Stacking-unstacking of the dinucleoside monophosphate guanylyl-3'',5''-uridine studied with molecular dynamics.

Molecular dynamics simulations were carried out on two conformations of the dinucleoside monophosphate guanylyl-3',5'-uridine (GpU) in aqueous solution with one sodium counterion. One stacked conformation and one with the C3'-O3'-P-O5' backbone torsion angle twisted 180 degrees to create an unstacked conformation. We observed a relatively stable behavior of the stacked conformation, which remained stacked throughout the simulation, whereas the unstacked conformation showed major changes in the backbone torsion and glycosidic angles. During the simulation the unstacked conformation transformed into a more stacked form and then back again to an unstacked one. The calculated correlation times for rotational diffusion from the molecular dynamics simulations are in agreement with fluorescence anisotropy and nuclear magnetic resonance data. As expected, the correlation times for rotational diffusion of the unstacked conformation were observed to be longer than for the stacked conformation. The 2'OH group may contribute in stabilizing the stacked conformation, where the O2'-H...O4' hydrogen bond occurred in 82.7% of the simulation.     

Trimers, dimers of trimers, and trimers of trimers are common building blocks of annexin a5 two-dimensional crystals

Annexin A5 is a member of a family of homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca(2+)-dependent manner. Annexin A5, as well as other annexins, self-assembles into two-dimensional (2D) ordered arrays upon binding to membranes, a property that has been proposed to have functional implications. Electron microscopy and atomic force microscopy experiments have revealed that annexin A5 forms two types of 2D crystals-with either p6 or p3 symmetry-that are both based on annexin trimers. In this study, we describe three other crystal forms that coexist with the p6 crystals. All crystal forms are made of the same building blocks, namely, dimers of trimers and trimers of trimers. A mechanistic model of the formation of the annexin A5 2D crystals is proposed.     

Protein organization in clathrin trimers

We have prepared a homogeneous, soluble 8.6S species (“8.6S clathrin”) from calf-brain coated vesicles. Crosslinking experiments show that this 8.6S clathrin is composed of three heavy chains (molecular weight 180,000) and three light chains (molecular weights 33,000 and 36,000). Each heavy chain is in close contact with a single light chain, and the light chains appear not to be in contact with each other. Intact 8.6S clathrin can reassemble into cages without participation of additional protein species.     

Chemical synthesis of oligodeoxynucleotide dumbbells

The chemical synthesis of DNA dumbbells is investigated by using two sequences, cyclo-d(GCG-T4-CGCCGC-T4-GCG) and cyclo-d(TTCC-T4-GGAATTCC-T4-GGAA). This method readily and inexpensively yields multimicromole quantities of circular DNA, allowing detailed structural and physical studies to be carried out. Linear oligomers of sequence d(GCG-T4-CGCCGC-T4-GCG) having either 3'- or 5'-phosphates were cyclized in 40% and 67% isolated yield, respectively, by using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide. Formation of the circular product is confirmed by a 28 degrees C increase in the optical melting temperature, anomalously rapid electrophoretic migration, sequential nuclear Overhauser enhancements between protons of G1 and G20, and observed nuclear coupling between the ligated phosphorus and protons of both G1 and G20. cyclo-d(TTCC-T4-GGAATTCC-T4-GGCC) was synthesized from the corresponding linear 3'-phosphate in 80% yield by the same procedure. Chemical ligation is most effective for 3'-phosphorylated nick sites flanked by two purine bases.     

DockTrina: Docking triangular protein trimers

In spite of the abundance of oligomeric proteins within a cell, the structural characterization of protein–protein interactions is still a challenging task. In particular, many of these interactions involve heteromeric complexes, which are relatively difficult to determine experimentally. Hence there is growing interest in using computational techniques to model such complexes. However, assembling large heteromeric complexes computationally is a highly combinatorial problem. Nonetheless the problem can be simplified greatly by considering interactions between protein trimers. After dimers and monomers, triangular trimers (i.e. trimers with pair‐wise contacts between all three pairs of proteins) are the most frequently observed quaternary structural motifs according to the three‐dimensional (3D) complex database. This article presents DockTrina, a novel protein docking method for modeling the 3D structures of nonsymmetrical triangular trimers. The method takes as input pair‐wise contact predictions from a rigid body docking program. It then scans and scores all possible combinations of pairs of monomers using a very fast root mean square deviation test. Finally, it ranks the predictions using a scoring function which combines triples of pair‐wise contact terms and a geometric clash penalty term. The overall approach takes less than 2 min per complex on a modern desktop computer. The method is tested and validated using a benchmark set of 220 bound and seven unbound protein trimer structures. DockTrina will be made available at http://nano‐d.inrialpes.fr/software/docktrina . Proteins 2014; 82:34–44. © 2013 Wiley Periodicals, Inc.     

