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Biomedical subjects

Holger Stark

Publications and source records attributed to Holger Stark.

At least 19 recordsLinked to original sources
PubMed2006

Organization of core spliceosomal components U5 snRNA loop I and U4/U6 Di-snRNP within U4/U6.U5 Tri-snRNP as revealed by electron cryomicroscopy.

In eukaryotes, pre-mRNA exons are interrupted by large noncoding introns. Alternative selection of exons and nucleotide-exact removal of introns are performed by the spliceosome, a highly dynamic macromolecular machine. U4/U6.U5 tri-snRNP is the largest and most conserved building block of the spliceosome. By 3D electron cryomicroscopy and labeling, the exon-aligning U5 snRNA loop I is localized at the center of the tetrahedrally shaped tri-snRNP reconstructed to approximately 2.1 nm resolution in vitrified ice. Independent 3D reconstructions of its subunits, U4/U6 and U5 snRNPs, show how U4/U6 and U5 combine to form tri-snRNP and, together with labeling experiments, indicate a close proximity of the spliceosomal core components U5 snRNA loop I and U4/U6 at the center of tri-snRNP. We suggest that this central tri-snRNP region may be the site to which the prespliceosomal U2 snRNA has to approach closely during formation of the catalytic core of the spliceosome.

Base Sequence↗
PubMed2006

Freezing and melting of a colloidal adsorbate on a 1D quasicrystalline substrate.

Using Monte Carlo simulations and an extended Landau-Alexander-McTague theory, we demonstrate that colloids in a one-dimensional quasicrystalline potential order in triangular and rhombic-alpha crystalline phases. Increasing the strength of the potential further, a new type of light-induced melting is discovered that has its origin in the nonperiodicity of the potential. In contrast to reentrant melting in periodic potentials, the quasicrystalline potential melts the crystalline phases even when they already exist at zero potential.

Journal Article↗
PubMed2006

Numerical study of a microscopic artificial swimmer.

We present a detailed numerical study of a microscopic artificial swimmer realized recently by Dreyfus in experiments [Dreyfus, Nature 437, 862 (2005)]. It consists of an elastic filament composed of superparamagnetic particles that are linked together by DNA strands. Attached to a load particle, the resulting swimmer is actuated by an oscillating external magnetic field so that it performs a nonreciprocal motion in order to move forward. We model the superparamagnetic filament by a bead-spring configuration that resists bending like a rigid rod and whose beads experience friction with the surrounding fluid and hydrodynamic interactions with each other. We show that, aside from finite-size effects, its dynamics is governed by the dimensionless sperm number, the magnitude of the magnetic field, and the angular amplitude of the field's oscillating direction. Then we study the mean velocity and the efficiency of the swimmer as a function of these parameters and the size of the load particle. In particular, we clarify that the real velocity of the swimmer is influenced by two main factors, namely the shape of the beating filament (determined by the sperm number and the magnetic-field strength) and the oscillation frequency. Furthermore, the load size influences the performance of the swimmer and has to be chosen as a compromise between the largest swimming velocity and the best efficiency. Finally, we demonstrate that the direction of the swimming velocity changes in a symmetry-breaking transition when the angular amplitude of the field's oscillating direction is increased, in agreement with experiments.

Biomimetics↗
PubMed2006

Superdiffusion in a honeycomb billiard.

We investigate particle transport in the honeycomb billiard which consists of connected channels placed on the edges of a honeycomb structure. The spreading of particles is superdiffusive due to the existence of ballistic trajectories which we term perfect paths. Simulations give a time exponent of 1.72 for the mean-square displacement and a starlike, i.e., anisotropic, particle distribution. We present an analytical treatment based on the formalism of continuous-time random walks and explain the anisotropic distribution under the assumption that the perfect paths follow the directions of the six lattice axes. Furthermore, we derive a relation between the time exponent and the exponent of the distribution function for trajectories close to a perfect path. In billiards with randomly distributed channels, conventional diffusion is always observed in the long-time limit, although for small disorder transient superdiffusional behavior exists. Our simulation results are again supported by an analytical analysis.

