The NMR structure of 31mer RNA domain of Escherichia coli RNase P RNA using its non-uniformly deuterium labelled counterpart [the 'NMR-window' concept].

The NMR structure of a 31mer RNA constituting a functionally important domain of the catalytic RNase P RNA from Escherichia coli is reported. Severe spectral overlaps of the proton resonances in the natural 31mer RNA (1) were successfully tackled by unique spectral simplifications found in the partially-deuterated 31 mer RNA analogue (2) incorporating deuterated cytidines [C5 (>95 atom % 2H), C2' (>97 atom % 2H), C3' (>97 atom % 2H), C4' (>65 atom % 2H) and C5' (>97 atom % 2H)] [for the 'NMR-window' concept see: Földesi,A. et al. (1992) Tetrahedron, 48, 9033; Foldesi,A. et al. (1993) J. Biochem. Biophys. Methods, 26, 1; Yamakage,S.-I. et al. (1993) Nucleic Acids Res., 21, 5005; Agback,P. et al. (1994) Nucleic Acids Res., 22, 1404; Földesi,A. et al. (1995) Tetrahedron, 51, 10065; Földesi,A. et al. (1996) Nucleic Acids Res., 24, 1187-1194]. 175 resonances have been assigned out of total of 235 non-exchangeable proton resonances in (1) in an unprecedented manner in the absence of 13C and 15N labelling. 41 out of 175 assigned resonances could be accomplished with the help of the deuterated analogue (2). The two stems in 31mer RNA adopt an A-type RNA conformation and the base-stacking continues from stem I into the beginning of the loop I. Long distance cross-strand NOEs showed a structured conformation at the junction between stem I and loop I. The loop I-stem II junction is less ordered and shows structural perturbation at and around the G11 -C22 base pair.

Solution structure of lariat RNA by 500 MHz NMR spectroscopy and molecular dynamics studies in water.

A 500 MHz NMR study of the lariat RNA tetramer 1 and pentamer 2 mimicking the naturally occurring lariat RNA is reported. The conformational properties of 1 and 2 were compared with those of a linear branched RNA tetramer 3, which show that the conformational features of the two lariat RNAs, 1 and 2, are quite constrained and significantly different from those observed for the linear branched RNA tetramer 3. The conformation of all sugar residues forming the lariat ring in 1 and 2 are locked in a rigid South-type conformation. All residues in both lariat RNAs have a high population of gamma+ (67-85%) and beta t (95-100%) rotamers except guanosine where the gamma+ population is low. The conformation around the glycosidic bond is anti for all residues except for guanosine where NOE data indicates an equilibrium of syn<-->anti. In both lariat RNAs, 1 and 2, the temperature dependent 1H and 31P chemical shifts as well as the oligomerization shifts, with respect to adenosine 2',3',5'-triethyl-phosphate (Sund et al., 1992, Tetrahedron 48, 695) suggests that the 3'-->5' linked U4 or C4 residue is stacked on guanosine. Subsequently, 1H-1H, 1H-31P and 13C-31P coupling constants derived torsional constraints were used for molecular dynamics study in water with counter sodium ions for a total of 226 ps. The MD simulations were first carried out with harmonic torsional constraints which were derived from J couplings (0-86 ps) and then completely without constraints (96-226 ps). The lack of any major changes in the conformation of the two lariat-RNA structures upon releasing the NMR constraints indicate that the conformers generated in the MD simulation in water agree well with the structural features suggested by experimental observables. This means that the ensemble of conformers generated during the MD trajectory of 226 ps are not artificially held in these conformations due to the NMR constraints, suggesting that these conformers can be considered to be good representatives of the actual NMR observed solution structures.

NMR spectroscopic properties (1H at 500 MHz) of deuterated* ribonucleotide-dimers ApU*, GpC*, partially deuterated 2'-deoxyribonucleotide-dimers d(TpA*), d(ApT*), d(GpC*) and their comparison with natural counterparts (1H-NMR window).

Pure 1'#,2',3',4'#,5',5''-2H6-ribonucleoside derivatives 10-14, 1'#,2',2'',3',4'#,5',5''-2H7-2'-deoxynucleoside blocks 15-18 and their natural-abundance counterparts were used to assemble partially deuterated ribonucleotide-dimers (* indicates deuteration at 1'#,2',3',4'#,5',5''(2H6)): ApU* 21, GpC* 22 and partially deuterated 2'-deoxyribonucleotide-dimers d(TpA*) 23, d(ApT*) 25, d(GpC*) 26 (* indicates deuteration at 1'#,2',2'',3',4'#,5',5''(2H7)) according to the procedure described by Földesi et al. (Tetrahedron, in press). These five partially deuterated oligonucleotides were subsequently compared with their corresponding natural-abundance counterparts by 500 MHz 1H-NMR spectroscopy to evaluate the actual NMR simplifications achieved in the non-deuterated part (1H-NMR window) as a result of specific deuterium incorporation. Detailed one-dimensional 1H-NMR (500 MHz), two-dimensional correlation spectra (DQF-COSY and TOCSY) and deuterium isotope effect on the chemical shifts of oligonucleotides have been presented.

A comparative conformational study of thymidylyl(3'----5')-thymidine, thymidylyl(3'----5')-5'-thio-5'- deoxythymidine and thymidinylacetamido-[3'(O)----5'(C)]-5'-deoxythymidine.

A comparative 270 MHz NMR spectroscopic study on the solution structure of the dimer d(TpT) 1, and its two analogues, namely, d(TpST) 2, and NH2d(TcmT) 4 has been reported. Analysis of chemical shifts and coupling constants indicate that: (i) The sugar moieties of the constituent nucleotides are not affected by modification of the internucleotide linkages and adopt preferentially an S-type conformation. (ii) The C4'-C5' bond in the pT part of the modified dimers 2 and 4 shows a large conformational freedom (gamma+ = 32% and 35%, respectively) compared to 1 (gamma+ = 75%). (iii) The population of the trans conformer about C5'-O5' is less important in d(TpST) 2 compared to d(TpT) 1. (iv) The C3'-O3' bond in 2 adopts a trans conformation as in 1. (v) The glycosidic bonds in the modified dimers 2 and 4 showed preferential syn conformation. UV and CD data show that the modified dimers 2 and 4 have poor tendency to stack intramolecularly, they also base pair less efficiently with d(ApA) as compared to d(TpT) 1.

Structure of a hepatotoxic pentapeptide from the cyanobacterium Nodularia spumigena.

The structure of a hepatotoxic peptide from the cyanobacterium Nodularia spumigena was determined using 1D and 2D proton nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The toxin was a cyclic pentapeptide (mol. wt 824.5) with the structure cyclo-(beta-methylisoAsp-Arg-Adda-isoGlu-N-methylde hydrobutyric acid) (Adda: 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid).

The 15N-NMR spectroscopy of wyosine-triacetate and its congener.

Assignments of the 15N-NMR resonances of wyosine-triacetate 1 and a new congener 2 are reported using NOE and INEPT modes.