Assembly Interface Research Articles

Installation of orthogonality to the interface that assembles two modular domains in the Tetrahymena group I ribozyme

Two modular elements (P5abc and ΔP5) in the Tetrahymena group I ribozyme can be separated physically to generate a two-piece ribozyme derivative consisting of a separately prepared P5abc (P5 RNA) and the rest of the intron (ΔP5 RNA). Molecular recognition in the interface assembling P5 RNA and ΔP5 RNA is strong and specific, and the catalytic ability of the two-piece ribozyme is comparable to that of the parent unimolecular ribozyme. We designed alternative P14 (L5c-L2) interacting modules participating in the assembly of P5 and ΔP5 and investigated their ability in the context of complex formation of the two-piece ribozyme and in vivo splicing of the unimolecular intron ribozyme. Combined use of alternative P14 and L5b-P6 interacting modules provided robust orthogonality to the P5/ΔP5 assembly interface of the bimolecular complex.

Functional Mapping of Human Dynamin-1-Like GTPase Domain Based on X-ray Structure Analyses

Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5′-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains.

Structure of Pt(111)/Ionomer Membrane Interface and Its Bias-Induced Change in Membrane Electrode Assembly

The structure of the perfluorosulfonated ionomer (PFSI)/Pt(111) interface in a membrane electrode assembly (MEA)-like configuration of a polymer electrolyte membrane (PEM) fuel cell, that is, a vacuum evaporated Pt layer/PEM(Nafion membrane)/PFSI(adhesion Nafion layer)/Pt(111) single crystal, and its bias-induced change were investigated by surface X-ray scattering measurement at an atomic level. Crystal truncation rod measurement shows that PFSI adsorbed on the Pt(111)-(1 × 1) surface without bias. When the Pt(111) electrode was positively biased to form Pt oxide, the PFSI layer was detached from the Pt surface and oxygen atoms penetrated into the Pt lattice.

Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43)

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.

The NTD-CTD intersubunit interface plays a critical role in assembly and stabilization of the HIV-1 capsid

BackgroundLentiviruses exhibit a cone-shaped capsid composed of subunits of the viral CA protein. The intrinsic stability of the capsid is critical for HIV-1 infection, since both stabilizing and destabilizing mutations compromise viral infectivity. Structural studies have identified three intersubunit interfaces in the HIV-1 capsid, two of which have been previously studied by mutational analysis. In this present study we analyzed the role of a third interface, that which is formed between the amino terminal domain (NTD) and carboxyl terminal domain (CTD) of adjacent subunits.ResultsWe provided evidence for the presence of the NTD-CTD interface in HIV-1 particles by engineering intersubunit NTD-CTD disulfide crosslinks, resulting in accumulation of disulfide-linked oligomers up to hexamers. We also generated and characterized a panel of HIV-1 mutants containing substitutions at this interface. Some mutants showed processing defects and altered morphology from that of wild type, indicating that the interface is important for capsid assembly. Analysis of these mutants by transmission electron microscopy corroborated the importance of this interface in assembly. Other mutants exhibited quantitative changes in capsid stability, many with unstable capsids, and one mutant with a hyperstable capsid. Analysis of the mutants for their capacity to saturate TRIMCyp-mediated restriction in trans confirmed that the unstable mutants undergo premature uncoating in target cells. All but one of the mutants were markedly attenuated in replication owing to impaired reverse transcription in target cells.ConclusionsOur results demonstrate that the NTD-CTD intersubunit interface is present in the mature HIV-1 capsid and is critical for proper capsid assembly and stability.

New cross-linked interfacial layer on hydrocarbon membrane to improve long-term stability of polymer electrolyte fuel cells

Abstract In order to enhance the chemical compatibility of membrane-electrode assembly (MEA) interface, the cross-linked interfacial layer was coated on the cathode side of the hydrocarbon membrane. The interfacial layer, which consists of the sulfonated poly(ether ether ketone) (sPEEK) and the Nafion, provided good adhesion between the sPEEK membrane and the Nafion based electrode. As a result, the interfacial layer coated MEA (i-MEA) showed distinct performance enhancement after wet–dry cycling test, compared with the pristine MEA. The improved cell performance is attributed to the interfacial layer that enhances dimensional stability of the membrane and makes interface chemically more compatible. Various physical property measurements and electrochemical characterizations consistently support the interfacial layer effect for the improved long-term stability.

