Assembly Motif Research Articles

Assembling viral channel forming proteins: Vpu from HIV‐1

Different routes of assembly are probed for the transmembrane domain (TMD) of the bitopic membrane protein Vpu from HIV-1. Vpu is responsible for the amplification of viral release from the host cell. The mode of action includes (i) heteroassembly with host factors and (ii) the formation of homo-oligomers, which are able to conduct ions across the lipid membrane. Two different routes of assembling short sequences of the N terminus, including the TMD of Vpu, Vpu1-32, and Vpu8-26, are presented by using a combination of classical molecular dynamics (MD) simulations combined with a docking approach. The rim of alanines (Ala-8, -11, -15, and -19) resembles an interlocking motif for the sequential assembly into a dimer and trimer. Simultaneous assembly results in oligomeric bundles (trimers to pentamers) with either tryptophans (Trp-23) or purely hydrophobic residues facing the center. Bundles, with serines facing the pore (Ser-24), are energetically not the lowest structures. For pentameric bundles with Ser-24 facing the pore, no water column develops during a short 25 ns MD simulation.

Structural transitions of tin clusters: Snn (n = 34–44)

Snn (n=34–44) clusters were studied using a combined method of the genetic algorithm/tight-binding search and the DFT–PBEPBE calculations. Snn (n=34–44) clusters are shown to favor an assembly motif composed of small stable Sn9 and Sn10 subunits which are connected by cohesive linkages of smaller clusters (Sn1–5). Snn (n=34–44) clusters were found to transform from a prolate pattern for Sn34,35 to a ‘Y’ pattern for Sn36–39 to a plate motif for Sn40–44. Obvious structural transitions would easily occur at the cross points of binding energy curves when isomers become competitive.

Disruption of gingipain oligomerization into non-covalent cell-surface attached complexes

RgpA and Kgp gingipains are non-covalent complexes of endoprotease catalytic and hemagglutinin-adhesin domains on the surface of Porphyromonas gingivalis. A motif conserved in each domain has been suggested to function as an oligomerization motif. We tested this hypothesis by mutating motif residues to hexahistidine or insertion of hexahistidine tag to disrupt the motif within the Kgp catalytic domain. All modifications led to the secretion of entire Kgp activity into the growth media, predominantly in a form without functional His-tag. This confirmed the role of the conserved motif in correct posttranslational proteolytic processing and assembly of the multidomain complexes.

Learning to capture the functions of genetic regulatory networks using graph motifs

Network motifs have been suggested as a means to understand Systems biological functions. Based on several arguments from computer science, logic, mathematics, chemistry and non-linear dynamics, we explain the difficulties encountered in capturing the evolving dynamical functions carried out by the genetic regulatory networks (GRN), based on the assembly of motifs.

X-Ray Crystallographic Investigation of Fully Acetylated N-(2-Deoxy-2-Acetamido-β-D-Glucopyranosyl)Alkanamides as N-Glycoprotein Linkage Region Analogs

To understand the structural significance of the linkage region of N-glycoproteins, three title sugar amides have been prepared as analogs and their molecular assembly and crystal structures have been solved to explore the effect of acetyl protection and aglycon variation on the conformation, particularly of the N-glycosidic linkage. Comparative analysis of these structures with those of free sugar amides reported earlier showed that conformation of the amido aglycon moiety is not altered significantly by the masking of hydroxyl groups in the form of acetate. The bifurcated antiparallel pattern involving N‒H…O and C‒H…O hydrogen bonds, a hallmark of the N-glycoprotein models GlcNAcβNHAc and GlcNAcβAsn, is absent in all of the fully protected title alkanamides. The asymmetric unit of the tri-O-acetylated GlcNAcβNHAc contains two different conformations, in one of which the double-pillared hydrogen bond network involving C1 and C2 acetamido groups is antiparallel, while it is parallel in the other. The co-occurrence of a molecular assembly motif—a double-pillared parallel and antiparallel hydrogen bonding pattern—is hitherto unknown in the crystal structures of N-glycoprotein linkage region models and analogs.

