Search

With few exceptions.sup.1-3, polydispersity or molecular heterogeneity in matter tends to impede self-assembly and state transformation. For example, shape transformations of liquid droplets with monodisperse ingredients have been reported in equilibrium.sup.4-7 and non-equilibrium studies.sup.8,9, and these transition phenomena were understood on the basis of homogeneous material responses. Here, by contrast, we study equilibrium suspensions of drops composed of polydisperse nematic liquid crystal oligomers (NLCOs). Surprisingly, molecular heterogeneity in the polydisperse drops promotes reversible shape transitions to a rich variety of non-spherical morphologies with unique internal structure. We find that variation of oligomer chain length distribution, temperature, and surfactant concentration alters the balance between NLCO elastic energy and interfacial energy, and drives formation of nematic structures that range from roughened spheres to 'flower' shapes to branched filamentous networks with controllable diameters. The branched structures with confined liquid crystal director fields can be produced reversibly over areas of at least one square centimetre and can be converted into liquid crystal elastomers by ultraviolet curing. Observations and modelling reveal that chain length polydispersity plays a crucial role in driving these morphogenic phenomena, via spatial segregation. This insight suggests new routes for encoding network structure and function in soft materials. Study of droplets containing nematic liquid crystal oligomers shows that a heterogeneous distribution of chain lengths plays a key part in driving reversible shape transformations with cooling and heating., Author(s): Wei-Shao Wei [sup.1] [sup.2] , Yu Xia [sup.2] [sup.3] , Sophie Ettinger [sup.1] [sup.2] , Shu Yang [sup.2] [sup.3] , A. G. Yodh [sup.1] [sup.2] Author Affiliations: (1) Department [...]

Biodegradable poly (ζ-caprolactone) (PCL)/polyhedral oligomeric silsesquioxanes (POSS) nanocomposites at various POSS loadings were prepared via solution casting method in this work. Scanning electron microscopy observation indicates that POSS are homogeneously dispersed in the PCL matrix. The experiments show that the crystallization peak temperature is higher in the nanocomposites than in neat PCL during nonisothermal melt crystallization; moreover, the overall crystallization rate is faster in the nanocomposites than in neat PCL during isothermal melt crystallization. Both nonisothermal and isothermal melt crystallization studies suggest that the crystallization of PCL is enhanced by the presence of POSS and influenced by the POSS loading. The effect of POSS on the crystallization is twofold: the presence of POSS may provide heterogeneous nucleation sites for the PCL crystallization while the aggregates of POSS may restrict large crystal growth of PCL. However, thecrystallization mechanism and crystal structure of PCL remain almost unchanged despite the presence of POSS in the nanocomposites. POLYM. ENG. SCI., 51:2159-2165, 2011. © 2011 Society of Plastics Engineers, INTRODUCTION Biodegradable poly(ζ -caprolactone) (PCL) has attracted more and more attention recently; however, the disadvantages, such as low glass transition temperature, low melting temperature, low modulus, poor abrasion, poor stability [...]

An extremely thermophilic bacterium, Thermus thermophilus, possesses two glutamate dehydrogenase (GDH) genes, gdhA and gdhB, putatively forming an operon on the genome. To elucidate the functions of these genes, the gene products were purified and characterized. GdhA showed no GDH activity, while GdhB showed GDH activity for reductive amination 1.3-fold higher than that for oxidative deamination. When GdhA was co-expressed with His-tag-fused GdhB, GdhA was co-purified with His-tagged GdhB. Compared with GdhB alone, co-purified GdhA-GdhB had decreased reductive amination activity and increased oxidative deamination activity, resulting in a 3.1-fold preference for oxidative deamination over reductive amination. Addition of hydrophobic amino acids affected the GDH activity of the co-purified GdhA-GdhB hetero-complex. Among the amino acids, leucine had the largest effect on activity: addition of 1 mM leucine elevated the GDH activity of the co-purified GdhA-GdhB by 974 and 245% for reductive amination and oxidative deamination, respectively, while GdhB alone did not show such marked activation by leucine. Kinetic analysis revealed that the elevation of GDH activity by leucine is attributable to the enhanced turnover number of GDH. In this hetero-oligomeric GDH system, GdhA and GdhB act as regulatory and catalytic subunits, respectively, and GdhA can modulate the activity of GdhB through hetero-complex formation, depending on the availability of hydrophobic amino acids. This study provides the first finding, to our knowledge, of a heterooligomeric GDH that can be regulated allosterically. DOI 10.1099/mic.0.042721-0

Coiled coils are extensively and successfully used nowadays to rationally design multistranded structures for applications, including basic research, biotechnology, nanotechnology, materials science, and medicine. The wide range of applications as well as the important functions these structures play in almost all biological processes highlight the need for a detailed understanding of the factors that control coiled-coil folding and oligomerization. Here, we address the important and unresolved question why the presence of particular oligomerization-state determinants within a coiled coil does frequently not correlate with its topology. We found an unexpected, general link between coiled-coil oligomerization-state specificity and trigger sequences, elements that are indispensable for coiled-coil formation. By using the archetype coiled-coil domain of the yeast transcriptional activator GCN4 as a model system, we show that well-established trimer-specific oligomerization-state determinants switch the peptide's topology from a dimer to a trimer only when inserted into the trigger sequence. We successfully confirmed our results in two other, unrelated coiled-coil dimers, ATF1 and cortexillin-1. We furthermore show that multiple topology determinants can coexist in the same trigger sequence, revealing a delicate balance of the resulting oligomerization state by position-dependent forces. Our experimental results should significantly improve the prediction of the oligomerization state of coiled coils. They therefore should have major implications for the rational design of coiled coils and consequently many applications using these popular oligomerization domains. protein folding | protein structure | protein-protein interactions doi/ 10.1073/pnas.1008502107