Comparative Dynamics of NMDA- and AMPA-Glutamate Receptor N-Terminal Domains

SummaryIonotropic glutamate receptors (iGluRs) harbor two extracellular domains: the membrane-proximal ligand-binding domain (LBD) and the distal N-terminal domain (NTD). These are involved in signal sensing: the LBD binds L-glutamate, which activates the receptor channel. Ligand binding to the NTD modulates channel function in the NMDA receptor subfamily of iGluRs, which has not been observed for the AMPAR subfamily to date. Structural data suggest that AMPAR NTDs are packed into tight dimers and have lost their signaling potential. Here, we assess NTD dynamics from both subfamilies, using a variety of computational tools. We describe the conformational motions that underly NMDAR NTD allosteric signaling. Unexpectedly, AMPAR NTDs are capable of undergoing similar dynamics; although dimerization imposes restrictions, the two subfamilies sample similar, interconvertible conformational subspaces. Finally, we solve the crystal structure of AMPAR GluA4 NTD, and combined with molecular dynamics simulations, we characterize regions pivotal for an as-yet-unexplored dynamic spectrum of AMPAR NTDs.

Small molecule inhibitors of influenza A and B viruses that act by disrupting subunit interactions of the viral polymerase

Influenza viruses are the cause of yearly epidemics and occasional pandemics that represent a significant challenge to public health. Current control strategies are imperfect and there is an unmet need for new antiviral therapies. Here, we report the identification of small molecule compounds able to effectively and specifically inhibit growth of influenza A and B viruses in cultured cells through targeting an assembly interface of the viral RNA-dependent RNA polymerase. Using an existing crystal structure of the primary protein-protein interface between the PB1 and PA subunits of the influenza A virus polymerase, we conducted an in silico screen to identify potential small molecule inhibitors. Selected compounds were then screened for their ability to inhibit the interaction between PB1 and PA in vitro using an ELISA-based assay and in cells, to inhibit nuclear import of a binary PB1-PA complex as well as transcription by the full viral ribonucleoprotein complex. Two compounds emerged as effective inhibitors with IC(50) values in the low micromolar range and negligible cytotoxicity. Of these, one compound also acted as a potent replication inhibitor of a variety of influenza A virus strains in Madin-Darby canine kidney (MDCK) cells, including H3N2 and H1N1 seasonal and 2009 pandemic strains. Importantly, this included an oseltamivir-resistant isolate. Furthermore, potent inhibition of influenza B viruses but not other RNA or DNA viruses was seen. Overall, these compounds provide a foundation for the development of a new generation of therapeutic agents exhibiting high specificity to influenza A and B viruses.

FE Analysis of Sif of Interfacial Crack Emanating from a Circular Notch in Ceramic/Metal Bi-Materials

Ceramic/metal bimaterials systems are increasingly used in industry owing to the synergetic association of opposite properties of the bonded materials. Discontinuity in these properties can result in crack initiation and stress concentration entailing the bimaterial system failure. A good understanding of damage mechanism at the metal/ceramic interface is crucial to the dimensioning of multilayered systems. In this work the finite element method is used to analyze the stress concentration effect in a linear interface and curved interface. The geometrical configuration of the interface has a great influence the stress concentration on distribution developed near or in the assembly interface. The effect of a circular notch on the behavior of an interfacial crack is highlighted. The results indicate that the interface generates normal and tangential stress concentration resondless of the assembly mechanical properties. The curved geometry of the interface strongly reduces stress concentration in comparison with the linear interface. The reduction is 90%.

