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T Hermann

Publications and source records attributed to T Hermann.

At least 19 recordsLinked to original sources
PubMed1999

Docking of cationic antibiotics to negatively charged pockets in RNA folds.

The binding of aminoglycosides to RNA provides a paradigm system for the analysis of RNA-drug interactions. The electrostatic field around three-dimensional RNA folds creates localized and defined negatively charged regions which are potential docking sites for the cationic ammonium groups of aminoglycosides. To explore in RNA folds the electronegative pockets suitable for aminoglycoside binding, we used calculations of the electrostatic field and Brownian dynamics simulations of cation diffusion. We applied the technique on those RNA molecules experimentally known to bind aminoglycosides, namely, two tobramycin aptamers (Wang, Y.; Rando, R. R. Chem. Biol. 1995, 2, 281-290): the aminoglycoside-binding region in 16S ribosomal RNA (Moazed, S.; Noller, H. F. Nature 1987, 327, 389-394) and the TAR RNA from human immunodeficiency virus (Mei, H.-Y.; et al. Bioorg. Med. Chem. Lett. 1995, 5, 2755-2760). For the aptamers and ribosomal RNA, for which the binding sites of the aminoglycosides are known, a good agreement between negatively charged pockets and the binding positions of the drugs was found. On the basis of variations between neomycin-like and kanamycin-like aminoglycosides in the interaction with the electrostatic field of ribosomal RNA, we propose a model for the different binding specificities of these two classes of drugs. The spatial congruence between the electronegative pockets in RNA folds and binding positions of aminoglycosides was used to dock aminoglycosides to ribosomal and TAR RNAs. Molecular dynamics simulations were used to analyze possible RNA-drug interactions. Aminoglycosides inhibit the binding of the viral Tat protein to TAR RNA; however, the drug-binding sites are still unknown. Thus, our docking approach provides first structural models for TAR-aminoglycoside complexes. The RNA-drug interactions observed in the modeled complexes support the view that the antibiotics might lock TAR in a conformation with low affinity for the Tat protein, explaining the experimentally found aminoglycoside inhibition of the Tat-TAR interaction (Mei, H.-Y.; et al. Bioorg. Med. Chem. Lett. 1995, 5, 2755-2760).

Anti-Bacterial Agents↗
PubMed1999

Simulations of the dynamics at an RNA-protein interface.

Molecular dynamics simulations of the RNA-binding domain of the U1A spliceosomal protein in complex with its cognate RNA hairpin, performed at low and high ionic strength in aqueous solution, suggest a pathway for complex dissociation. First, cations condense around the RNA and compete with the protein for binding sites. Then solvated ions specifically destabilize residues at the RNA-protein interface. For a discrete cluster of residues at the complex interface, the simulations reveal an increased deviation from the crystal structure at high salt concentrations while the remaining protein scaffold is stabilized under these conditions. The microscopic picture of salt influence on the complex suggests guidelines for rational design of interface inhibitors targeted at RNA-protein complexes.

Amino Acid Sequence↗
PubMed1999

Mixed reconstitution of mutated subunits of HIV-1 reverse transcriptase coexpressed in Escherichia coli - two tags tie it up.

The active form of HIV-1 reverse transcriptase (RT) is a p66/p51 heterodimer, in which the p51 subunit is generated by C-terminal proteolytic cleavage of p66. A well-known problem of p66 recombinant expression is partial cleavage of a 15-kDa peptide from the C-terminus by host proteases that can not be completely suppressed. In order to analyse the contribution of specific residues to a particular function in one distinct subunit, an expression and purification system is required that selects for the combination of the two individual subunits with the desired substitutions. We reconstituted the p66/p51 heterodimer from subunits coexpressed in Escherichia coli as an N-terminal fusion protein of glutathione S-transferase (GST) with p51 and a C-terminally His-tagged p66, respectively. The two-plasmid coexpression system ensures convenience for gene manipulation while degradation is reduced to a minimum, as dimerization protects the protein from further proteolysis. The combination of glutathione-agarose, phenyl-superose and Ni/nitrilotriacetate affinity chromatography allows rapid and selective purification of the desired subunit combination. Truncated forms of p51 are efficiently removed. Mobility-shift assay revealed that the preparations are free of p66 homodimer. In a successful test of the novel expression system, mixed reconstituted RTs with p51 selectively mutated in a putative nucleic acid binding motif (the so called helix clamp) show reduced binding of dsDNA in mobility-shift assays. This indicates the p51 subunit has an active role in DNA binding

Base Sequence↗
PubMed1998

Exploration of metal ion binding sites in RNA folds by Brownian-dynamics simulations.