Because oligomers and aggregates of the protein [alpha]-synuclein ([alpha]S) are implicated in the initiation and progression of Parkinson's disease, investigation of various aS aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, [beta]-sheet-rich fibrillar species by incubation of aS in the presence. of intermediate (10-20% v/v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient production of these TFE fibrils is strongly correlated with the TFE-induced formation of a monomeric, partly helical intermediate conformation of aS, which exists in equilibrium with the natively disordered state at low [TFE] and with a highly a-helical conformation at high [TFE]. This partially helical intermediate is on-pathway to the TFE-induced formation of both the highly helical monomeric conformation and the fibrillar species. TFE-induced conformational changes in the monomer protein are similar for wild-type aS and the C-terminal truncation mutant [alpha]S1-102, indicating that TFE-induced structural transitions involve the N terminus of the protein. Moreover, the secondary structural transitions of three Parkinson's disease-associated mutants, A30P, A53T, and E46K, are nearly identical to wild-type [alpha]S, but oligomerization rates differ substantially among the mutants. Our results add to a growing body of evidence indicating the involvement of helical intermediates in protein aggregation processes. Given that aS is known to populate both highly and partially helical states upon association with membranes, these TFE-induced conformations imply relevant pathways for membrane-induced aS aggregation both in vitro and in vivo. amyloid | circular dichroism | Parkinson's disease | principal component analysis | protein misfolding doi/ 10.1073/pnas.1012336107

B lymphocytes are activated by many different antigens to produce specific antibodies protecting higher organisms from infection. To detect its cognate antigen, each B cell contains up to 120,000 B-cell antigen receptor (BCR) complexes on its cell surface. How these abundant receptors stay silent on resting B cells and how they can be activated by a molecularly diverse set of ligands is poorly understood (1). Here we show, with the use of a quantitative bifluorescence complementation assay (BiFC) (2,3), that the BCR has an intrinsic ability to form oligomers on the surface of living cells. A BCR mutant that fails to form oligomers is more active and cannot be expressed stably on the B-cell surface, whereas BiFC-stabilized BCR oligomers are less active and more strongly expressed on the surface. We propose that oligomers are the autoinhibited form of the BCR and that it is the shift from closed BCR oligomers to clustered monomers that drives B-cell activation in a way that is independent of the structural input from the antigen., The BCRs of all major immunoglobulin classes (IgM, IgD, IgG, IgE and IgA) form a complex comprising the transmembrane-bound immunoglobulin (mIg) molecule and the Igα-Igβ heterodimer signalling subunits (4). The [...]

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), an intracellular signaling pathway that adjusts the protein folding capacity of the ER according to need. If homeostasis in the ER protein folding environment cannot be reestablished, cells commit to apoptosis. The ERresident transmembrane kinase-endoribonuclease inositol-requiring enzyme 1 (IRE1) is the best characterized UPR signal transduction molecule. In yeast, Ire1 oligomerizes upon activation in response to an accumulation of misfolded proteins in the ER. Here we show that the salient mechanistic features of IRE1 activation are conserved: mammalian IRE1 oligomerizes in the ER membrane and oligomerization correlates with the onset of IRE1 phosphorylation and RNase activity. Moreover, the kinase/RNase module of human IRE1 activates cooperatively in vitro, indicating that formation of oligomers larger than four IRE1 molecules takes place upon activation. Highorder IRE1 oligomerization thus emerges as a conserved mechanism of IRE1 signaling. IRE1 signaling attenuates after prolonged ER stress. IRE1 then enters a refractive state even if ER stress remains unmitigated. Attenuation includes dissolution of IRE1 clusters, IRE1 dephosphorylation, and decline in endoribonuclease activity. Thus IRE1 activity is governed by a timer that may be important in switching the UPR from the initially cytoprotective phase to the apoptotic mode. receptor | oligomerization | kinase | RNase | fluorescent microscopy www.pnas.org/cgi/doi/10.1073/pnas.1010580107

One of the great challenges in surface chemistry is to assemble aromatic building blocks into ordered structures that are mechanically robust and electronically interlinked--i.e., are held together by covalent bonds. We demonstrate the surface-confined growth of ordered arrays of poly(3,4-ethylenedioxythiophene) (PEDOT) chains, by using the substrate (the 110 facet of copper) simultaneously as template and catalyst for polymerization. Copper acts as promoter for the Ullman coupling reaction, whereas the inherent anisotropy of the fcc 110 facet confines growth to a single dimension. High resolution scanning tunneling microscopy performed under ultrahigh vacuum conditions allows us to simultaneously image PEDOT oligomers and the copper lattice with atomic resolution. Density functional theory calculations confirm an unexpected adsorption geometry of the PEDOToligomers, which stand on the sulfur atom of the thiophene ring rather than lying flat. This polymerization approach can be extended to many other halogen-terminated molecules to produce epitaxially aligned conjugated polymers. Such systems might be of central importance to develop future electronic and optoelectronic devices with high quality active materials, besides representing model systems for basic science investigations. metal-catalyzed coupling reaction | molecular wires | cis-polythiophene | scanning probe microscopy | polymerization mechanism doi/ 10.1073/pnas.1000726107