Journal Article↗
PubMed2006

Ether derivatives of 3-piperidinopropan-1-ol as non-imidazole histamine H3 receptor antagonists.

A series of aliphatic and aromatic ether derivatives of 3-piperidinopropan-1-ol has been prepared by four different methods. The ethers obtained were evaluated for their affinities at recombinant human histamine H3 receptor, stably expressed in CHO-K1 or HEK 293 cells. All compounds investigated show from moderate to high in vitro affinities in the nanomolar concentration range. Selected compounds were investigated under in vivo conditions after oral administration to mice. Some proved to be highly potent and orally available histamine H3 receptor antagonists. The most potent antagonists in this series have been in vitro the 4-(1,1-dimethylpropyl)phenyl ether 19 (hH3R K(i) = 8.4 nM) and in vivo the simple ethyl ether 2 (ED50 = 1.0mg/kg).

1-Propanol↗
PubMed2006

Fluorescent non-imidazole histamine H3 receptor ligands with nanomolar affinities.

Omega-piperidinoalkanamine derivatives with fluorescent moieties (2-cyanoisoindol-1-yl, 7-nitrobenzofurazan-4-yl) have been synthesized starting from piperidine in three steps. The compounds display moderate to good histamine hH(3) receptor affinities with K(i) values ranging from 178 to 11nM. The new compounds may act as tools for identification and understanding of the binding site on the histamine H(3) receptor.

Fluorescent Dyes↗
PubMed2006

Crystal structure of a core spliceosomal protein interface.

The precise excision of introns from precursor mRNAs (pre-mRNAs) in eukaryotes is accomplished by the spliceosome, a complex assembly containing five small nuclear ribonucleoprotein (snRNP) particles. Human p14, a component of the spliceosomal U2 and U11/U12 snRNPs, has been shown to associate directly with the pre-mRNA branch adenosine early in spliceosome assembly and within the fully assembled spliceosome. Here we report the 2.5-A crystal structure of a complex containing p14 and a peptide derived from the p14-associated U2 snRNP component SF3b155. p14 contains an RNA recognition motif (RRM), the surface of which is largely occluded by a C-terminal alpha-helix and a portion of the SF3b155 peptide. An analysis of RNA.protein crosslinking to wild-type and mutant p14 shows that the branch adenosine directly interacts with a conserved aromatic within a pocket on the surface of the complex. This result, combined with a comparison of the structure with known RRMs and pseudoRRMs as well as model-building by using the electron cryomicroscopy structure of a spliceosomal U11/U12 di-snRNP, suggests that p14.SF3b155 presents a noncanonical surface for RNA recognition at the heart of the mammalian spliceosome.

Adenosine↗
PubMed2006

Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions.

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including approximately 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.

Affinity Labels↗
PubMed2006

Cryo-electron microscopy of spliceosomal components.

Splicing is an essential step of gene expression in which introns are removed from pre-mRNA to generate mature mRNA that can be translated by the ribosome. This reaction is catalyzed by a large and dynamic macromolecular RNP complex called the spliceosome. The spliceosome is formed by the stepwise integration of five snRNPs composed of U1, U2, U4, U5, and U6 snRNAs and more than 150 proteins binding sequentially to pre-mRNA. To study the structure of this particularly dynamic RNP machine that undergoes many changes in composition and conformation, single-particle cryo-electron microscopy (cryo-EM) is currently the method of choice. In this review, we present the results of these cryo-EM studies along with some new perspectives on structural and functional aspects of splicing, and we outline the perspectives and limitations of the cryo-EM technique in obtaining structural information about macromolecular complexes, such as the spliceosome, involved in splicing.

Cryoelectron Microscopy↗
PubMed2005

Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C.