BACKGROUND: Metal ions participate in the three-dimensional folding of RNA and provide active centers in catalytic RNA molecules. The positions of metal ions are known for a few RNA structures determined by X-ray crystallography. In addition to the crystallographically identified sites, solution studies point to many more metal ion binding sites around structured RNAs. Metal ions are also present in RNA structures determined by nuclear magnetic resonance (NMR) spectroscopy, but the positions of the ions are usually not revealed. RESULTS: A novel method for predicting metal ion binding sites in RNA folds has been successfully applied to a number of different RNA structures. The method is based on Brownian-dynamics simulations of cations diffusing under the influence of random Brownian motion within the electrostatic field generated by the static three-dimensional fold of an RNA molecule. In test runs, the crystallographic positions of Mg2+ ions were reproduced with deviations between 0.3 and 2.7 A for several RNA molecules for which X-ray structures are available. In addition to the crystallographically identified metal ions, more binding sites for cations were revealed: for example, tRNAs were shown to bind more than ten Mg2+ ions in solution. Predictions for metal ion binding sites in four NMR structures of RNA molecules are discussed. CONCLUSIONS: The successful reproduction of experimentally observed metal ion binding sites demonstrates the efficiency of the prediction method. A promising application of the method is the prediction of cation-binding sites in RNA solution structures, determined by NMR.

Base Sequence↗
PubMed1998

The transcriptional activator GvpE for the halobacterial gas vesicle genes resembles a basic region leucine-zipper regulatory protein.

The GvpE protein involved in the regulation of gas vesicles synthesis in halophilic archaea has been identified as the transcriptional activator for the promoter located upstream of the gvpA gene encoding the major gas vesicle structural protein GvpA. A closer inspection of the GvpE protein sequence revealed that GvpE resembles basic leucine-zipper proteins typically involved in the gene regulation of eukarya. A molecular modelling study of the C-terminal part implied a cluster of basic amino acid residues constituting the DNA-binding site (DNAB) followed by an amphiphilic helix, suitable for the formation of a leucine-zipper structure within a GvpE dimer. The model of a GvpE dimer docked onto DNA indicated that the side-chains of the basic residues could perfectly interact with the negatively charged phosphate groups of the DNA backbone. Substitution of three basic amino acid residues of this putative DNAB by alanine and/or glutamate generated mutated GvpE proteins. None of these was able to activate the c-gvpA promoter in vivo, indicating that these basic residues are required for GvpE activity. This identification of an archaeal gene regulator displaying similarity to eukaryal regulatory proteins implies that the basic transcription machinery of eukarya and archaea are closely related, and that the regulatory proteins have evolved according to common principles.

Amino Acid Sequence↗
PubMed1998

Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA.

A variety of drugs inhibit biological key processes by binding to a specific RNA component. We focus here on the well-analysed hammer-head ribozyme RNA that is inhibited by aminoglycoside antibiotics, a process considered as a paradigm for studying drug/RNA interactions. With insight gained from molecular dynamics simulations of the ribozyme in the presence of Mg2+ identified by crystallography and of aminoglycosides in solution, a general model for aminoglycoside binding to RNA is proposed. A striking structurally based complementarity between the charged ammonium groups of the aminoglycosides and the metal binding sites in the hammerhead was uncovered. Despite dynamical flexibility of the aminoglycosides, several of the intramolecular distances between the charged ammonium groups of the drugs were found to be rather constant. Intramolecular ammonium distances of the aminoglycosides span ranges similar to the interionic distances between Mg2+ in the hammerhead. Successful docking of aminoglycosides to the hammerhead ribozyme could be achieved by positioning the ammonium groups at the sites occupied by Mg2+. The covalently linked ammonium groups of the aminoglycosides are thus able to complement in space the negative electrostatic potential created by a three-dimensional RNA fold. Consequently, it is suggested that aminoglycoside-derived sugars could constitute a basic set of yardstick synthons ideal for rational and combinatorial synthesis of drugs targeted at biologically relevant RNA folds.

