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RNA tertiary structure motifs are stabilized by a wide variety of hydrogen-bonding interactions. Protonated A and C nucleotides are normally not considered to be suitable building blocks for such motifs since their p Ka values are far from physiological pH. Here, we report the NMR solution structure of an in vitro selected GTP-binding RNA aptamer bound to GTP with an intricate tertiary structure. It contains a novel kind of base quartet stabilized by a protonated A residue. Owing to its unique structural environment in the base quartet, the p Ka value for the protonation of this A residue in the complex is shifted by more than 5 pH units compared to the p Ka for A nucleotides in single-stranded RNA. This is the largest p Ka shift for an A residue in structured nucleic acids reported so far, and similar in size to the largest p Ka shifts observed for amino acid side chains in proteins. Both RNA pre-folding and ligand binding contribute to the p Ka shift. [ABSTRACT FROM AUTHOR]

Oxidation of methionine to methionine sulphoxide (MetSO) may lead to loss of molecular integrity and function. This oxidation can be 'repaired' by methionine sulphoxide reductases (MSRs), which reduce MetSO back to methionine. Two structurally unrelated classes of MSRs, MSRA and MSRB, show stereoselectivity towards the S and the R enantiomer of the sulphoxide respectively. Interestingly, these enzymes were even maintained throughout evolution in anaerobic organisms. Here, the activity and the nuclear magnetic resonance (NMR) structure of MTH711, a zinc containing MSRB from the thermophilic, methanogenic archaebacterium Methanothermobacter thermoautotrophicus, are described. The structure appears more rigid as compared with similar MSRBs from aerobic and mesophilic organisms. No significant structural differences between the oxidized and the reduced MTH711 state can be deduced from our NMR data. A stable sulphenic acid is formed at the catalytic Cys residue upon oxidation of the enzyme with MetSO. The two non-zinc-binding cysteines outside the catalytic centre are not necessary for activity of MTH711 and are not situated close enough to the active-site cysteine to serve in regenerating the active centre via the formation of an intramolecular disulphide bond. These findings imply a reaction cycle that differs from that observed for other MSRBs. [ABSTRACT FROM AUTHOR]

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 H1 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ribonucleic acids (RNA) and RNA–protein complexes are essential components of biological information transfer, catalytic processes and are associated with regulatory functions. This broad range of biological functions is paralleled at the conformational level by a large number of non-canonical structural elements or sequences with non-standard backbone conformations, e.g., loops, bulges, pseudo-knots and complex tertiary folds. NMR spectroscopy has evolved to a powerful tool for the determination of ribonucleic acid structures of up to 20 kDa. Uniform or selective stable isotope labelling aids in solving assignment problems arising from the inherently limited chemical shift dispersion and overlap of resonances for larger nucleotide sequences. Recent developments of multi-dimensional heteronuclear NMR pulse sequences allow e.g., to directly observe the hydrogen bonding pattern of canonical Watson–Crick base pairs as well as of unusual types of base pairs, thereby opening up a fast access to secondary structure screening of RNA. Detailed conformational descriptions are obtained using conventional NOE and J coupling-derived data, nowadays supplemented by information from residual dipolar couplings. The latter method also provides a new means for the probing of dynamical features of ribonucleic acids. [ABSTRACT FROM AUTHOR]

With a view to obtain 13C chemical shift correlation spectra of uniformly labelled peptides/proteins at high magnetic fields and high magic angle spinning frequencies (ωr/2π ≤ 20 kHz), the efficacy of RFDR with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements employing different adiabatic pulse phasing schemes, shapes and durations. It is demonstrated that homonuclear dipolar recoupling with superior performance under resonance offset and H1 inhomogeneity effects and without strong dependence on the 13C chemical shift differences can be achieved with adiabatic pulses. It is shown that 13C chemical shift correlation spectra in the entire range of carbon chemical shifts can be obtained efficiently with short adiabatic inversion pulses. In situations where correlation spectra of only the aliphatic region are required, the possibility for minimising the interference between the recoupling and decoupling RF fields with long adiabatic pulses, at low recoupling power levels and without compromising the broadband RFDR characteristics, is also indicated. [ABSTRACT FROM AUTHOR]

Structural and dynamic properties of [8-arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3-mercapto-3,3-cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two-dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The conformation of vasopressin and the V1 antagonists differ mainly in the region of the mutated residue. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic β-turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2 -Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different β-turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocycic part attributed to cis/trans isomerization. The X-ray structure of deamino-oxytocin [Wood, S. P., Tickle, I. I, Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, J. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633 -636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca¹, Arg8]vasopressin. [ABSTRACT FROM AUTHOR]

The performance of symmetry-based adiabatic RF pulse schemes for 1H decoupling at moderate magic angle spinning (MAS) frequencies has been evaluated via numerical simulations and experimentally compared with other decoupling techniques. Our studies reveal that in MAS solid state NMR studies of polycrystalline specimens, in contrast to symmetry-based RF pulse schemes involving rectangular RF pulses, adiabatic decoupling sequences can be effectively implemented to make use of all the available decoupling RF field strength and to eliminate the deleterious effects of H1 inhomogeneity and resonance offset. Symmetry-based adiabatic decoupling sequences lead to improved decoupling performance compared to a continuous train of adiabatic inversion pulses that are phase cycled as in solution state NMR studies. [Copyright &y& Elsevier]

Abstract: The venom of marine cone snails contains a variety of conformationally constrained peptides utilized by the animal to capture prey. Besides numerous conotoxins, which are characterized by complex disulfide patterns, other peptides with only a single disulfide bridge were isolated from different conus species. Here, we report the synthesis, structure elucidation and biological evaluation of the novel C-terminally amidated decapeptide CCAP-vil, PFc[CNSFGC]YN-NH2, from Conus villepinii. The linear precursor peptide was generated by standard solid phase synthesis. Oxidation of the cysteine residues to yield the disulfide-bridged peptide was investigated under different conditions, including several ionic liquids (ILs) as new biocompatible reaction media. Among the examined ILs, 1-ethyl-3-methylimidazolium tosylate ([C2mim][OTs]) was most efficient for CCAP-vil oxidative folding, since oxidation occurred without any byproduct formation. The structure of CCAP-vil was determined by NMR methods in aqueous solution and revealed a loop structure adopting a type(I) β-turn between residues 4–7 imposed by the flanking disulfide bridge. The amino acid side chains of Pro1, Phe2, Phe6 and Tyr9 point in three directions away from the cyclic core into the solvent creating a rather hydrophobic surface of the molecule. Based on sequence homology to cardioactive peptides (CAPs) from gastropods and arthropods, such as PFc[CNAFTGC]-NH2 (CCAP), the influence of CCAP-vil on heart rate using zebrafish embryos was investigated. CCAP-vil reduced the heart rate immediately upon injection into the heart as well as upon indirect application indicating an opposite effect to the cardioaccelerating CCAP. [Copyright &y& Elsevier]

The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central α-helix and a short two-stranded antiparallel β-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases. [Copyright &y& Elsevier]