Glucose Autoxidation Induces Functional Damage to Proteins via Modification of Critical Arginine Residues

Nonenzymatic modification of proteins in hyperglycemia is a major mechanism causing diabetic complications. These modifications can have pathogenic consequences when they target active site residues, thus affecting protein function. In the present study, we examined the role of glucose autoxidation in functional protein damage using lysozyme and RGD-α3NC1 domain of collagen IV as model proteins in vitro. We demonstrated that glucose autoxidation induced inhibition of lysozyme activity as well as NC1 domain binding to α(V)β(3) integrin receptor via modification of critical arginine residues by reactive carbonyl species (RCS) glyoxal (GO) and methylglyoxal while nonoxidative glucose adduction to the protein did not affect protein function. The role of RCS in protein damage was confirmed using pyridoxamine which blocked glucose autoxidation and RCS production, thus protecting protein function, even in the presence of high concentrations of glucose. Glucose autoxidation may cause protein damage in vivo since increased levels of GO-derived modifications of arginine residues were detected within the assembly interface of collagen IV NC1 domains isolated from renal ECM of diabetic rats. Since arginine residues are frequently present within protein active sites, glucose autoxidation may be a common mechanism contributing to ECM protein functional damage in hyperglycemia and oxidative environment. Our data also point out the pitfalls in functional studies, particularly in cell culture experiments, that involve glucose treatment but do not take into account toxic effects of RCS derived from glucose autoxidation.

ArcheoTUI—Driving virtual reassemblies with tangible 3D interaction

ArcheoTUI is a new tangible user interface for the efficient assembly of the 3D scanned fragments of fractured archeological objects. An efficient user interaction for the complex task to orientate or position two 3D objects relative to each other is essential, eventually in addition to automatic matching techniques. Our key idea is to use tangible props for the manipulation of the virtual fragments. In each hand, the user manipulates an electromagnetically tracked prop, and the translations and rotations are directly mapped to the corresponding virtual fragments on the display. For each hand, a corresponding foot pedal is used to clutch the movements of the hands. Hence, the user's hands can be repositioned, or the user can be switched. The software of ArcheoTUI is designed to easily change assembly hypotheses, beyond classical undo/redo, by using a scene graph. We designed ArcheoTUI on the demand of archeaologists and in a direct collaboration with them, and we conducted two user studies on site at their workplace. The first user study revealed that the interface, and especially the foot pedal, was accepted, and that all the users managed to solve simple assembly tasks. In a second user study, we compare a different clutching mechanism with buttons on the props to the foot pedal mechanism. This second user study revealed that the movement of the hands is more similar to real-world assembly scenarios when using the foot pedals, and that the users can keep on concentrating on the actual assembly task. Finally, we show how the virtual assembly is used for a fractured archeological finding.

Native Electrospray and Electron-Capture Dissociation in FTICR Mass Spectrometry Provide Top-Down Sequencing of a Protein Component in an Intact Protein Assembly

The intact yeast alcohol dehydrogenase (ADH) tetramer of 147 kDa was introduced into a FTICR mass spectrometer by native electrospray. Electron capture dissociation of the entire 23+ to 27+ charge state distribution produced the expected charge-reduced ions and, more unexpectedly, 39 c-type peptide fragments that identified N-terminus acetylation and the first 55 amino acids. The results are in accord with the crystal structure of yeast ADH, which shows that the C-terminus is buried at the assembly interface, whereas the N-terminus is exposed, allowing ECD to occur. This remarkable observation shows promise that a top-down approach for intact protein assemblies will be effective for characterizing their components, inferring their interfaces, and obtaining both proteomics and structural biology information in one experiment.

Elliptical seal interface for a filter assembly

Elliptical seal interface for a filter assembly

Structure of the gating ring from the human large-conductance Ca(2+)-gated K(+) channel.