Anti-Bacterial Agents↗
PubMed1998

Saccharide-RNA recognition.

Among saccharides, the antibiotics of the aminoglycoside family are the best-studied class of molecules interacting with RNA. By binding to RNA targets, aminoglycosides act as inhibitors of protein biosynthesis, they interfere with protein-RNA interaction of retroviral regulatory elements, and they inhibit the catalytic action of ribozymes. Here, we survey the available data on molecular structural details of aminoglycoside-RNA interaction.

Aminoglycosides↗
PubMed1998

Local and systemic delivery of low molecular weight heparin following PTCA: acute results and 6-month follow-up of the initial clinical experience with the porous balloon (PILOT-study). Preliminary Investigation of Local Therapy Using Porous PTCA Balloons.

The purpose of this study was to assess safety and feasibility of intracoronary delivery of reviparin using a porous balloon following percutaneous transluminal coronary angioplasty. The 2.7 mm porous balloon used in this study had 35 holes arranged in a spiral pattern. Eighteen patients (male n = 10, female n = 8, age 63 +/- 9 years) undergoing successful PTCA in coronary arteries with a vessel diameter of 2.5 to 3.0 mm determined by online QCA (LAD = 11, RCX = 3, RCA = 4) were included. They received a bolus of 7,000 anti-Xa-IU reviparin followed by local delivery of 1,500 anti-Xa-IU in 4 ml with an injection pressure of 2 atm. The patients received additionally 10500 anti-Xa-units intravenously during the following 24 hours and a daily dose of 7000 anti-Xa-units reviparin subcutaneously for the following 28 days. Angiograms were obtained before and after PTCA, directly after local delivery, at 24 hours postintervention and after 6 months. The primary success rate was 100%. Quantitative coronary angiography showed a minimum luminal diameter of 0.42 +/- 0.14 mm before PTCA, 1.87 +/- 0.45 after PTCA, 1.67 +/- 0.43 after LDD, 1.63 +/- 0.46 after 24 hours, and 1.06 +/- 0.6 after 6 months. Angiographic follow-up was obtained in all patients. No major complications occurred during the 6-month follow-up period. The angiographic restenosis rate was 28% (5/18) at follow-up. This study demonstrates safety and feasibility of local intracoronary delivery of reviparin with a porous balloon following PTCA even in smaller diameter coronary arteries.

Aged↗
PubMed1998

Mapping and identification of Corynebacterium glutamicum proteins by two-dimensional gel electrophoresis and microsequencing.

As a prerequisite for proteome analyses of Corynebacterium glutamicum separation of the cytoplasm and the membrane fraction was optimized and two-dimensional (2-D) gel electrophoresis was established. The resulting 2-D protein maps revealed over 1000 silver-stained protein spots separated by isoelectric point and molecular mass for cytoplasmic proteins and approximately 700 silver-stained spots for proteins of the membrane fraction. Proposing a mean size of 1 kbp per gene the complete C. glutamicum genome of 3 Mbp encodes 3000 different proteins; more than half of these can be located using the maps which are presently available. In this study 10 proteins were identified by N-terminal microsequencing, namely the 35 kDa antigen, antigen 84, ATP synthase subunits alpha, gamma and delta, cysteine synthase, elongation factor G and Ts, enolase, and rotamase. For seven sequences, corresponding proteins could not be identified. Additionally, two proteins were specifically detected by immunoblotting, a corynebacterial porin and the cytoplasmic protein threonine dehydratase. The methods and 2-D maps established in this study will be the basis for comparative studies of protein expression and a detailed proteome analysis of C. glutamicum.