Protein misfolding and formation of [beta]-sheet-rich amyloid fibrils or aggregates is related to cellular toxicity and decay in various human disorders including Alzheimer's and Parkinson's disease. Recently, we demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibits [alpha]-synuclein and amyloid-[beta] fibrillogenesis. It associates with natively unfolded polypeptides and promotes the self-assembly of unstructured oligomers of a new type. Whether EGCG disassembles preformed amyloid fibrils, however, remained unclear. Here, we show that EGCG has the ability to convert large, mature [alpha]-synuclein and amyloid-[beta] fibrils into smaller, amorphous protein aggregates that are nontoxic to mammalian cells. Mechanistic studies revealed that the compound directly binds to [beta]-sheet-rich aggregates and mediates the conformational change without their disassembly into monomers or small diffusible oligomers. These findings suggest that EGCG is a potent remodeling agent of mature amyloid fibrils. Alzheimer | Parkinson | catechine | misfolding | oligomer doi: 10.1073/pnas.0910723107

Replication proteins encoded by nonconjugative plasmids from the hyperthermophilic archaea of the order Sulfolobales show great diversity in amino acid sequence. We have biochemically characterized ORF735, a replication protein from pSSVi, an integrative nonconjugative plasmid from Sulfolobus solfataricus P2. We show that ORF735 is a DNA helicase of superfamily 3. It unwound double-stranded DNA (dsDNA) in a 3'-to-5' direction in the presence of ATP over a wide range of temperatures, from 37[degrees]C to 75[degrees]C, and possessed DNA-stimulated ATPase activity. ORF735 existed in solution as a salt-stable dimer and was capable of assembling into a salt-sensitive oligomer that was significantly larger than a hexamer in the presence of a divalent cation ([Mg.sup.2+]) and an adenine nucleotide (ATP, dATP, or ADP) or its analog (ATP[gamma]S or AMPPNP). Both N-terminal and C-terminal portions of ORF735 (87 and 160 amino acid residues, respectively, in size) were required for protein dimerization but dispensable for the formation of the higher-order oligomer. The protein unwound DNA only as a large oligomer. Yeast two-hybrid and coimmunoprecipitation assays revealed that ORF735 interacted with the noncatalytic subunit of host primase. These findings provide clues to the functional role of ORF735 in pSSVi DNA replication. doi:10.1128/JB.01300-09

Although actin filaments can form by oligomer annealing in vitro, they are assumed to assemble exclusively from actin monomers in vivo. In this study, we show that a pool of actin resistant to the monomer-sequestering drug latrunculin A (lat A) contributes to filament assembly in vivo. Furthermore, we show that the cofilin accessory protein Aip1 is important for establishment of normal actin monomer concentration in cells and efficiently converts cofilin-generated actin filament disassembly products into monomers and short oligomers in vitro. Additionally, in alp 1[DELTA] mutant cells, lat A--insensitive actin assembly is significantly enhanced. We conclude that actin oligomer annealing is a physiologically relevant actin filament assembly pathway in vivo and identify Aip1 as a crucial factor for shifting the distribution of short actin oligomers toward monomers during disassembly. doi/10.1083/jcb.200909176

The kinase domain of human epidermal growth factor receptor (HER) 3/ErbB3, a member of the EGF receptor (EGFR) family, lacks several residues that are critical for catalysis. Because catalytic activity in EGFR family members is switched on by an allosteric interaction between kinase domains in an asymmetric kinase domain dimer, HER3 might be specialized to serve as an activator of other EGFR family members. We have determined the crystal structure of the HER3 kinase domain and show that it appears to be locked into an inactive conformation that resembles that of EGFR and HER4. Although the crystal structure shows that the HER3 kinase domain binds ATP, we confirm that it is catalytically inactive but can serve as an activator of the EGFR kinase domain. The HER3 kinase domain forms a dimer in the crystal, mediated by hydrophobic contacts between the N-terminal lobes of the kinase domains. This N--lobe dimer closely resembles a dimer formed by inactive HER4 kinase domains in crystal structures determined previously, and molecular dynamics simulations suggest that the HER3 and HER4 N-lobe dimers are stable. The kinase domains of HER3 and HER4 form similar chains in their respective crystal lattices, in which N-lobe dimers are linked together by reciprocal exchange of C-terminal tails. The conservation of this tiling pattern in HER3 and HER4, which is the closest evolutionary homolog of HER3, might represent a general mechanism by which this branch of the HER receptors restricts ligand-independent formation of active heterodimers with other members of the EGFR family. EGFR | human epidermal growth factor receptor 3 | human epidermal growth factor receptor4 | receptor oligomerization doi/10.1073/pnas.0912101106

The mouse [gamma]-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mOAT1 designs incorporating fluorescent proteins were functionally characterized by [[sup.3]H] GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Forster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [[sup.3]H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ~30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GATI: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst. doi/ 10.1085/jgp.200910314

