Monitoring of reversible boronic acid-diol interactions by fluorine NMR spectroscopy in aqueous media.

Recognition and sensing of various biologically relevant species using boronic acid-based chemosensors have become increasingly popular over the last few years. Herein, we describe a new convenient method for monitoring boronic acid-diol interactions in aqueous media based on (19)F NMR spectroscopy with fluorinated boronic acid probes.

Base-type-selective high-resolution 13C edited NOESY for sequential assignment of large RNAs.

Extensive spectral overlap presents a major problem for the NMR study of large RNAs. Here we present NMR techniques for resolution enhancement and spectral simplification of fully 13C labelled RNA. High-resolution 1H-13C correlation spectra are obtained by combining TROSY-type experiments with multiple-band-selective homonuclear 13C decoupling. An additional C-C filter sequence performs base-type-selective spectral editing. Signal loss during the filter is significantly reduced because of TROSY-type spin evolution. These tools can be inserted in any 13C-edited multidimensional NMR experiment. As an example we have chosen the 13C-edited NOESY which is a crucial experiment for sequential resonance assignment of RNA. Application to a 33-nucleotide RNA aptamer and a 76-nucleotide tRNA illustrates the potential of this new methodology.

Heteronuclear NMR studies of the interaction of tRNA(Lys)3 with HIV-1 nucleocapsid protein.

Reverse transcription of HIV-1 viral RNA uses human tRNA(Lys)3 as a primer. Recombinant tRNA(Lys)3 was previously overexpressed in Escherichia coli, 15N-labelled and purified for NMR studies. It was shown to be functional for priming of HIV-1 reverse transcription. Using heteronuclear 2D and 3D NMR, we have been able to assign almost all the imino groups in the helical regions and involved in the tertiary base interactions of tRNA(Lys)3. This crucial step enabled us to address the question of the annealing mechanism of tRNA(Lys)3 by the nucleocapsid protein (NC) using heteronuclear NMR. Moreover, structural aspects of the tRNA(Lys)3/(12-53)NCp7 interaction have been characterised. The (12-53)NCp7 protein binds preferentially to the inside of the L-shape of the tRNA(Lys)3, on the acceptor and D stems, and at the level of the tertiary interactions between the D and T-psi-C loops. (12-53)NCp7 binding does not induce the melting of any single base-pair or unwinding of the tRNA(Lys)3 helical domains. Moreover, NMR provides a unique means to investigate the base-pairs that are destabilised by (12-53)NCp7 binding. Indeed, the measurements of the longitudinal relaxation time T1 and of the exchange time of the imino protons revealed two major regions sensitive to catalysis by the protein, namely the G6-U67 and T54(A58) pairs. It is interesting that for the biological role of the NC protein, these pairs could be the starting points of the tRNA melting required for the hybridisation to the viral RNA.

NMR and biochemical characterization of recombinant human tRNA(Lys)3 expressed in Escherichia coli: identification of posttranscriptional nucleotide modifications required for efficient initiation of HIV-1 reverse transcription.

Reverse transcription of HIV-1 viral RNA uses human tRNA(Lys)3 as a primer. Some of the modified nucleotides carried by this tRNA must play a key role in the initiation of this process, because unmodified tRNA produced in vitro is only marginally active as primer. To provide a better understanding of the contribution of base modifications in the initiation complex, we have designed a recombinant system that allows tRNA(Lys)3 expression in Escherichia coli. Because of their high level of overexpression, some modifications are incorporated at substoichiometric levels. We have purified the two major recombinant tRNA(Lys)3 subspecies, and their modified nucleotide contents have been characterized by a combination of NMR and biochemical techniques. Both species carry psis, Ds, T, t6A, and m7G. Differences are observed at position 34, within the anticodon. One fraction lacks the 5-methylaminomethyl group, whereas the other lacks the 2-thio group. Although the s2U34-containing recombinant tRNA is a less efficient primer, it presents most of the characteristics of the mammalian tRNA. On the other hand, the mnm5U34-containing tRNA has a strongly reduced activity. Our results demonstrate that the modifications that are absent in E. coli (m2G10, psi27, m5C48, m5C49, and m1A58) as well as the mnm5 group at position 34 are dispensable for initiation of reverse transcription. In contrast, the 2-thio group at position 34 seems to play an important part in this process.

How NF-kappaB can be attracted by its cognate DNA.

