[Show abstract][Hide abstract] ABSTRACT: NMR chemical shifts are highly sensitive probes of local molecular conformation and environment and form an important source of structural information. In this study, the relationship between the NMR chemical shifts of nucleic acids and the glycosidic torsion angle, χ, has been investigated for the two commonly occurring sugar conformations. We have calculated by means of DFT the chemical shifts of all atoms in the eight DNA and RNA mono-nucleosides as a function of these two variables. From the DFT calculations, structures and potential energy surfaces were determined by using constrained geometry optimizations at the BP86/TZ2P level of theory. The NMR parameters were subsequently calculated by single-point calculations at the SAOP/TZ2P level of theory. Comparison of the (1) H and (13) C NMR shifts calculated for the mono-nucleosides with the shifts determined by NMR spectroscopy for nucleic acids demonstrates that the theoretical shifts are valuable for the characterization of nucleic acid conformation. For example, a clear distinction can be made between χ angles in the anti and syn domains. Furthermore, a quantitative determination of the χ angle in the syn domain is possible, in particular when (13) C and (1) H chemical shift data are combined. The approximate linear dependence of the C1' shift on the χ angle in the anti domain provides a good estimate of the angle in this region. It is also possible to derive the sugar conformation from the chemical shift information. The DFT calculations reported herein were performed on mono-nucleosides, but examples are also provided to estimate intramolecularly induced shifts as a result of hydrogen bonding, polarization effects, or ring-current effects.
Chemistry - A European Journal 08/2012; 18(39):12372-87. · 5.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located 12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A., Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol., 310, 1109-1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar contacts. A mutational analysis was performed to test the functional importance of the triple helix for -1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of the triple helix in -1 frameshifting.
Nucleic Acids Research 11/2010; 38(21):7665-72. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Complexes of lanthanides with 1,4,7,10-tetrakis(phosphonomethyl)-1,4,7,10-tetraazacyclododecane (DOTP) were synthesized for use as shift reagents in a study on the three-dimensional structure of the gene-5 protein encoded by Coli-phage M13. The detailed synthesis is an improvement of methods scattered throughout the literature on three main aspects, and involves a series of three steps. The first step consists of condensation of diethylenetriamine and diethanolamine, yielding the twelve-membered ring 1,4,7,10-tetraazacyclododecane (“cyclen”). Ring closure was performed by reaction of N-(4-toluenesulfonyl)diethanolamine bis-4-toluenesulfonic ester (the synthesis of which was improved), and the disodium salt of tris(4-toluenesulfonyl)diethylenetriamine. Following hydrolysis, a simplified work-up procedure was devised. Subsequently, in the second step, phosphonomethyl groups were introduced in the amino functions by condensation with formaldehyde and phosphorous acid to give DOTP. Since DOTP failed to crystallize spontaneously, the previously described work-up procedure was modified. In the third step, a lanthanide ion (Ln3 +) was added to a small excess of DOTP at 80 °C and pH 8-9. The identity of each desired Ln(DOTP) complex was determined by: (1)1H NMR and 31PNMR spectroscopy and (2) lanthanide ion fluorescence experiments. Preliminary 1H NMR studies were performed to determine the activity of the Ln(DOTP) complexes as shift reagents for the gene-5 protein.
Recueil des Travaux Chimiques des Pays-Bas. 09/2010; 110(4):124 - 128.
[Show abstract][Hide abstract] ABSTRACT: Plant cells are enclosed by a rigid cell wall that counteracts the internal osmotic pressure of the vacuole and limits the rate and direction of cell enlargement. When developmental or physiological cues induce cell extension, plant cells increase wall plasticity by a process called loosening. It was demonstrated previously that a class of proteins known as expansins are mediators of wall loosening. Here, we report a type of cell wall-loosening protein that does not share any homology with expansins but is a member of the lipid transfer proteins (LTPs). LTPs are known to bind a large range of lipid molecules to their hydrophobic cavity, and we show here that this cavity is essential for the cell wall-loosening activity of LTP. Furthermore, we show that LTP-enhanced wall extension can be described by a logarithmic time function. We hypothesize that LTP associates with hydrophobic wall compounds, causing nonhydrolytic disruption of the cell wall and subsequently facilitating wall extension.
The Plant Cell 08/2005; 17(7):2009-19. · 9.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 13C NMR spectra of free bleomycin A2 at pH 4 and 6.7 and of its zinc complex at pH 6.7 were completely assigned by making use of two-dimensional 1H13C correlated spectroscopy. Differences in resonance positions between the spectra of the free bleomycin at pH 4 and 6.7 are observed for the β-hydroxyhistidine and the β-aminoalanine residues, obviously because of protonation/deprotonation of these moieties in the pH region considered. Also, shifts are observed for the carbon resonances of the gulose residue. Upon complex formation between zinc(II) and bleomycin large 13C shifts are observed for the carbon atoms in the pyrimidinepropionamide, β-hydroxyhistidine, β-aminoalanine, -L-gulose and -D-mannose fragments. Although for some residues the observed shifts correspond with the position of the zinc(II) binding sites in bleomycin proposed on the basis of earlier 1H NMR experiments, a simple correlation between the location of the zinc(II) binding sites and the 13C shifts cannot be drawn.