Interleaved dimers and higher order symmetric oligomers are ubiquitous in biology but present a challenge to de novo structure prediction methodology: The structure adopted by a monomer can be stabilized largely by interactions with other monomers and hence not the lowest energy state of a single chain. Building on the Rosetta framework, we present a general method to simultaneously model the folding and docking of multiple-chain interleaved homo-oligomers. For more than a third of the cases in a benchmark set of interleaved homo-oligomers, the method generates near-native models of large q-helical bundles, interlocking [beta] sandwiches, and interleaved [alpha]/[beta] motifs with an accuracy high enough for molecular replacement based phasing. With the incorporation of NMR chemical shift information, accurate models can be obtained consistently for symmetric complexes with as many as 192 total amino acids; a blind prediction was within 1 [Angstrom] rmsd of the traditionally determined NMR structure, and fit independently collected RDC data equally well. Together, these results show that the Rosetta 'fold-and-dock' protocol can produce models of homo-oligomeric complexes with nearatomic-level accuracy and should be useful for crystallographic phasing and the rapid determination of the structures of multimers with limited NMR information. homo-oligomers | molecular replacement | NMR structure inference | protein structure prediction I symmetry www.pnas.org/cgi/doi/10.1073/pnas.0904407106

The heptameric Nup84 complex constitutes an evolutionarily conserved building block of the nuclear pore complex. Here, we present the crystal structure of the heterotrimeric Sec13 x Nup145C x Nup84 complex, the centerpiece of the heptamer, at 3.2-[Angstrom] resolution. Nup84 forms a U-shaped a-helical solenoid domain, topologically similar to two other members of the heptamer, Nup145C and Nup85. The interaction between Nup84 and Nup145C is mediated via a hydrophobic interface located in the kink regions of the two solenoids that is reinforced by additional interactions of two long Nup84 loops. The Nup84 binding site partially overlaps with the homo-dimerization interface of Nup145C, suggesting competing binding events. Fitting of the elongated Z-shaped heterotrimer into electron microscopy (EM) envelopes of the heptamer indicates that structural changes occur at the Nup145C x Nup84 interface. Docking the crystal structures of all heptamer components into the EM envelope constitutes a major advance toward the completion of the structural characterization of the Nup84 complex. electron microscopy docking | nuclear pore complex | protein-protein interaction | X-ray crystallography | binding promiscuity doi/ 10.1073/pnas.0909373106

Oligomerization of members of the p53 family of transcription factors (p53, p63, and p73) is essential for their distinct functions in cell-cycle control and development. To elucidate the molecular basis for tetramer formation of the various family members, we solved the crystal structure of the human p73 tetramerization domain (residues 351-399). Similarly to the canonical p53 tetramer, p73 forms a tetramer with [D.sub.2] symmetry that can be described as a dimer of dimers. The most striking difference between the p53 and p73 tetramerization domain is the presence of an additional C-terminal helix in p73. This helix, which is conserved in p63, is essential for stabilizing the overall architecture of the tetramer, as evidenced by the different oligomeric structures observed for a shortened variant lacking this helix. The helices act as clamps, wrapping around the neighboring dimer and holding it in place. In addition, we show by mass spectrometry that the tetramerization domains of p63 and p73, but not p53, fully exchange, with different mixed tetramers present at equilibrium, albeit at a relatively slow rate. Taken together, these data provide intriguing insights into the divergent evolution of the oligomerization domain within the p53 family, from the ancestral p63/p73-like protein toward smaller, less promiscuous monomeric building blocks in human p53, allowing functional separation of the p53 pathway from that of its family members. crystallography | mass spectrometry | tetramer | transcription factor doi/ 10.1073/pnas.0905867106

The structural and dynamical properties of the metal-free form of WT human superoxide dismutase 1 (SOD1) and its familial amyotrophic lateral sclerosis (fALS)-related mutants, T54R and I113T, were characterized both in solution, through NMR, and in the crystal, through X-ray diffraction. We found that all 3 X-ray structures show significant structural disorder in 2 loop regions that are, at variance, well defined in the fully-metalated structures. Interestingly, the apo state crystallizes only at low temperatures, whereas all 3 proteins in the metalated form crystallize at any temperature, suggesting that crystallization selects one of the most stable conformations among the manifold adopted by the apo form in solution. Indeed, NMR experiments show that the protein in solution is highly disordered, sampling a large range of conformations. The large conformational variability of the apo state allows the free reduced cysteine Cys-6 to become highly solvent accessible in solution, whereas it is essentially buried in the metalated state and the crystal structures. Such solvent accessibility, together with that of Cys-111, accounts for the tendency to oligomerization of the metal-free state. The present results suggest that the investigation of the solution state coupled with that of the crystal state can provide major insights into SOD1 pathway toward oligomerization in relation to fALS. amyotrophic lateral sclerosis | NMR | X-ray I mobility | [H.sub.2]O/[D.sub.2]O exchange

NLRP1 (NLR family, pyrin domain-containing 1) is a contributor to innate immunity involved in intracellular sensing of pathogens, as well as danger signals related to cell injury. NLRP1 is one of the core components of caspase-1-activating platforms termed 'inflammasomes,' which are involved in proteolytic processing of interleukin-1[beta] (IL-1[beta]) and in cell death. We previously discovered that anti-apoptotic proteins Bcl-2 and Bcl-[X.sub.L] bind to and inhibit NLRP1 in cells. Using an in vitro reconstituted system employing purified recombinant proteins, we studied the mechanism by which Bcl-2 and Bcl-[X.sub.L] inhibit NLRP1. Bcl-2 and BcI-[X.sub.L] inhibited caspase-1 activation induced by NLRP1 in a concentration-dependent manner, with [K.sub.i] [approximately equal to] 10 nM. Bcl-2 and Bcl-[X.sub.L] were also determined to inhibit ATP binding to NLRP1, which is required for oligomerization of NLRP1, and BcL-[X.sub.L] was demonstrated to interfere with NLRP1 oligomerization. Deletion of the flexible loop regions of Bcl-2 and Bcl-[X.sub.L], which ate Iocated between the first and second [alpha]-helices of these anti-apoptotic proteins and which were previously shown to be required for binding NLRP1, abrogated ability to inhibit caspase-1 activation, ATP binding and oligomerization of NLRP1. Conversely, synthetic peptides corresponding to the loop region of Bcl-2 were sufficient to potently inhibit NLRP1. These findings thus demonstrate that the loop domain is necessary and sufficient to inhibit NLRP1, providing insights into the mechanism by which anti-apoptotic proteins Bcl-2 and Bcl-[X.sub.L] inhibit NLRP1. apoptosis | innate immunity