NF-kappaB is involved in the transcriptional regulation of a large number of genes, in particular those of human immunodeficiency virus (HIV). Recently, we used NMR spectroscopy and molecular modelling to study the solution structure of a native duplex related to the HIV-1 kappaB site, together with a mutated duplex for which a three base-pair change abolishes NF-kappaB binding. The native duplex shows unusual dynamics of the four steps surrounding the kappaB site. Here, we explore the intrinsic properties of the NMR-refined structures of both duplexes in order to understand why the native sequence is recognised by NF-kappaB among other DNA sequences. We establish that only the native kappaB site can adopt a conformation where its structure (curvature and base displacement), the accessibility and the electrostatic potentials of key atoms become very favourable for binding the large loops of NF-kappaB, in contrast to the mutated duplex. Finally, we show that the neutralisation of phosphate groups contacted by NF-kappaB favours a more canonical DNA structure. These findings lead to a new hypothesis for specific recognition through the phosphodiester backbone dynamics of the sequences flanking a binding site. Such unusual behaviour confers upon the overall duplex properties that can be used by NF-kappaB to select its binding site. Thus, the selectivity determinants for NF-kappaB binding appear to depend on deformability of an "extended" consensus sequence.

NF-kappa B binding mechanism: a nuclear magnetic resonance and modeling study of a GGG --> CTC mutation.

We present the solution structure of the nonpalindromic 16 bp DNA 5'd(CTGCTCACTTTCCAGG)3'. 5'd(CCTGGAAAGTGAGCAG)3' containing a mutated kappaB site for which the mutation of a highly conserved GGG tract of the native kappaB HIV-1 site to CTC abolishes NF-kappaB binding. 1H and 31P NMR spectroscopies have been used together with molecular modeling to determine the fine structure of the duplex. NMR data show evidence for a BI-BII equilibrium of the CpA.TpG steps at the 3'-end of the oligomer. Models for the extreme conformations reached by the mutated duplex (denoted 16M) are proposed in agreement with the NMR data. Since the distribution of BII sites is changed in the mutated duplex compared to that of the native duplex (denoted 16N), large differences are induced in the intrinsic structural properties of both duplexes. In particular, in BII structures, 16M shows a kink located at the 3'-end of the duplex, and in contrast, 16N exhibits an intrinsic global curvature toward the major groove. Whereas 16N can reach a conformation very favorable for the interaction with NF-kappaB, 16M cannot mimic such a conformation and, moreover, its deeper and narrower major groove could hinder the DNA-protein interactions.

Solution structure of a non-palindromic 16 base-pair DNA related to the HIV-1 kappa B site: evidence for BI-BII equilibrium inducing a global dynamic curvature of the duplex.

1H and 31P NMR spectroscopy have been used together with molecular modelling to determine the fine structure of a non-palindromic 16 bp DNA containing the NF-kappa B binding site. Much emphasis has been placed upon NMR optimization of both two-dimensional 31P NMR techniques to extract structural information defining the phosphodiester backbone conformation and selective homonuclear 2D COSY experiments to determine sugar conformations. NMR data show evidence for a dynamic behaviour of steps flanking the ten base-pairs of the NF-kappa B binding site. A BI-BII equilibrium at these steps is demonstrated and two models for each extreme conformation are proposed in agreement with NMR data. In the refined BII structures, the NF-kappa B binding site exhibits an intrinsic curvature towards the major groove that is magnified by the four flanking steps in the BII conformation. Furthermore, the base-pairs are translated into the major groove. Thus, we present a novel mode of dynamic intrinsic curvature compatible with the DNA curvature observed in the X-ray structure of the p50-DNA complex.

Solution conformation of alpha, beta or gamma-methylglutamyl-containing derivatives as probes of vitamin K-dependent carboxylase using molecular modelling and nuclear magnetic resonance.

In the present study, the conformational behaviour of methyl substituted N-BOC glutamic acid methyl esters (2M, 3T, 3E, 4T, 4E) has been completely characterized through combined NMR and molecular modeling studies. Hetero- and homonuclear coupling constants were measured in order to assign the remaining diastereotopic methylene protons at C(3) and/or C(4), and used for comparison with theoretical data. In parallel, the complete conformational analysis of these analogues has been achieved using molecular mechanics and molecular dynamics (MD) methods. The conformation of the glutamyl residue is established by the excellent agreement between the experimental and calculated side chain scalar coupling constants. The theoretical NMR data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for internal motions. There is a significant influence of the methyl group on the conformational behaviour and on the biological relevance of these structures. Steric effect or electrostatic interaction may also have a considerable influence in stabilizing a conformational population in D2O solution. The conformational preferences of those different analogues in aqueous and methanol solution are discussed in the light of biological results obtained on the vitamin K-dependent carboxylase system.

[Marshall syndrome. 2 new cases]. / Syndrome de Marshall. Deux nouveaux cas.

Two new familial cases of Marshall syndrome are reported. The main features of this rare syndrome are outlined. Its possible relationship with Stickler syndrome is discussed.