C W Hilbers

Radboud University Nijmegen, Nymegen, Gelderland, Netherlands

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Publications (216)901.97 Total impact

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    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 09/2012; 18(39):12372-87. DOI:10.1002/chem.201103593 · 5.73 Impact Factor
  • C I Nabuurs · C W Hilbers · B Wieringa · A Heerschap
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    ABSTRACT: To the editor:
    AJP Cell Physiology 04/2012; 302(10):C1566-7. DOI:10.1152/ajpcell.00409.2011 · 3.78 Impact Factor
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    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. DOI:10.1093/nar/gkq629 · 9.11 Impact Factor
  • C. I. H. Nabuurs · C. W. Hilbers · A. Heerschap
    Neuromuscular Disorders 10/2010; 20(9):667-668. DOI:10.1016/j.nmd.2010.07.228 · 2.64 Impact Factor
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    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.
    09/2010; 110(4):124 - 128. DOI:10.1002/recl.19911100406
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    ABSTRACT: Essentially complete (96%) sequence-specific assignments were made for the backbone and side-chain 1H, 13C, and 15N resonances of Fusarium solani pisi cutinase, produced as a 214-residue heterologous protein in Escherichia coli, using heteronuclear NMR techniques. Three structural features were noticed during the assignment. (1) The secondary structure in solution corresponds mostly with the structure from X-ray diffraction, suggesting that both structures are globally similar. (2) The HN of Ala32 has a strongly upfield-shifted resonance at 3.97 ppm, indicative of an amide-aromatic hydrogen bond to the indole ring of Trp69 that stabilizes the N-terminal side of the parallel beta-sheet. (3) The NMR data suggest that the residues constituting the oxyanion hole are quite mobile in the free enzyme in solution, in contrast to the existence of a preformed oxyanion hole as observed in the crystal structure. Apparently, cutinase forms its oxyanion hole upon binding of the substrate like true lipases.
    Protein Science 11/2008; 6(11):2375-84. DOI:10.1002/pro.5560061111 · 2.85 Impact Factor
  • Cornelis W. Hilbers · Sybren S. Wijmenga
    Encyclopedia of Magnetic Resonance, 03/2007; , ISBN: 9780470034590
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    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. DOI:10.1105/tpc.105.032094 · 9.34 Impact Factor
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    R.C.L. OLSTHOORN · M Laurs · F Sohet · C W Hilbers · HA Heus · C W A Pleij
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    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.
    RNA 12/2004; 10(11):1702-3. DOI:10.1261/rna.7139704 · 4.94 Impact Factor
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    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.
    Biochemistry 09/2004; 43(31):10237-46. DOI:10.1021/bi049524s · 3.02 Impact Factor
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    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. DOI:10.1074/jbc.M308304200 · 4.57 Impact Factor
  • Hans A Heus · Cornelis W Hilbers
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    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.
    Nucleosides Nucleotides &amp Nucleic Acids 10/2003; 22(5-8):559-71. DOI:10.1081/NCN-120021955 · 1.02 Impact Factor
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    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. DOI:10.1016/S0022-2836(03)00787-3 · 4.33 Impact Factor
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    Slot · K.A.E · van 't · Burg · H.A · van den · C.P.A.M. Kloks · C W Hilbers · W Knogge · C.H.M. Papavoine
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    ABSTRACT: Biomolecular structures provide the basis for many studies in several research areas such as homology modelling, structure-based drug design and functional genomics. It is an important prerequisite that the structure is reliable in terms of accurate description of the experimental data, and in terms of good quality of local- and overall geometry. Recent surveys indicate that structures solved by NMR-spectroscopy normally are of lower precision than high-resolution X-ray structures. Here, we present a refinement protocol that improves the quality of protein structures determined by NMR-spectroscopy to the level of those determined by high resolution X-ray crystallography in terms of local geometry. The protocol was tested on experimental data of the proteins IL4 and Ubiquitin and on simulated data of the protein Crambin. In almost all aspects, the protocol yielded better results in terms of accuracy and precision. Independent validation of the results for Ubiquitin, using residual dipolar couplings, indicates that the ensemble of NMR structure is substantially improved by the protocol.
    Journal of Biomolecular NMR 04/2002; 22(3):281-9. DOI:10.1023/A:1014971029663 · 3.14 Impact Factor
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    ABSTRACT: The human Y-box protein 1 (YB-1) is a member of the Y-box protein family, a class of proteins involved in transcriptional and translational regulation of a wide range of genes. Here, we report the solution structure of the cold-shock domain (CSD) of YB-1, which is thought to be responsible for nucleic acid binding. It is the first structure solved of a eukaryotic member of the cold-shock protein family and consists of a closed five-stranded anti-parallel beta-barrel capped by a long flexible loop. The structure of CSD is similar to the OB-fold and a comparison with bacterial cold-shock proteins shows that its structural properties are conserved from bacteria to man. Our data suggest the presence of a DNA-binding site consisting of a patch of positively charged and aromatic residues on the surface of the beta-barrel. Further, it is shown that CSD, which has a preference for binding single-stranded pyrimidine-rich sequences, binds weakly and hardly specifically to DNA. Binding affinities reported for intact YB-1 indicate that domains other than the CSD play a role in DNA binding of YB-1.