Ion mobility (IM) combined with tandem mass spectrometry (MS/MS) has been employed to separate and differentiate between polyether oligomers with the same nominal molecular weights. Poly(ethylene glycol)s with the same nominal mass-to-charge ratio (m/z), but with differing structures, were separated using ion mobility. IM-MS/MS data were able to aid identification of the backbone and end groups of the four individual polyethers in the two sets of isobaric mixtures. The MS/MS data from the resolved oligomers enabled a detailed structural characterization of the polyether mixtures to be completed in one experiment.

The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coil [TyrR.sup.E274Q], a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for [TyrR.sup.N316D] and [TyrR.sup.N316R] mutants. Gel filtration analyses revealed that the ligand-mediated oligomer formation was enhanced and diminished for [TyrR.sup.N316D] and [TyrR.sup.N316R], respectively, compared with the wild-type TyrR. ADP was substituted for ATP in the oligomer formation of [TyrR.sup.N316D].

[mu] opioid receptors are G protein--coupled receptors that mediate the pain-relieving effects of clinically used analgesics, such as morphine. Accumulating evidence shows that [mu]-[delta] opioid heterodimers have a pharmacologic profile distinct from those of the [mu] or [delta] homodimers. Because the heterodimers exhibit distinct signaling properties, the protein and mechanism regulating their levels have significant effects on morphine-mediated physiology. We report the characterization of RTP4, a Golgi chaperone, as a regulator of the levels of heterodimers at the cell surface. We show that the association with RTP4 protects [mu]-[delta] receptors from ubiquitination and degradation. This leads to increases in surface heterodimer levels, thereby affecting signaling. Thus, the oligomeric organization of opioid receptors is controlled by RTP4, and this governs their membrane targeting and functional activity. This work is the first report of the identification of a chaperone involved in the regulation of the biogenesis of a family A GPCR heterodimer. The identification of such factors as RTP4 controlling dimerization will provide insight into the regulation of heterodimers in vivo. This has implications in the modulation of pharmacology of their endogenous ligands, and in the development of drugs with specific therapeutic effects. enkephalin | G protein-coupled receptors | heroin | hetero-oligomerization

Abstract: G protein-coupled receptors (GPCRs), including endothelin receptor A ([ET.sub.A]) and B ([ET.sub.B]), may form dimers or higher-order oligomers that profoundly influence signaling. Here we examined a PDZ finger motif [...]

The genome of zebrafish (Danio rerio) encodes two unlinked genes equally closely related to the SLC4A2/AE2 anion exchanger genes of mammals. One of these is the recently reported zebrafish ae2 gene (Shmukler BE, Kurschat CE, Ackermann GE, Jiang L, Zhou Y, Barut B, Stuart-Tilley AK, Zhao J, Zon LI, Drummond IA, Vandorpe DH, Paw BH, Alper SL. Am J Physiol Renal Physiol Renal Physiol 289: F835-F849, 2005), now called ae2.1. We now report the structural and functional characterization of Ae2.2, the product of the second zebrafish Ae2 gene, ae2.2. The ae2.2 gene of zebrafish linkage group 24 encodes a polypeptide of 1,232 aa in length, sharing 70% amino acid identity with zebrafish Ae2.1 and 67% identity with mouse AE2a. Zebrafish Ae2.2 expressed in Xenopus oocytes encodes a 135-kDa polypeptide that mediates bidirectional, DIDS-sensitive [Cl.sup.-]/[Cl.sup.-] exchange and [Cl.sup.-]/HC[O.sup.-.sub.3] exchange. Ae2.2-mediated [Cl.sup.-]/[Cl.sup.-] exchange is cation independent, voltage insensitive, and electroneutral. Acute regulation of anion exchange mediated by Ae2.2 includes activation by N[H.sup.+.sub.4] and independent inhibition by acidic intracellular pH and by acidic extracellular pH. In situ hybridization reveals low-level expression of Ae2.2 mRNA in zebrafish embryo, most notably in posterior tectum, eye, pharynx, epidermal cells, and axial vascular structures, without notable expression in the Ae2.1-expressing pronephric duct. Knockdown of Ae2.2 mRNA, of Ae2.1 mRNA, or of both with nontoxic or minimally toxic levels of N-morpholino oligomers produced no grossly detectable morphological phenotype, and preserved normal structure of the head and the pronephric duct at 24 h postfertilization. chloride/bicarbonate exchanger; Xenopus oocyte; isotopic flux; in situ hybridization; two-electrode voltage clamp; N-morpholino oligomer

A synthetic strategy is described for the preparation of well-defined [epsilon]-caprolactone oligomers up to the 64-mer by using an iterative divergent-convergent approach. The results have shown a distinct structure/property relationship with a close correlation between the number of repeat units and physical properties.