Amino Acid Sequence↗
PubMed1998

Structural changes of the Escherichia coli GroEL-GroES chaperonins upon complex formation in solution: a neutron small angle scattering study.

We applied neutron scattering in conjunction with deuterium (D-) labeling in order to obtain information about the domain structure of GroEL and GroES isolated and in the complex. Each subunit of the heptameric GroES consists of two domains, a core domain (Met1 to Lys15 and Lys34 to Ala97) and an intervening loop region (Glu16 to Ala33). Neutron scattering shows that both regions change their conformation upon GroEL/GroES complex formation. The interdomain angle between the core regions of the heptameric GroES increases from 120 to 140 degrees, leading to a less dome-like shape of GroES, and the loop regions turn inwards by 75 degrees. The 23 C-terminal amino acids of the 14 GroEL subunits (Lys526 to Met548), which are unresolved in the crystal structure, are located either at the bottom of the cavity formed by the seven-membered GroEL ring or at the inner wall of the cavity. Upon complex formation the apical domains of GroEL move outwards, which facilitates binding of GroES at a Gro-EL-GroES center-to-center distance of (87 +/- 8) A. These structural changes may be important for the dissociation of the unfolded protein bound to the central cavity upon GroES binding. The overall structure determined by neutron scattering in solution tallies with the crystallographic model published after completion of this study. Differences in the conformation of GroES observed in the complex by the two methods support the view that the chaperonin complex is a flexible molecule which might switch in solution between different conformations.

Chaperonin 10↗
PubMed1998

Isoleucine uptake in Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene product.

By complementation analysis of an isoleucine-uptake-deficient Escherichia coli strain, it was shown that a 1.6-kb HindIII-StuI fragment of Corynebacterium glutamicum ATCC 13032, located downstream of the aecD gene, encodes an isoleucine uptake system. Sequence analysis revealed that the complementing fragment carried an open reading frame, termed brnQ, that encodes a protein with sequence similarities to branched-chain amino acid carriers of gram-positive and gram-negative bacteria. The brnQ gene specifies a predominantly hydrophobic protein of 426 amino acid residues with a calculated molecular mass of 44.9 kDa. A topology prediction by neural network computer analysis suggests the existence of 12 hydrophobic segments that most probably form transmembrane alpha-helices. A C. glutamicum mutant strain harboring a defined deletion of brnQ in the chromosome showed a considerably lower isoleucine uptake rate of 0.04 nmol min-1 mg (dry mass)-1 as compared to the wild-type strain rate of 1.2 nmol min-1 mg (dry mass)-1. Overexpression of brnQ by means of a tac promotor resulted in an elevated uptake rate for isoleucine of 11.3 nmol min-1 mg (dry mass)-1. Evidently, the brnQ gene encodes the only transport system in C. glutamicum directing isoleucine uptake.

Amino Acid Sequence↗
PubMed1998

RNA as a drug target: chemical, modelling, and evolutionary tools.

Dramatic technical progress in RNA synthesis and structure determination has allowed several difficulties inherent to the preparation, handling and structural analysis of RNA to be overcome, and this has led to a wealth of information about RNA structure and its relationship with biological function. It is now fully recognized that RNA molecules intervene at all stages of cell life, not only because of key sequence motifs but also because of intricate three-dimensional folds. This realization has promoted RNA to a potential therapeutic target. As in protein motifs recognizing nucleic acids, groups of the molecule interacting with RNA contribute to specific binding through defined hydrogen bonds and van der Waals docking, while other parts contribute to the driving force of binding via less specific electrostatic interactions accompanied by water and ion displacement.

Aminoglycosides↗
PubMed1998

Deciphering RNA recognition: aminoglycoside binding to the hammerhead ribozyme.

Aminoglycoside antibiotics inhibit protein biosynthesis and various ribozymes. Structural electrostatic complementarity can explain the inhibition mechanism of the hammerhead ribozyme: positively charged ammonium groups match the negatively charged metal-ion-binding pockets created by the RNA fold's electrostatic field.

