J. H. Van Boom

Molecular structure of a left-handed double helical DNA fragment at atomic resolution.

The DNA fragment d(CpGpCpGpCpG) crystallises as a left-handed double helical molecule with Watson–Crick base pairs and an antiparallel organisation of the sugar phosphate chains. The helix has two nucleotides in the asymmetric unit and contains twelve base pairs per turn. It differs significantly from right-handed B-DNA.

Iodonium ion promoted reactions at the anomeric centre. II An efficient thioglycoside mediated approach toward the formation of 1,2-trans linked glycosides and glycosidic esters☆

Abstract N-iodosuccinimide (NIS) in the presence of an organic acid was found to be effective for the activation of fully acylated thioglycosides leading to 1,2-trans linked esters. On the other hand, NIS together with a catalytic amount of trifluoromethanesulfonic acid proved to be very convenient for the rapid, high-yielding and stereoselective (1,2-trans) glycosidation of esterified thioglycosides with glycosyl acceptors.

An efficient thioglycoside-mediated formation of α-glycosidic linkages promoted by iodonium dicollidine perchlorate

Abstract Chemospecific glycosidation of partially-benzoylated thioglycosides (“disarmed” acceptors) with perbenzylated thioglycosides (“armed” donors) can be realized in the presence of the promotor iodonium dicollidine perchlorate. The reaction results predominantly in the formation of α-linked saccharides and is compatible with the use of various protecting groups.

Iterative procedure for structure determination from proton-proton NOEs using a full relaxation matrix approach. Application to a DNA octamer

Abstract A method is proposed, called the iterative relaxation matrix approach (IRMA), for the structure determination of biomolecules in solution based on 2D NOE data. Proton-proton distances are determined in a way in which indirect magnetization transfer (spin diffusion) is taken fully into account. In this method experimental NOEs are combined with calculated NOE values based on a molecular model. Back-transformation of this mixed NOE matrix gives a relaxation matrix which provides a better estimation of the cross-relaxation rates than can be obtained directly from the NOE cross peaks. From the cross-relaxation rates distance constraints can be derived, which are used in restrained molecular dynamics calculations to obtain an improved molecular model. The iteration cycle can be repeated until all experimental NOE values are satisfactorily explained. The method was tested with a DNA octamer, d(GCGTTGCG)·d(CGCAACGC).

Topogenesis of Microbody Enzymes - a Sequence Comparison of the Genes for the Glycosomal (microbody) and Cytosolic Phosphoglycerate Kinases of Trypanosoma-brucei

To determine how microbody enzymes enter microbodies, we are studying the genes for cytosolic and glycosomal (microbody) isoenzymes in Trypanosoma brucei. We have found three genes (A, B and C) coding for phosphoglycerate kinase (PGK) in a tandem array in T. brucei. Gene B codes for the cytosolic and gene C for the glycosomal isoenzyme. Genes B and C are 95% homologous, and the predicted protein sequences share approximately 45% amino acid homology with other eukaryote PGKs. The microbody isoenzyme differs from the cytosolic form and other PGKs in two respects: a high positive charge and a carboxy‐terminal extension of 20 amino acids. Our results show that few alterations are required to redirect a protein from cytosol to microbody. From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into glycosomes.

Molecular Structure of the Netropsin-d(CGCGATATCGCG) Complex: DNA Conformation in an Alternating AT Segment

The molecular structure of the complex between a minor groove binding drug (netropsin) and the DNA dodecamer d(CGCGATATCGCG) has been solved and refined by single-crystal X-ray diffraction analysis to a final R factor of 20.0% to 2.4-A resolution. The crystal is similar to that of the other related dodecamers with unit cell dimensions of a = 25.48 A, b = 41.26 A, and c = 66.88 A in the space group P2(1)2(1)2(1). In the complex, netropsin binds to the central ATAT tetranucleotide segment in the narrow minor groove of the dodecamer B-DNA double helix as expected. However, in the structural refinement the drug is found to fit the electron density in two orientations equally well, suggesting the disordered model. This agrees with the results from solution studies (chemical footprinting and NMR) of the interactions between minor groove binding drugs (e.g., netropsin and distamycin A) and DNA. The stabilizing forces between drug and DNA are provided by a combination of ionic, van der Waals, and hydrogen-bonding interactions. No bifurcated hydrogen bond is found between netropsin and DNA in this complex due to the unique dispositions of the hydrogen-bond acceptors (N3 of adenine and O2 of thymine) on the floor of the DNA minor groove. Two of the four AT base pairs in the ATAT stretch have low propeller twist angles, even though the DNA has a narrow minor groove. Alternating helical twist angles are observed in the ATAT stretch with lower twist in the ApT steps than in the TpA step.

Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under magic angle spinning.

Resonance assignments recently obtained on immobilized polypeptides and a membrane protein aggregate under Magic Angle Spinning are compared to random coil values in the liquid state. The resulting chemical shift differences (secondary chemical shifts) are evaluated in light of the backbone torsion angle ψ previously reported using X-ray crystallography. In all cases, a remarkable correlation is found suggesting that the concept of secondary chemical shifts, well established in the liquid state, can be of similar importance in the context of multiple-labelled polypeptides studied under MAS conditions.

A new approach to the synthesis of phosphotriester intermediates of nucleosides and nucleic acids

Abstract Aryl phosphorodichloridates can be converted by means of 1-hydroxybenzotriazole into an effective phosphorylating agent, which can be applied to the synthesis of phosphotriester intermediates of nucleic acids.

A convenient and general approach to the synthesis of properly protected d-nucleoside-3′-hydrogenphosphonates via phosphite intermediates

Abstract Evidence will be presented to show that the monofunctional phosphitylating reagents bis(N,N-di-ethylamino)chlorophosphine and salicylchlorophosphine are very effective for the preparation of 5′-0,N-protected d-nucleoside-3′-hydrogenphosphonates.

Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for glycosomal (microbody) enzymes in Trypanosoma brucei. Here we present our results for triosephosphate isomerase (TIM), which is found exclusively in the glycosome. We found a single TIM gene without introns, having one major polyadenylated transcript of 1500 nucleotides with a long untranslated tail of approximately 600 nucleotides. By a novel method, suitable for low abundance transcripts, we demonstrate that TIM mRNA contains the 35‐nucleotide leader sequence (mini‐exon) also found on several other trypanosome mRNAs. The TIM gene and a DNA segment of at least 6 kbp upstream of the gene are transcribed at an equal rate in isolated nuclei, suggesting that the gene is part of a much larger transcription unit. The predicted protein is of the same size as TIMs from other organisms and shares approximately 50% amino acid homology with other eukaryote TIMs, somewhat less with prokaryote TIMs. Trypanosome TIM is the most basic of all TIMs sequenced thus far. This is, in part, due to the presence of two clusters of positively charged residues in the molecule which may act as a signal for entry into glycosomes.