Using optical microscopy, we have studied the phase behavior of mixtures of 12- to 22-bp-long nanoDNA oligomers. The mixtures are chosen such that only a fraction of the sample is composed of mutually complementary sequences, and hence the solutions are effectively mixtures of single-stranded and double-stranded (duplex) oligomers. When the concentrations are large enough, such mixtures phase-separate via the nucleation of duplex-rich liquid crystalline domains from an isotropic background rich in single strands. We find that the phase separation is approximately complete, thus corresponding to a spontaneous purification of duplexes from the single-strand oligos. We interpret this behavior as the combined result of the energy gain from the end-to-end stacking of duplexes and of depletion-type attractive interactions favoring the segregation of the more rigid duplexes from the flexible single strands. This form of spontaneous partitioning of complementary nDNA offers a route to purification of short duplex oligomers and, if in the presence of ligation, could provide a mode of positive feedback for the preferential synthesis of longer complementary oligomers, a mechanism of possible relevance in prebiotic environments. condensation | nucleation | depletion | prebiotic | PEG

A generic method has been developed for the analysis of polar compounds and oligomers in secondary organic aerosol (SOA) formed during atmospheric simulation chamber experiments. The technique has been successfully applied to SOA formed in a variety of systems, ranging from ozonolysis of biogenic volatile organic compounds to aromatic photooxidation. An example application of the method is described for the SOA produced from the reaction of ozone with cis-3-hexenyl acetate, an important biogenic precursor. A range of solvents were tested as extraction media, and water was found to yield the highest recovery. Extracts were analyzed using reversed-phase liquid chromatography coupled to ion trap mass spectrometry. In order to determine correct molecular weight assignments and increase sensitivity for less polar species, a series of low-concentration mobile-phase additives were used (NaCl, LiBr, N[H.sub.4]OH). Lithium bromide produced better fragmentation patterns, with more structural information than in the other cases with no reduction in sensitivity. The main reaction products identified in the particle-phase were 3-acetoxypropanal, 3-acetoxypropanoic acid, and 3-acetoxypropane peroxoic acid and a series of dimers and trimers up to 500 Da. Structural identification of oligomers indicates the presence of linear polyesters possibly formed via esterfication reactions or decomposition of peroxyhemiacetals.

The [[M + Ag].sup.+] ions of polystyrene (PS) oligomers are formed by matrix-assisted laser desorption/ionization, and their fragmentation characteristics are determined by tandem mass spectrometry experiments in a quadrupole/ time-of-flight mass spectrometer. Collisionally activated dissociation (CAD) of [[M + Ag].sup.+] starts with random homolytic C-C bond cleavages in the PS chain, which generate radical ions carrying either the initiating ([a.sub.n]*, [b.sub.n]*) or the terminating ([y.sub.n]*, [z.sub.n]*) chain end and primary ([a.sub.n]*, [y.sub.n]*) or benzylic ([b.sub.n]*, [z.sub.n]*) radical centers. The fragments ultimately observed arise by consecutive, radical-induced dissociations. The primary radical ions mainly decompose by monomer evaporation and, to a lesser extent, by [beta]-H* loss. The benzylic radical ions primarily decompose by 1,5-H rearrangement (backbiting) followed by [beta] C-C bond scissions; this pathway leads to either closed-shell fragments with C[H.sub.2] end groups, internal fragments with 2-3 repeat units, or truncated benzylic [b.sub.n]/[z.sub.n]* radical ions that can undergo anew backbiting. The same internal fragments are produced in all backbiting steps; hence, these fragments and small benzylic radical ions (which cannot undergo backbiting) dominate the low-mass region of the CAD spectra, while the less abundant closed-shell fragments with C[H.sub.2] end groups ([a.sub.n]/[y.sub.n]) dominate the medium-and high-mass regions. The latter fragments are suitable for determining the individual initiating and terminating end groups, whereas the internal ions could be valuable in sequence analyses of styrene copolymers.

The [Ag.sup.+] adducts of polystyrene (PS) oligomers with different sizes (6-19 repeat units) and initiating ([alpha] or terminating ([omega]) end groups mainly decompose via free radical chemistry pathways upon collisionally activated dissociation. This reactivity is observed for ions formed by matrix-assisted laser desorption/ionization as well as electrospray ionization. With end groups lacking weak bonds (robust end groups), dissociation starts with random homolytic C-C bond cleavages along the PS chain, which lead to primary and benzylic radical ions containing either of the chain ends. The primary radical ions mainly depolymerize by successive [beta] C-C bond scissions. For the benzylic radical ions, two major pathways are in competition, namely, depolymerization by successive p C-C bond scissions and backbiting via 1,5-H rearrangement followed by [beta] C-C bond scissions. The extent of backbiting decreases with internal energy. With short PS chains, the primary radical ions also undergo backbiting involving 1,4- and 1,6-H rearrangements; however, this process becomes negligible with longer chains. If the polystyrene contains a labile substituent at a chain end, this substituent is eliminated easily and, thus, not contained in the majority of observed fragments. Changes in the PS backbone structure can have a dramatic effect on the resulting dissociation chemistry. This is demonstrated for poly([alpha]-methylstyrene), in which backbiting is obstructed due to the lack of benzylic H atoms; instead, this backbone connectivity promotes 1,2-phenyl shifts in the primary radical ions formed after initial C-C bond homolyses as well as H atom transfers between the incipient primary and benzylic radicals emerging from these homolyses.