Aminoglycosides↗
PubMed1997

Isoalloxazine derivatives promote photocleavage of natural RNAs at G.U base pairs embedded within helices.

We have recently shown that isoalloxazine derivatives are able to photocleave RNA specifically at G.U base pairs embedded within a helical stack. The reaction involves the selective molecular recognition of G.U base pairs by the isoalloxazine ring and the removal of one nucleoside downstream of the uracil residue. Divalent metal ions are absolutely required for cleavage. Here we extend our studies to complex natural RNA molecules with known secondary and tertiary structures, such as tRNAs and a group I intron (td). G.U pairs were cleaved in accordance with the phylogenetically and experimentally derived secondary and tertiary structures. Tandem G.U pairs or certain G.U pairs located at a helix extremity were not affected. These new cleavage data, together with the RNA crystal structure, allowed us to perform molecular dynamics simulations to provide a structural basis for the observed specificity. We present a stable structural model for the ternary complex of the G. U-containing helical stack, the isoalloxazine molecule and a metal ion. This model provides significant new insight into several aspects of the cleavage phenomenon, mechanism and specificity for G. U pairs. Our study shows that in large natural RNAs a secondary structure motif made of an unusual base pair can be recognized and cleaved with high specificity by a low molecular weight molecule. This photocleavage reaction thus opens up the possibility of probing the accessibility of G.U base pairs, which are endowed with specific structural and functional roles in numerous structured and catalytic RNAs and interactions of RNA with proteins, in folded RNAs.

Base Composition↗
PubMed1997

Stimulation of DNA inversion by FIS: evidence for enhancer-independent contacts with the Gin-gix complex.

Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein. Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology. Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS. Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question. Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro. We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner. Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.

Base Sequence↗
PubMed1997

Evidence for a hydroxide ion bridging two magnesium ions at the active site of the hammerhead ribozyme.

In the presence of magnesium ions, cleavage by the hammerhead ribozyme RNA at a specific residue leads to 2'3'-cyclic phosphate and 5'-OH extremities. In the cleavage reaction an activated ribose 2'-hydroxyl group attacks its attached 3'-phosphate. Molecular dynamics simulations of the crystal structure of the hammerhead ribozyme, obtained after flash-freezing of crystals under conditions where the ribozyme is active, provide evidence that a mu-bridging OH-ion is located between two Mg2+ions close to the cleavable phosphate. Constrained simulations show further that a flip from the C3'- endo to the C2'- endo conformation of the ribose at the cleavable phosphate brings the 2'-hydroxyl in proximity to both the attacked phosphorous atom and the mu-bridging OH-ion. Thus, the simulations lead to a detailed new insight into the mechanism of hammerhead ribozyme cleavage where a mu-hydroxo bridged magnesium cluster, located on the deep groove side, provides an OH-ion that is able to activate the 2'-hydroxyl nucleophile after a minor and localized conformational change in the RNA.

Binding Sites↗
PubMed1997

A three-dimensional working model for a guide RNA from Trypanosoma brucei.

RNA editing in protozoan parasites is a mitochondrial RNA processing reaction in which exclusively uridylate residues are inserted into, and less frequently deleted from, pre-mRNAs. Molecules central to the process are so-called guide RNAs (gRNAs) which function as templates in the reaction. For a detailed molecular understanding of the mechanism of the editing process knowledge of structural features of gRNAs will be essential. Here we report on a computer-assisted molecular modelling approach to construct the first three-dimensional gRNA model for gND7-506, a ND7-specific gRNA from Trypanosoma brucei. The modelling process relied on chemical modification and enzymatic probing data and was validated by in vitro mutagenesis experiments. The model predicts a reasonably compact structure, where two stem/loop secondary structure elements are brought into close proximity by a triple A tertiary interaction, forming a core element within the centre of the molecule. The model further suggests that the surface of the gRNA is primarily made up of the sugar-phoshate backbone. On the basis of the model, footprinting experiments of gND7-506 in a complex with the gRNA binding protein gBP21 could successfully be interpreted and provide a first picture for the assembly of gRNAs within a ribonucleoprotein complex.

Animals↗