Extracellular conversion of adiponectin hexamers into trimers

Adiponectin is an adipocyte-secreted hormone that exists as trimers, hexamers and larger species collectively referred to as HMW (high-molecular-weight) adiponectin. Whether hexamers or HMW adiponectin serve as precursors for trimers outside the circulation is currently unknown. Here, we demonstrate that adiponectin trimers can be generated from larger oligomers secreted from primary rat adipose cells or differentiated 3T3-L1 adipocytes. Purified hexameric, but not HMW, adiponectin converted into trimers in conditioned media separated from 3T3-L1 adipocytes or, more efficiently, when enclosed in the dialysis membrane in the presence of adipocytes. Several lines of evidence indicate that the conversion is mediated by an extracellular redox system. First, N-terminal epitope-tagged hexamers converted into trimers without proteolytic removal of the tag. Secondly, appearance of trimers was associated with conversion of disulfide-bonded dimers into monomers. Thirdly, thiol-reactive agents inhibited conversion into trimers. Consistent with a redox-based mechanism, purified hexamers reductively converted into trimers in defined glutathione redox buffer with reduction potential typically found in the extracellular environment while the HMW adiponectin remained stable. In addition, conversion of hexamers into trimers was enhanced by NADPH, but not by NADP⁺. Collectively, these data strongly suggest the presence of an extracellular redox system capable of converting adiponectin oligomers.     

Synthesis of light-activated antisense oligodeoxynucleotide

The activity of a 20-mer antisense oligodeoxynucleotide (asODN) is transiently blocked by attaching a partially complementary sense strand (sODN) via a heterobifunctional photocleavable linker (PL). The asODN-PL-sODN conjugate forms a DNA hairpin-like structure that is considerably more stable than the corresponding asODN/sODN duplex. In conjugate form, the asODN is prevented from hybridizing to exogenous RNA or DNA molecules. Activity is restored after modest exposure to UV light (lambda approximately 365 nm). Here, we provide a detailed procedure for synthesizing photoactive asODNs in good yields. Synthesis, purification and analysis of the light-activated asODN can be completed within 1-2 weeks.     

Random sequential adsorption of trimers and hexamers

Adsorption of trimers and hexamers built of identical spheres was studied numerically using the random sequential adsorption (RSA) algorithm. Particles were adsorbed on a two-dimensional, flat and homogeneous surface. Numerical simulations allowed us to determine the maximal random coverage ratio, RSA kinetics as well as the available surface function (ASF), which is crucial for determining the kinetics of the adsorption process obtained experimentally. Additionally, the density autocorrelation function was measured. All the results were compared with previous results obtained for spheres, dimers and tetramers.     

Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs

Nanodiscs, phospholipid bilayer assemblies of controlled size, were used to self-assemble bacteriorhodopsin (bR) into single trimers. Self-assembly at optimal bR to Nanodisc and phospholipid stoichiometry yielded particles containing three bR molecules. Analysis of solution small angle X-ray scattering indicated that bacteriorhodopsin is embedded in a discoidal phospholipid bilayer structure. Formation of trimers, as evidenced by visible circular dichroism of the retinal absorbance bands, is facilitated in Nanodiscs at a specific size threshold, suggesting that a critical bilayer area or amount of lipid is necessary to maintain a native oligomeric state. The lipid to bR ratio in the assembly process was also found to be an important factor in determining oligomerization state. These nanoscale bilayers offer the opportunity to understand and control the assembly of oligomeric integral membrane proteins critical to macromolecular recognition and cellular signaling.     