Glucocorticoid-induced TNF receptor ligand (GITRL) is a member of the TNF super family (TNFSF). GITRL plays an important role in controlling regulatory T cells. The crystal structure of the mouse GITRL (mGITRL) was determined to 1.8-[Angstrom] resolution. Contrary to the current paradigm that all ligands in the TNFSF are trimeric, mGITRL associates as dimer through a unique C terminus tethering arm. Analytical ultracentrifuge studies revealed that in solution, the recombinant mGITRL exists as monomers at low concentrations and as dimers at high concentrations. Biochemical studies confirmed that the mGITRL dimer is biologically active. Removal of the three terminal residues in the C terminus resulted in enhanced receptor-mediated NF-[kappa]B activation than by the wild-type receptor complex. However, deletion of the tethering C-terminus arm led to reduced activity. Our studies suggest that the mGITRL may undergo a dynamic population shift among different oligomeric forms via C terminus-mediated conformational changes. We hypothesize that specific oligomeric forms of GITRL may be used as a means to differentially control GITR receptor signaling in diverse cells. costimulation | oligomerization | regulatory | T cell | TNF

This work describes an integrated microfluidic ([micro]-fl) device that can be used to effect separations that discriminate single-nucleotide polymorphisms (SNP) based on kinetic differences in the lability of perfectly matched (PM) and mismatched (MM) DNA duplexes during alkaline dehybridization. For this purpose a 21-base singlestranded DNA (ssDNA) probe sequence was immobilized on agarose-coated magnetic beads, that in turn can be localized within the channels of a poly(dimethylsiloxane) microfluidic device using an embedded magnetic separator. The PM and MM ssDNA targets were hybridized with the probe to form a mixture of PM and MM DNA duplexes using standard protocols, and the hydroxide ions necessary for mediating the dehybridizafion were generated electrochemically in situ by performing the oxygen reduction reaction (ORR) using Ou that passively permeates the device at a Pt working electrode (Pt-WE) embedded within the microfluidic channel system. The alkaline DNA dehybridization process was followed using fluorescence microscopy. The results of this study show that the two duplexes exhibit different kinetics of dehybridization, rate profiles that can be manipulated as a function of both the amount of the hydroxide ions generated and the masstransfer characteristics of their transport within the device. This system is shown to function as a durable platform for effecting hybridization/dehybridization cycles using a nonthermal, electrochemical actuation mechanism, one that may enable new designs for lab-on-a-chip devices used in DNA analysis.

Recent knowledge supports the idea that early protein aggregates share basic structural features and are responsible for cytotoxicity underlying neurodegeneration; in most cases, early aggregate cytotoxicity apparently proceeds through similar molecular mechanisms and results in similar biochemical modifications. Data suggest that aggregate cytotoxicity may be considered a generic property of the oligomers preceding fibril appearance. Oligomers can interact with cell membranes, impairing their structural organization and destroying their selective ion permeability, eventually culminating with cell death. This process can be influenced by the physicochemical features and aggregation state of amyloids as well as by the physical and biochemical features of cell surfaces. The roles of synthetic and biological surfaces in affecting protein folding and misfolding, in speeding up aggregate nucleation, and as targets of aggregate toxicity is gaining consideration. Recent research has highlighted the involvement of surfaces as protein-misfolding chaperones and aggregation catalysts and their effects in these phenomena. NEUROSCIENTIST 13(5):519-531, 2007. DOI: 10.1177/1073858407303428 KEY WORDS Amyloid, Protein aggregation, Prefibrillar aggregates, Neurodegenerative diseases, Amyloidoses, Amyloid diseases are by far the most clinically relevant protein-misfolding pathologies because of the high prevalence of some of them, including type II diabetes mellitus and Alzheimer's and Parkinson's diseases. [...]

In the current model of conjugal plasmid transfer in mycelium-forming streptomycetes, plasmid transfer by the FtsK-like TraB protein is followed by the subsequent spreading of the newly transferred plasmid within the neighbouring mycelial compartments. Several plasmid-encoded Spd proteins are involved in the plasmid spreading by an unknown mechanism, spdB2 of the conjugative pSVH1 plasmid of Streptomyces venezuelae was heterologously expressed in Escherichia coli and Streptomyces lividans, with a C-terminal His-tag-encoding sequence. Induction of spdB2-His expression affected viability in both species. The integral membrane protein SpdB2-His was eluted from the membrane fraction of S. lividans with Triton X-100, and purified as a soluble protein by Ni-NTA affinity chromatography. Cross-linking experiments with glutaraldehyde showed that SpdB2-His formed oligomers. SpdB2-His had a nonspecific DNA-binding activity: while all types of dsDNA were bound, single-stranded M13-DNA was not recognized. The spd genes of the spdB3-spd79-spdB2 operon of pSVH1 were simultaneously expressed in E. coli with different affinity tags. While expression of Strepll-SpdB3 was not detected, Spd79-flag and SpdB2-His were localized in the membrane fraction of E. coli. In the absence of SpdB2, most of the Spd79-flag protein was found in the cytoplasmic fraction, indicating that SpdB2 affects localization of Spd79. Pulldown assays with StreplI-TraB protein of pSVH1 demonstrated that TraB interacted with SpdB2, suggesting that the septal DNA translocator TraB is also involved in intramycelial plasmid spreading.

