydrodynamic calculations. For the nucleobase carbons, the validity of different reported 13C chemical shift anisotropy values (Stueber, D. and Grant, D. M., 2002 J. Am. Chem. Soc. 124, 10539-10551; Fiala et al., 2000 J. Biomol. NMR 16, 291-302; Sitkoff, D. and Case, D. A., 1998 Prog. NMR Spectroscopy 32, 165-190) is discussed. The resulting dynamics are in agreement with the structural features of the cUUCGg motif in that all residues are mostly rigid (0.82 < S2 < 0.96) in both the nucleobase and the ribose moiety except for the nucleobase of U7, which is protruding into solution (S2 = 0.76). In general, ribose mobility follows nucleobase dynamics, but is less pronounced. Nucleobase dynamics resulting from the analysis of 13C relaxation rates were found to be in agreement with 15N relaxation data derived dynamic information (Akke et al., 1997 RNA 3, 702-709).

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.

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.

Long-range interactions within a nonnative protein.

Protein folding and unfolding are coupled to a range of biological phenomena, from the regulation of cellular activity to the onset of neurodegenerative diseases. Defining the nature of the conformations sampled in nonnative proteins is crucial for understanding the origins of such phenomena. We have used a combination of nuclear magnetic resonance (NMR) spectroscopy and site-directed mutagenesis to study unfolded states of the protein lysozyme. Extensive clusters of hydrophobic structure exist within the wild-type protein even under strongly denaturing conditions. These clusters involve distinct regions of the sequence but are all disrupted by a single point mutation that replaced residue Trp62 with Gly located at the interface of the two major structural domains in the native state. Thus, nativelike structure in the denatured protein is stabilized by the involvement of Trp62 in nonnative and long-range interactions.