Naphthoquinone dimers and trimers from Euclea natalensis

The naphthoquinone dimers natalenone, 8′-hydroxydiospyrin and euclanone and the trimers galpinone and a compound with MW 562 were isolated from E. natalensis roots. Natalenone is a dehydrodimer of 7-methyl- juglone with the two moieties linked by two CC bonds to give a fused tetracyclic structure, one ring bearing a methylene bridge. Galpinone is a 7-methyljuglone linear trimer, the three units probably being linked C-8-C-6′and C-3′-C-3″. Euclanone is a new dimer of 7-methyljuglone and methylnaphthazarin, isomeric with 8′-hydroxydiospyrin.     

Antisense oligodeoxynucleotide and ribozyme design

The overwhelming advances of the last few years in the field of nucleic acid-based technologies laid the basis for the development of this new technology as a frontier method not only to combat diseases and infections but also to study gene function. The development of antisense strategies has generated considerable expectations in the neurosciences and, in particular, behavioral neurobiology. Antisense application in the brain has become a technology with tremendous impact, especially for determining the molecular pathways and substrates of behavior of an organism controlled by independent stimuli. The antisense agents, either oligodeoxynucleotides or ribozymes, interfere in the genetic flow of information from DNA via RNA to protein. According to the literature it seems clear that appropriately modified antisense compounds successfully and stably bind to their target ribonucleic acid molecules. This antisense binding leads to a decrease in the corresponding protein levels. If the targeted protein exerts detrimental effects on the cell or tissue, its reduction should be beneficial from a therapeutic point of view. If the investigator wants to study the function of a specific gene product the selective and transient downregulation of the corresponding target protein will help in functional analysis. In the following article I describe the chemical nature of the antisense oligodeoxynucleotides and some of the most commonly used derivatives and give some guidelines on antisense construction and application. The possible mode of action is discussed, as is expansion of the oligonucleotide-based application to ribozyme-mediated gene inhibition. Finally, problems that may be encountered during antisense application are discussed.     

Structural properties of trimers and tetramers of ribonuclease A

Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) x poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) x poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.     

Alpha-oxo semicarbazone peptide or oligodeoxynucleotide microarrays

We describe in this paper the preparation and characterization of semicarbazide glass slides and their use for the fabrication of microarrays using site-specific alpha-oxo semicarbazone ligation. The functional density and homogeneity of the semicarbazide glass slides were optimized by analyzing the reactivity of the layer toward a synthetic glyoxylyl fluorescent probe. Oligonucleotide microarrays were prepared by site-specific immobilization of glyoxylyl oligodeoxynucleotides. The slides were directly used in the hybridization assays using fluorescence detection and displayed a significant gain in sensibility as compared to the aldehyde glass slide/amino oligodeoxynucleotide chemistry. Semicarbazide slides were also used for the immobilization of a biotinylated peptide alpha-oxo aldehyde. The peptide microarrays allowed model interaction studies with streptavidin or an anti-biotin antibody.     

Synthesis and micellar mimic properties of bile acid trimers

Two fan-shaped bile acid trimers have been synthesized via Cu^I-catalyzed azide–alkyne cycloaddition (CuAAC) ‘click chemistry’, and their extraction experiments of cresol red sodium (CR) and pyrene were investigated in the polar and non-polar solvents, respectively. The transmission electron microscopy (TEM) results showed that the homogenous hollow capsules formed with the diameter size range of 40–70 nm in a solution of water and acetone. Thus the amphiphilicity of fan-shaped bile acid trimers might be used as the promising candidate in biological and drug delivery applications.     

Chromatographic analysis of photodynamically significant porphyrin dimers and trimers

Two porphyrin dimers, dihematoporphyrin dieter (DHD) and divinyl dieter (DVD), and two porphyrin trimers, dihematoporphyrin trimer (DHT) and divinyl trimer (DVT), have been analyzed utilizing isocratic ion-pair reversed-phase high-performance liquid chromatography. Results indicate that the vinyl porphyrins can be distinguished by three peaks appearing near 15, 38, and 42 min. The hematoporphyrin complexes are identified by the appearance of a peak located at 35 min. The DVT and DVD complexes present unique chromatographic markers at 28 and 15 min, respectively. Based on the location of these chromatographic markers, it was found that the Photofrin® drug must contain the DVD and the DHT complexes, but does not contain the DVT complex. The purity of the DVT complex is compromised by the presence of DHD and DHT impurities.     