High-conductance Ca2+-gated K+ (BK) channels are essential for many biological processes such as smooth muscle contraction and neurotransmitter release1-4. This group of channels can be activated synergistically by both voltage and intracellular Ca2+, with the large C-terminal intracellular portion being responsible for Ca2+ sensing5-13. Here we present the crystal structure of the entire cytoplasmic region of the human BK channel in a Ca2+ free state. The structure reveals four intracellular subunits, each comprising two tandem RCK domains, assembled into a gating ring similar to that seen in the MthK channel14 and likely representing its physiological assembly. Three Ca2+ binding sites including the Ca2+ bowl are mapped onto the structure based on mutagenesis data. The Ca2+ bowl, located within the second RCK domain, forms an EF-hand like motif and is strategically positioned close to the assembly interface between two subunits. The other two Ca2+ (or Mg2+) binding sites, Asp367 and Glu374/Glu399, are located on the first RCK domain. The Asp367 site has high Ca2+ sensitivity and is positioned in the groove between the N- and C- terminal subdomains of RCK1, whereas the low affinity Mg2+-binding Glu374/Glu399 site is positioned on the upper plateau of the gating ring and close to the membrane. Our structure also contains the linker connecting the transmembrane and intracellular domains, allowing us to dock a voltage-gated K+ channel pore of known structure onto the gating ring with reasonable accuracy and generate a structural model for the full BK channel.

Singularities at Solder Joint Interfaces and Their Effects on Fracture Models

In this study, we focus on investigating the nature of the stress and strain behavior in solder joints and their effect on the hybrid damage modeling approach, which is inspired by cohesive zone modeling and Weibull functions [Towashiraporn, et al., 2005, “A Hybrid Model for Computationally Efficient Fatigue Fracture Simulations at Microelectronic Assembly Interfaces,” Int. J. Solids Struct., 42(15), pp. 4468–4483]. We review well understood principles in elastic-plastic fracture mechanics and more recent work in cohesive zone modeling, that address the nature of the singular solutions at the crack tip and provide insight when dealing with the more complex problem of solder joint fracture. Using three-dimensional finite element analysis of a chip scale package, we systematically examine the stress-strain behavior at the edge of the solder joint along the interface. The singular nature of the behavior manifests itself as mesh dependence of the predicted crack front shape and the cycles to failure. We discuss the conditions under which the predicted crack growth rate is of reasonable accuracy by incorporating a characteristic length measure. We validate predictions made by the hybrid damage modeling approach against a companion experimental study in which crack growth was tracked in packages subjected to accelerated thermal cycling.

Fire Performance Testing of Building Element Interfaces and Connections

Abstract The minimum fire-resistance ratings for vertical and horizontal assemblies and structural members are provided in the prescriptive requirements of the building codes. National and international test methods have been developed to provide a standardized means for evaluating the performance of these building elements. Building elements such as horizontal assemblies (floors), vertical assemblies (walls), and structural members (beams and columns) are generally tested as discrete elements. In an actual building, the walls intersect with the floors and beams and girders are connected to columns. In light of recent high-profile fire events, the fire community has been questioning if these connections and interfaces are being properly tested and evaluated. In an actual building, these building elements are interconnected, and their performance is affected by all elements that comprise the building structure during a real fire event. This paper will review common structural building interface and connection construction features, how they are currently being evaluated, and how their large-scale fire performance evaluations can be conducted. The results of the testing of a simulated wall/floor assembly interface assembly will be presented to highlight one potential means for testing these features along with the limitations mainly due to the available test equipment.

Designer Peptides: Attempt to Control Peptide Structure by Exploiting Ferrocene as a Scaffold

AbstractThe design of peptides that display a β‐sheet‐like secondary structure remains a challenge. Molecular scaffolds, such as ferrocene (Fc) can help to overcome some of these challenges. Monosubstituted Fc derivatives offer no control over the self‐assembly of the conjugates, and the formation of supramolecular structures is entirely serendipitous. 1,n′‐disubstituted Fc derivatives provide a significant level of control over the direction of the peptide, their relative orientation, and the intramolecular hydrogen‐bonding patterns, which increases the rigidity of the molecule and in many cases, generates a new H‐bonding interface for intermolecular assembly. In this Microreview, we explore the use of ferrocene as a scaffold in the design of structurally well‐defined peptide conjugates. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Orthogonal Enzymatic Reactions for the Assembly of Proteins at Electrode Addresses