Mapping GABAA Receptor Single Nucleotide Polymorphisms (SNPs) Linked to Epilepsy: Insights into the Receptor Gating and Assembly

Missense polymorphisms or variants that affect function and/or expression of GABAA receptors (GABARs) have been associated with idiopathic generalized epilepsies (IGEs) in GABRA1, GABRA6, GABRB3, GABRG2 and GABRD genes. Three separate IGE-associated mutations were identified in GABAR subunits (β3(G32R), α6(Q237R) and γ2(K328M)), which represent a large range of GABARs in the nervous system. We sought to investigate the contributions of these mutations into the assembly and function of GABARs. Homology modeling suggested that G32R is located within the N-terminal α-helix β3 subunit domain, and Q237R and K328M are located within the pre-M1 segment of the N-terminal α6 subunit domain and the M2-M3 loop of the N-terminal γ2 subunit domain, respectively. We studied gating properties and surface expression of wild type (wt) α1β3γ2, α6β2γ2, α1β2γ2 and mutant α1β3(G32R)γ2, α6(Q237R)β2γ2, α1β2γ2(K328M) receptors expressed in HEK293T cells. We found that the mutations share common gating defects, but distinctive trafficking defects. Thus, mutant β3(G32R) subunits displayed a mixed profile, causing both gating and trafficking defects of α1β3γ2 receptors, whereas mutant α6(Q237R) and γ2(K328M) subunits caused exclusive channel gating defects of α6β2γ2 and α1β2γ2 receptors. Unexpected, homology modeling predicted that the β3(G32R) mutation affects a salt bridge across the γ2/β3 subunit interface, which underlies an assembly motif reported to be essential for inter-subunit interactions in assembled receptors. In contrast, α6(Q237R) and γ2(K328M) subunit mutations are predicted to interact with residues in Pre-M1 domain, M2-M3 loop and Cys-loop of α6 and γ2 subunits that are critical for desensitization-deactivation coupling of GABARs.NIH funding NS33300 to RLM.

Aminophenylnitronylnitroxides: Highly Networked Hydrogen-Bond Assembly in Organic Radical Materials

2-(Meta-aminophenyl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl (mAPN) and 2-(para-aminophenyl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl (pAPN) were synthesized and subjected to magnetostructural analysis. Both form extended hydrogen bonding networks involving both amino NH bonds to radical spin-density bearing nitronylnitroxide NO groups. Their crystallographic assembly motifs and magnetic exchange properties are compared to those of tert-butoxylcarbonyl (BOC) and amide derivatives having only one NH bond. The conversion of pAPN to acid salt derivatives gives a solid that is essentially diamagnetic, although dissolution of the solid shows the radical spin units to be preserved.

Anticlinic smectic phase formed by calamitic hydrazide derivatives with terminal hydroxyl group

A new series of liquid crystalline hydrazide derivatives N-4-(alkoxyphenyl)-N'-4-[(4-hydroxyphenyl)azophenyl] benzohydrazide (BNB-n, n = 8, 12, 14) was designed and synthesised. Based on the results of variable temperature 1H NMR and FT-IR spectroscopy, a supramolecular assembly motif of BNB-n through both axial intermolecular hydrogen bonds between hydroxyl groups and lateral hydrogen bonds between hydrazide groups was proposed. Interestingly, BNB-n (n = 12, 14) with longer terminal chains displayed anticlinic bilayer smectic C mesophase (SmC2a). In order to reveal the effect of axial hydrogen bonding between hydroxyl groups on the formation of this unique mesophase, the hydroxyl group was replaced by a methoxy group to form N-4-(alkoxyphenyl)-N'-4-[(4-methoxyphenyl)azophenyl] benzohydrazide (BNBC-n, n = 12, 14). However, BNBC-n showed synclinic monolayer smectic C (SmCs) features, suggesting that the hydroxyl groups in BNB-n have an important role in the formation of anticlinic molecular packing in the smectic layers.

Role for a Conserved Structural Motif in Assembly of a Class I Aminoacyl-tRNA Synthetase Active Site

The catalytic domains of class I aminoacyl-tRNA synthetases are built around a conserved Rossmann nucleotide binding fold, with additional polypeptide domains responsible for tRNA binding or hydrolytic editing of misacylated substrates. Structural comparisons identified a conserved motif bridging the catalytic and anticodon binding domains of class Ia and Ib enzymes. This stem contact fold (SCF) has been proposed to globally orient each enzyme's cognate tRNA by interacting with the inner corner of the L-shaped tRNA. Despite the structural similarity of the SCF among class Ia/Ib enzymes, the sequence conservation is low. We replaced amino acids of the MetRS SCF with portions of the structurally similar glutaminyl-tRNA synthetase (GlnRS) motif or with alanine residues. Chimeric variants retained significant tRNA methionylation activity, indicating that structural integrity of the helix-turn-strand-helix motif contributes more to tRNA aminoacylation than does amino acid identity. In contrast, chimeras were significantly reduced in methionyl adenylate synthesis, suggesting a role for the SCF in formation of a structured active site domain. A highly conserved aspartic acid within the MetRS SCF is proposed to make an electrostatic interaction with an active site lysine; these residues were replaced with alanines or conservative substitutions. Both methionyl adenylate formation and methionine transfer were impaired, and activity was not significantly recovered by making the compensatory double substitution.