Singlet-singlet annihilation kinetics in aggregates and trimers of LHCII

Singlet-singlet annihilation experiments have been performed on trimeric and aggregated light-harvesting complex II (LHCII) using picosecond spectroscopy to study spatial equilibration times in LHCII preparations, complementing the large amount of data on spectral equilibration available in literature. The annihilation kinetics for trimers can well be described by a statistical approach, and an annihilation rate of (24 ps)(-1) is obtained. In contrast, the annihilation kinetics for aggregates can well be described by a kinetic approach over many hundreds of picoseconds, and it is shown that there is no clear distinction between inter- and intratrimer transfer of excitation energy. With this approach, an annihilation rate of (16 ps)(-1) is obtained after normalization of the annihilation rate per trimer. It is shown that the spatial equilibration in trimeric LHCII between chlorophyll a molecules occurs on a time scale that is an order of magnitude longer than in Photosystem I-core, after correcting for the different number of chlorophyll a molecules in both systems. The slow transfer in LHCII is possibly an important factor in determining excitation trapping in Photosystem II, because it contributes significantly to the overall trapping time.     

Monitoring antisense oligodeoxynucleotide activity in hematopoietic cells

Traditionally, methods designed to impair translation through direct interactions with target messenger RNA (mRNA) have been designated as "antisense" strategies because of their reliance on the formation of reverse complementary (antisense) Watson-Crick base pairs between the targeting oligodeoxynucleotide (ODN) and the mRNA whose function is to be disrupted. Proof of putative "antisense effects," and other mechanistic studies, would be greatly facilitated by the ability to directly demonstrate hybridization between an antisense (AS) ODN and its mRNA target in vivo. In addition, evidence of AS activity by demonstrating reduced levels of RNA or protein or by showing cleaved target molecules would lend proof of the concept. In this article we discuss how AS ODN may be used to down-regulate target gene expression with an emphasis on those targets chosen for our investigations, and we summarize the methods employed for this type of study.     

Serine acetyltransferase from Escherichia coli is a dimer of trimers

Equilibrium sedimentation studies show that the serine acetyltransferase (SAT) of Escherichia coli is a hexamer. The results of velocity sedimentation and quasi-elastic light scattering experiments suggest that the identical subunits are loosely packed and/or arranged in an ellipsoidal fashion. Chemical cross-linking studies indicate that the fundamental unit of quaternary structure is a trimer. The likelihood, therefore, is that in solution SAT exists as an open arrangement of paired trimers. Crystals of SAT have 32 symmetry, consistent with such an arrangement, and the cell density function is that expected for a hexamer. Electron microscopy with negative staining provides further evidence that SAT has an ellipsoidal subunit organization, the dimensions of the particles consistent with the proposed paired trimeric subunit arrangement. A bead model analysis supports the view that SAT has a low packing density and, furthermore, indicates that the monomers may have an ellipsoidal shape. Such a view is in keeping with the ellipsoidal subunit shape of trimeric LpxA, an acyltransferase with which SAT shares contiguous repeats of a hexapeptide motif.     

Structures of the two 3D domain-swapped RNase A trimers

When concentrated in mildly acidic solutions, bovine pancreatic ribonuclease (RNase A) forms long-lived oligomers including two types of dimer, two types of trimer, and higher oligomers. In previous crystallographic work, we found that the major dimeric component forms by a swapping of the C-terminal beta-strands between the monomers, and that the minor dimeric component forms by swapping the N-terminal alpha-helices of the monomers. On the basis of these structures, we proposed that a linear RNase A trimer can form from a central molecule that simultaneously swaps its N-terminal helix with a second RNase A molecule and its C-terminal strand with a third molecule. Studies by dissociation are consistent with this model for the major trimeric component: the major trimer dissociates into both the major and the minor dimers, as well as monomers. In contrast, the minor trimer component dissociates into the monomer and the major dimer. This suggests that the minor trimer is cyclic, formed from three monomers that swap their C-terminal beta-strands into identical molecules. These conclusions are supported by cross-linking of lysyl residues, showing that the major trimer swaps its N-terminal helix, and the minor trimer does not. We verified by X-ray crystallography the proposed cyclic structure for the minor trimer, with swapping of the C-terminal beta-strands. This study thus expands the variety of domain-swapped oligomers by revealing the first example of a protein that can form both a linear and a cyclic domain-swapped oligomer. These structures permit interpretation of the enzymatic activities of the RNase A oligomers on double-stranded RNA.