The development of organic semiconductors, specifically small molecule or oligomeric materials is presented. Synthetic approaches to produce organic semiconductors with a wide variety of functional groups and physical properties and the resulting structure-property relationships are discussed.

Atmospheric aerosols are subject to be responsible for human health effects. In this context, besides mass and number concentration of particles, their chemical composition has gained interest recently. However, knowledge about the organic content of particulate matter is still relatively scarce; i.e., only 10-40% of compounds present in the aerosol are as yet identified. By means of a newly developed measurement technique, thermal desorption/ photoionization time-of-flight mass spectrometry (TOFMS), organic species evolved from urban aerosol samples collected at Augsburg, Germany, are analyzed. Thereby, compounds desorbed according to a temperature protocol following procedures for OC/EC analysis (120,250, and 340 [degrees]C as desorbing temperatures) are ionized by soft, fragmentationless resonance mulfiphoton ionization (REMPI) and single photon ionization (SPI), respectively. With REMPI-TOFMS, a large variety of PAH is detectable. A comprehensive analysis is enabled by adding SPI-TOFMS, which gives access to aliphatic and carbonylic hydrocarbons as well as alkanoic acids and esters. Analysis of the data showed a high abundance of phenol and guiacol as well as retene, which are known markers for wood combustion. Similar patterns were found with ash from spruce wood combustion. An increase of volatile substances at 340 [degrees]C gave rise to the suggestion that these compounds are re-formed by pyrolytic decomposition reactions from oligomeric, polymeric, and polyfunctional oxygenated species. This was corroborated by the investigation of the behavior of cellulose acetate, which exhibited a similar pattern in its SPI-TOFMS spectrum at 340 [degrees]C as the aerosol. More thorough investigations of urban aerosol and source material with respect to problems such as the mass closure of carbonaceous material, indications for source apportionment, and allotment of organic species on a molecular level to fractions of organic and elemental carbon seem feasible with this measurement method.

Structure-property correlations of a several model compounds for oligomer liquid crystals (LCs) are investigated. The compounds investigated are monomers- C6H5O(CH2)xCH3 (x=4 and 5), dimers- C6H5O(CH2)OC6H5 (x=3, 4, 5, and 6) and tetramers- C6H5O(CH2)xOC6H4O(CH2)xOC6H5 (x= 5 and 6).

The diagonal and off-diagonal vibrational interactions in the amide I subspace were examined for the oligomers and hydration clusters of N-methylacetamide (NMA). The average partial vector method was developed for constructing the force constants matrix (F matrix) in the amide I subspace from that in the full Cartesian space.

A study to investigate how the distance between the positively charged trimethylalkylammonium group and the conjugated chromophore influences the fluorescence resonance energy transfer (FRET) to flurophore-tagged DNA is presented. FRET experiments in water between the tetracationic C3-C8 and pentaanionic chromophore reveal that the most efficient energy transfer occurs with C8.

Molecular orbital calculations are performed on thiophene oligomers bearing heterocyclic substituents at the third position in order to study the interplay of effects such as the nature of substituent, skeletal substitution pattern and heteroatom identity on electronic transitions. The experimentally determined optical transitions are found to follow the predicted trend of electronic transitions of monomers and polymers.

A series of pseudodi- (1-3) and trisaccharides (4-6) oligomers bearing alternating urea, thiourea, and guanidinium segments were prepared and evaluated as phopshate binder in water. Result point out that the binding strength of the oligomers depends strongly on the nature of the pseudoamide group, especially the acidity of the NH protons and the solvation properties.

Trends obtained from systematic studies based on chain-length variation have provided valuable insight and understanding into the behavior of m-phenylene ethynylene foldamers. The generalization of this experimental approach, the chain-length dependence test, is useful for studying solution conformation, packing in the solid state, specific intrachain interactions, and the contributions of end groups to a particular property.

The folding behavior of different amine-functionalized m-poly(phenyleneethynylene) (m-PPE) oligomers containing 24 phenyl rings in water was examined by using a combination of molecular dynamics (MD) and replica exchange molecular dynamics (REMD) simulation techniques. The result showed that the REMD method was more effective in predicting the helical conformation of the m-PPE in water, from an extended structure, than canonical MD methods in the same simulation time.

The electron-vibrational coupling for the oligomeric systems is examined and is found to behave in a well-defined manner with increased conjugation length. The Stokes shift can be correlated with chain length and number of calculated and vibrational mode.

A study is used to examine the folding behavior of an amine-fictionalized m-poly(phenylenetheynylene) (m-PPE) oligomer in aqueous environment. Simulation results showed that the helix is the preferred minimum energy conformation of a single oligomer in water and that Lennard-Jones interactions are the dominant forces for the stabilization of the helix.

Fluoropolyol, a sorbent chemoselective oligomer, was deposited using a matrix-assisted laser-based deposition technique. Photochemical and/or photothermal modification of the oligomer that occurs for the UV-deposited films is shown by comparing films deposited with infrared (2.94 mum) and ultraviolet (UV) (193nm) radiation.

A comprehensive photophysical study is carried out on series of platinum(II)-containing phenyl-ethnyl oligomers. The effect of increased conjugation is a red shift of So-S1 and T1-Tn and an increase in both the So-S1 and T1-Tn molar extinction coefficients.