The ability to interface proteins to device surfaces is important for a range of applications. Here, we enlist the unique capabilities of enzymes and biologically derived polymers to assemble target proteins to electrode addresses. First, the stimuli-responsive aminopolysaccharide chitosan is directed to assemble at the electrode address in response to electrode-imposed signals. The electrodeposited chitosan film serves as the biodevice interface for subsequent protein assembly. Next, tyrosinase is used to catalyze grafting of a protein or peptide tether to the chitosan film. Finally, microbial transglutaminase (mTG) catalyzes the assembly of target proteins to the tether. mTG covalently links proteins through their glutamine (Gln) and lysine (Lys) residues. Since Gln and Lys residues of globular proteins are often inaccessible to mTG, we engineered our target proteins to have fusion tags with added Gln or Lys residues. This assembly method employs the electrical signal to confer spatial selectivity (during chitosan electrodeposition) and employs the enzymes to confer chemical selectivity (i.e., amino acid residue selectivity). Further, this method is mild, since no reactive reagents or protection steps are required, and all steps are performed in aqueous solution. These results demonstrate the potential for employing biological materials and mechanisms to biofabricate the biodevice interface.

Web-based modular interface geometries with constraints in assembly models

The use of a modular technique in modeling for assembly can improve design efficiency and reduce the cost of product development. This paper presents an approach to an assembly model that was built using modular interface geometries. This paper proposes a novel, hybrid modular design strategy instead of the traditional, top-down design process. The curve-joint method is used as a simplified process for converting a 3D solid model to a skeleton model with interface geometries in modeling for assembly. This research builds assembly interface geometries with their constraints in the assembly model instead of using information about individual assembly parts for the product. These interface geometries are easy to share, and they deliver the design requirements properly. They also ensure that minimal efforts will be required in the design change process. By implementing this method, the constraints of the features in modular assembly parts can be transferred to interface geometries. Designers can easily add, replace, and delete design parts in the modular product. Module interaction for application programmed interface (MIAPI) is developed using HTML and JavaScript. The module structure of products can be verified via the web-based Internet in VRML format. These simplified assembly models that have fewer constraints allow design project managers to simulate the functioning of the product in the modularized design before the prototype is built. By using the assembly models, customers can easily choose various modules to assemble the exact products they are seeking via the Internet process. A desk lamp model is used as the example for implementation to validate the feasibility of this research.

Modulation of the Conductance-Voltage Relationship of the BK Ca Channel by Mutations at the Putative Flexible Interface between Two RCK Domains

Calcium-dependent gating of the large-conductance Ca 2+-activated K + (BK Ca) channel is conferred by the large cytosolic carboxyl terminus containing two domains of the regulator of K + conductance (RCK) and the high-affinity Ca 2+-binding site (the Ca 2+-bowl). In our previous study, we located the putative second RCK domain (RCK2) and demonstrated that it interacts directly with RCK1 via a hydrophobic “assembly interface”. In this study, we tested the structural model of the other interface, the “flexible interface”, by strategically positioning charge pairs across the putative interface. Several charge mutations on RCK2 affected the voltage-dependent activation of the channel. In particular, the Gly-to-Asp substitution at position 803 profoundly affected channel activation by stabilizing the open conformation of the channel with minimal effects on its Ca 2+ affinity and voltage sensitivity. Various mutations at Gly-803 shifted the channel's conductance-voltage curve either left or right over a 145-mV range. Since this residue is predicted to be in the first loop of RCK2 these results strongly suggest that this loop plays a critical role in determining the intrinsic equilibrium constant for channel opening, and they support the hypothesis that this loop is part of an interface that mediates conformational coupling between RCK1 and RCK2.