    Journal of Molecular Biology 03/2002; 316(2):317-26. DOI:10.1006/jmbi.2001.5334 · 4.33 Impact Factor
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    J.A. Cromsigt · C W Hilbers · S S Wijmenga
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    ABSTRACT: An analysis is presented of experimental versus calculated chemical shifts of the non-exchangeable protons for 28 RNA structures deposited in the Protein Data Bank, covering a wide range of structural building blocks. We have used existing models for ring-current and magnetic-anisotropy contributions to calculate the proton chemical shifts from the structures. Two different parameter sets were tried: (i) parameters derived by Ribas-Prado and Giessner-Prettre (GP set) [(1981) J. Mol. Struct., 76, 81-92.]; (ii) parameters derived by Case [(1995) J. Biomol. NMR, 6, 341-346]. Both sets lead to similar results. The detailed analysis was carried using the GP set. The root-mean-square-deviation between the predicted and observed chemical shifts of the complete database is 0.16 ppm with a Pearson correlation coefficient of 0.79. For protons in the usually well-defined A-helix environment these numbers are, 0.08 ppm and 0.96, respectively. As a result of this good correspondence, a reliable analysis could be made of the structural dependencies of the 1H chemical shifts revealing their physical origin. For example, a down-field shift of either H2' or H3' or both indicates a high-syn/syn chi-angle. In an A-helix it is essentially the 5'-neighbor that affects the chemical shifts of H5, H6 and H8 protons. The H5, H6 and H8 resonances can therefore be assigned in an A-helix on the basis of their observed chemical shifts. In general, the chemical shifts were found to be quite sensitive to structural changes. We therefore propose that a comparison between calculated and observed 1H chemical shifts is a good tool for validation and refinement of structures derived from NOEs and J-couplings.
    Journal of Biomolecular NMR 10/2001; 21(1):11-29. DOI:10.1023/A:1011914132531 · 3.14 Impact Factor
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    ABSTRACT: RNA pseudoknots play important roles in many biological processes. In the simian retrovirus type-1 (SRV-1) a pseudoknot together with a heptanucleotide slippery sequence are responsible for programmed ribosomal frameshifting, a translational recoding mechanism used to control expression of the Gag-Pol polyprotein from overlapping gag and pol open reading frames. Here we present the three-dimensional structure of the SRV-1 pseudoknot determined by NMR. The structure has a classical H-type fold and forms a triple helix by interactions between loop 2 and the minor groove of stem 1 involving base-base and base-sugar interactions and a ribose zipper motif, not identified in pseudoknots so far. Further stabilization is provided by a stack of five adenine bases and a uracil in loop 2, enforcing a cytidine to bulge. The two stems of the pseudoknot stack upon each other, demonstrating that a pseudoknot without an intercalated base at the junction can induce efficient frameshifting. Results of mutagenesis data are explained in context with the present three-dimensional structure. The two base-pairs at the junction of stem 1 and 2 have a helical twist of approximately 49 degrees, allowing proper alignment and close approach of the three different strands at the junction. In addition to the overwound junction the structure is somewhat kinked between stem 1 and 2, assisting the single adenosine in spanning the major groove of stem 2. Geometrical models are presented that reveal the importance of the magnitude of the helical twist at the junction in determining the overall architecture of classical pseudoknots, in particular related to the opening of the minor groove of stem 1 and the orientation of stem 2, which determines the number of loop 1 nucleotides that span its major groove.
    Journal of Molecular Biology 08/2001; 310(5):1109-23. DOI:10.1006/jmbi.2001.4823 · 4.33 Impact Factor
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    ABSTRACT: The cleavage site of the Neurospora VS RNA ribozyme is located in a separate hairpin domain containing a hexanucleotide internal loop with an A-C mismatch and two adjacent G-A mismatches. The solution structure of the internal loop and helix la of the ribozyme substrate hairpin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The 2 nt in the internal loop, flanking the cleavage site, a guanine and adenine, are involved in two sheared G.A base pairs similar to the magnesium ion-binding site of the hammerhead ribozyme. Adjacent to the tandem G.A base pairs, the adenine and cytidine, which are important for cleavage, form a noncanonical wobble A+-C base pair. The dynamic properties of the internal loop and details of the high-resolution structure support the view that the hairpin structure represents a ground state, which has to undergo a conformational change prior to cleavage. Results of chemical modification and mutagenesis data of the Neurospora VS RNA ribozyme can be explained in context with the present three-dimensional structure.
    RNA 01/2001; 6(12):1821-32. DOI:10.1017/S1355838200001394 · 4.94 Impact Factor

Publication Stats

6k Citations
901.97 Total Impact Points


  • 1977–2012
    • Radboud University Nijmegen
      • • Faculty of Science
      • • Department of Biophysical Chemistry
      Nymegen, Gelderland, Netherlands
  • 2000
    • TNO
      Delft, South Holland, Netherlands
  • 1998
    • Utrecht University
      • Department of Chemistry
      Utrecht, Utrecht, Netherlands
  • 1977–1994
    • Leiden University
      Leyden, South Holland, Netherlands
  • 1990
    • University of Toronto
      • Department of Biochemistry
      Toronto, Ontario, Canada
  • 1989
    • Université Libre de Bruxelles
      Bruxelles, Brussels Capital, Belgium
  • 1982
    • University of Groningen
      • Chemical Physics Group
      Groningen, Groningen, Netherlands
  • 1973–1976
    • Yale University
      • Department of Chemistry
      New Haven, Connecticut, United States