Side‐by‐Side and End‐to‐End Gold Nanorod Assemblies for Environmental Toxin Sensing

Controllable assembly of nanoscale building blocks (monomers) is a necessary part of practical realization of the unique optical, electrical, magnetic, and chemical properties of nanoscale matter in macroscale materials. Such assemblies also contain much fundamental information about collective behavior of nanocolloids, which we are just beginning to understand. The key decisive factors for the successful assembly of nanocolloids is the anisotropy of nanoscale interactions, which stems from both the shape of nanocolloids and unequal distribution of organic molecules on their surface. Gold nanorods (Au NRs) have both geometrical and chemical anisotropy components and demonstrate strong optical extinction in the range of visible and near-infrared (NIR) wavelengths convenient for both research and practical purposes. Au NRs can be assembled by interactions with organic molecules, polymers, an antibody–antigen reaction, biotin–strepavidin connectors, and DNA, leading to superstructures with different degree of organization and complexity of collective behavior. Besides the utilization of NR monomers in non-linear optics, cellular imaging, and cancer therapy, optical effects corresponding to monomer!superstructure transitions allowed preparation of excellent biosensors because of large changes in oscillation frequencies of plasmons when NR pairs are formed. These studies mostly targeted biomedical applications. Simultaneously, their unique sensing capabilities have been virtually unexplored for the needs of environmental detection and monitoring. These challenges and impact can equal or exceed those encountered in detection of cancer. A better understanding of methods for the realization of speed/selectivity/sensitivity detection of common environmental pollutants is thus of great importance. Therefore, we decided to explore the potential of NR assemblies taking a pervasive environmental toxin, namely microcystin-LR (MC-LR), as the model while also addressing the general questions about the choice of different assembly motif for different sensing tasks. MC-LR is common in both developed and developing countries, with recorded cases of mass poisoning. MC-LR originates from common bluegreen algae and causes rapid liver failure; prolonged exposure to small concentrations of MC-LR in drinking water causes liver cancer. Herein, we describe the successful use of Au NRs for detection of MC-LR, which is significantly more sensitive than the traditional techniques, such as ELISA, yielding detection limit of 5 pgmL . It is also much simpler and faster than any other methods. These two factors are critical for environmental monitoring and have been a long-standing challenge. The pattern of the assembly strongly affects the sensitivity parameters for MC-LR detection. To realize different modes of assembly, such as side-byside and end-to-end motifs, with a degree of control sufficient for conclusive evaluation of sensing implications, two kinds of protein-carrying Au NRs were synthesized (Figure 1). One type of NR carried MC-LR antibodies (ABs) preferentially on the sides, while the other type carried antibodies located almost exclusively in the ends. These motifs were formed by using either electrostatic binding or covalent attachment of the antibodies mediated by a bifinctional linker, thioctic acid (TA).When electrostatic forces govern the placement of ABs, they attach primarily to the sides of NRs due to the larger area of contact and thus stronger electrostatic interactions. When a TA anchor covalently binds by a S Au bond, the conjugation of the ABs occurs predominantly in the ends of the rods due to better accessibility of the gold surface to the reactive thiol end. Variation of pH also allows varying repulsion or attraction of NRs and MC-LR, modality of attachment, and geometrical characteristics of assemblies (see the Supporting Information). The number of AB molecules on the surface of one Au NR was estimated to be 31 and 10 for the side-by-side [*] L. Wang, Y. Zhu, L. Xu, Dr. W. Chen, H. Kuang, L. Liu, Prof. C. Xu School of Food Science and Technology, State Key Laboratory of Food Science and Technology Jiangnan University, Wuxi, 214122 (China) E-mail: xcl@jiangnan.edu.cn Dr. W. Chen, A. Agarwal, Prof. N. A. Kotov Department of Chemical Engineering, Department of Biomedical Engineering Department of Materials Science and Engineering University of Michigan, Ann Arbor, MI 48109 (USA) E-mail: kotov@umich.edu [] These authors contributed equally to this paper.