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase with essential functions in mitosis, meiosis, and G1 phase of the cell cycle. APC/C recognizes substrates via coactivator proteins such as Cdh1, and bound substrates are ubiquitinated by E2 enzymes that interact with a hetero-dimer of the RING subunit Apc11 and the cullin Apc2. We have obtained three-dimensional (3D) models of human and Xenopus APC/C by angular reconstitution and random conical tilt (RCT) analyses of negatively stained cryo-electron microscopy (cryo-EM) preparations, have determined the masses of these particles by scanning transmission electron microscopy (STEM), and have mapped the locations of Cdh1 and Apc2. These proteins are located on the same side of the asymmetric APC/C, implying that this is where substrates are ubiquitinated. We have further identified a large flexible domain in APC/C that adopts a different orientation upon Cdh1 binding. Cdh1 may thus activate APC/C both by recruiting substrates and by inducing conformational changes.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

Diffusive transport of light in a Kelvin foam.

Although diffusing-wave spectroscopy has already been successfully applied to study dynamic properties of foams, we still lack a clear understanding of the diffusive transport of photons in foams. In this paper, we present a thorough study of photon diffusion in the Kelvin structure as an example for a three-dimensional model foam. We consider the photons' random walk as they are reflected or transmitted by the liquid films according to the rules of ray optics. For constant reflectance and special one- and two-dimensional photon paths, we are able to calculate diffusion constants analytically. Extensive numerical simulations reveal a remarkable similarity with our previous two-dimensional investigations. To implement a more realistic model, we use thin-film reflectances. The simulated diffusion constants exhibit oscillations for varying film thickness d which vanish when disorder is introduced in d. Absolute values and the behavior at small d agree with measurements in very dry foams providing a strong argument for the importance of liquid films in the diffusive photon transport. An analytical theory with a minimum of input parameters reproduces the numerical results.

Journal Article↗
PubMed2005

Friction drag of a spherical particle in a liquid crystal above the isotropic-nematic transition.

We study the friction drag of a spherical particle in the isotropic phase of a nematic liquid crystal close to the isotropic-nematic transition point. To describe the orientational order in the liquid crystal, the second-rank tensor order parameter Qalphabeta is employed. We solve the hydrodynamic equations for Qalphabeta and the fluid velocity nu in order to determine the friction drag. In our discussion of the friction drag, we concentrate on four parameters: the temperature, the surface order parameter, the particle radius, and the Ericksen number Er (characterizing the ratio of the viscous force to the elastic force). The temperature dependence of the friction drag agrees well with experiments that show an increasing friction drag when the isotropic-nematic phase transition is approached from above. Furthermore the friction drag increases with the surface order parameter due to the more pronounced surface nematic layer, and for larger particles it is less affected by this layer. Finally, we observe that in the range of Er we study, the friction drag is almost independent of Er although flow-induced order occurs for sufficiently large Er and surface order parameter.

Journal Article↗
PubMed2005

Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation.

The L7/12 stalk of the large subunit of bacterial ribosomes encompasses protein L10 and multiple copies of L7/12. We present crystal structures of Thermotoga maritima L10 in complex with three L7/12 N-terminal-domain dimers, refine the structure of an archaeal L10E N-terminal domain on the 50S subunit, and identify these elements in cryo-electron-microscopic reconstructions of Escherichia coli ribosomes. The mobile C-terminal helix alpha8 of L10 carries three L7/12 dimers in T. maritima and two in E. coli, in concordance with the different length of helix alpha8 of L10 in these organisms. The stalk is organized into three elements (stalk base, L10 helix alpha8-L7/12 N-terminal-domain complex, and L7/12 C-terminal domains) linked by flexible connections. Highly mobile L7/12 C-terminal domains promote recruitment of translation factors to the ribosome and stimulate GTP hydrolysis by the ribosome bound factors through stabilization of their active GTPase conformation.

Amino Acid Sequence↗