Magnetic Resonance in Chemistry 04/2005; 26(9):793 - 802. · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Small RNAs play an important role in regulation of gene expression in eukaryotic and eubacterial cells by modulating gene expression both at the level of transcription and translation. Here, we show that short complementary RNAs can also affect gene expression by stimulating ribosomal frameshifting in vitro. This finding has important implications for understanding the process of ribosomal frameshifting and for the potential application of small RNAs in the treatment of diseases that are due to frameshift mutations.
[Show abstract][Hide abstract] ABSTRACT: The three-dimensional structure of the central cold shock domain (CSD) of the human Y-box protein (YB-1 CSD) is virtually identical to those available for the bacterial cold shock proteins (Csp's). We have further characterized YB-1 CSD by studying its dynamics by nuclear magnetic resonance. The observed structural similarity is reflected in the backbone dynamics, which for YB-1 CSD is very similar to that of the Escherichia coli protein CspA. The rotational correlation time of YB-1 CSD shows that it is a monomer. This indicates that the dimerization observed for the YB-1 protein is not caused by its CSD, but involves other parts of this protein. The YB-1 CSD is only marginally stable as are the mesophilic bacterial Csp's. In contrast to the rapid two-state folding of the bacterial Csp's, the formation of the native form of YB-1 CSD is slow and at least a three-state process. The NMR experiments revealed the presence of a second state of YB-1 CSD in equilibrium with the native form. The exchange rates from and to the folded state are in the order of 0.2 and 0.5 s(-1), respectively. Relaxation experiments indicated that the second state is a highly flexible, partly structured molecule.
[Show abstract][Hide abstract] ABSTRACT: Activation of the disease resistance response in a host plant frequently requires the interaction of a plant resistance gene product with a corresponding, pathogenderived signal encoded by an avirulence gene. The products of resistance genes from diverse plant species show remarkable structural similarity. However, due to the general paucity of information on pathogen avirulence genes the recognition process remains in most cases poorly understood. NIP1, a small protein secreted by the fungal barley pathogen Rhynchosporium secalis, is one of only a few fungal avirulence proteins identified and characterized to date. The defense-activating activity of NIP1 is mediated by barley resistance gene Rrs1. In addition, a role of the protein in fungal virulence is suggested by its nonspecific toxicity in leaf tissues of host and non-host cereals as well as its resistance gene-independent stimulatory effect on the plant plasma membrane H+-ATPase. Four naturally occurring NIP1 isoforms are characterized by single amino acid alterations that affect the different activities in a similar way. As a step toward unraveling the signal perception/transduction mechanism, the solution structure of NIP1 was determined. The protein structure is characterized by a novel fold. It consists of two parts containing beta-sheets of two and three anti-parallel strands, respectively. Five intramolecular disulfide bonds, comprising a novel disulfide bond pattern, stabilize these parts and their position with respect to each other. A comparative analysis of the protein structure with the properties of the NIP1 isoforms suggests two loop regions to be crucial for the resistance-triggering activity of NIP1.
Journal of Biological Chemistry 12/2003; 278(46):45730-6. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The structures of tandem non-canonical base pairs, a frequently recurring motif in RNA molecules, are reviewed and analysed. The tandem non-canonical base pair motifs can be roughly divided in three groups, containing seven subgroups based on their base pairing patterns and local geometries. Structural details and helical parameters that can be used to numerically distinguish between the subgroups are tabulated. Remarkably, while the individual helical twists of the tandem and adjacent base pair steps can be substantially smaller or larger than the typical A-form value of 32.7 degrees, the average value is close to A-form. This and other striking regularities resulting from compensating geometrical adjustments, important for understanding and predicting the configurations of non-canonical base pairs geometries are discussed.
[Show abstract][Hide abstract] ABSTRACT: Formation of non-canonical base-pairs in RNA often plays a very important functional role. In addition they frequently serve as factors in stabilizing the secondary structure elements that provide the frame of large compact RNA structures. Here we describe the structure of an internal loop containing a 5'CU3'/5'UU3' non-canonical tandem base-pair motif, which is conserved within the 3'-UTR of poliovirus-like enteroviruses. Structural details reveal striking regularities of the local helix geometry, resulting from alternating geometrical adjustments, which are important for understanding and predicting stabilities and configurations of tandem non-canonical base-pairs. The C-U and U-U base-pairs severely contract the minor groove of the sugar-phosphate backbone, which might be important for protein recognition or binding to other RNA elements.
Journal of Molecular Biology 09/2003; 331(4):759-69. · 3.96 Impact Factor