Non-Traditional Aromatic Topologies and Biomimetic Assembly Motifs as Components of Functional Pi-Conjugated Oligomers

This article will highlight our recent work using conjugated oligomers as precursors to electroactive polymer films and self-assembling nanomaterials. One area of investigation has focused on nonbenzenoid aromaticity in the context of charge delocalization in conjugated polymers. In these studies, polymerizable pi-conjugated units were coupled onto unusual aromatic cores such as methano[10]annulene. This article will also show how biologically-inspired assembly of molecularly well-defined oligopeptides that flank pi-conjugated oligomers has resulted in the aqueous construction of 1-dimensional nanomaterials that encourage electronic delocalization among the pi-electron systems.

Structural Transitions Associated with the Assembly Dynamics of Transthyretin Amyloid Protofibrils

Transthyretin (TTR) is a serum protein involved in the transport of thyroxine and co-transport of retinol. Several neurodegenerative diseases are associated with the aggregation of TTR into amyloid fibrils, but the molecular mechanisms of the assembly remain unknown. Here we used AFM to identify intermediates along the assembly-disassembly of TTR amyloid protofibrils. By using dynamic force spectroscopy, we also characterized the mechanical response of protofibrils at different growth stages before and after their partial disassembly.During TTR assembly, annular oligomers with 15.8 ± 2.3 nm in diameter appeared after 3-5 days of incubation. These annular oligomers displayed a tendency to associate laterally, forming linear structures that preceded the appearance of amyloid protofibrils. In other proteins, similar annular oligomeric structures have been implicated in amyloid cytotoxicity, but it remains unclear to which extent they represent an on pathway intermediate. Upon solvent exchange, dramatic morphological rearrangements occurred leading to the partial disassembly of protofibrils. During disassembly, annular oligomers with 7.0 ± 0.6 nm appeared, suggesting that the annular arrangement is a common motif in the TTR assembly and disassembly pathways. Force spectroscopy of both native TTR and early protofibrils contained a sawtooth pattern with ∼4 nm periodicity. Conceivably, this pattern corresponds to successive structural transitions related to the sequential unfolding of the s-strands within the TTR monomer. Force spectra of TTR protofibrils also revealed a time-dependent increase in the length of the manipulated structure, indicating that the axial association between monomers stabilizes with time. Thus, stabilization of intermonomeric contacts appears to be a much slower process than that of mere assembly, possibly involving additional structural rearrangements of the monomer within the protofibrils.

Size and geometry dependent protein–nanoparticle self-assembly

Fundamentally different assembly motifs are observed when proteins of different sizes are complexed with monolayer-protected nanoparticles.

Examination of the Structural Features That Favor the Columnar Self-Assembly of Bis-urea Macrocycles

Second-generation self-assembling bis-urea macrocycles were designed that form columnar structures in the solid state. The new macrocycles were constructed from more flexible building blocks yielding greater solubility and a more efficient synthesis. In addition, heteroatoms in the form of ether oxygens were incorporated in the walls of the macrocycles to provide additional recognition sites for guest encapsulation. We observed reduced fidelity of the stacking motif and in some cases the intermolecular urea-urea hydrogen bonds were disrupted by the formation of intramolecular hydrogen bonds. We also observed new offset assembly motifs that maintained the urea-urea interaction. These results suggest that the stacking of the arene units in the rigid first-generation systems was an important factor in guiding the formation of the columnar stacks.

Specific RNA Self-Assembly with Minimal Paranemic Motifs

The paranemic crossover (PX) is a motif for assembling two nucleic acid molecules using Watson-Crick (WC) basepairing without unfolding preformed secondary structure in the individual molecules. Once formed, the paranemic assembly motif comprises adjacent parallel double helices that crossover at every possible point over the length of the motif. The interaction is reversible as it does not require denaturation of basepairs internal to each interacting molecular unit. Paranemic assembly has been demonstrated for DNA but not for RNA and only for motifs with four or more crossover points and lengths of five or more helical half-turns. Here we report the design of RNA molecules that paranemically assemble with the minimum number of two crossovers spanning the major groove to form paranemic motifs with a length of three half turns (3HT). Dissociation constants (Kd's) were measured for a series of molecules in which the number of basepairs between the crossover points was varied from five to eight basepairs. The paranemic 3HT complex with six basepairs (3HT_6M) was found to be the most stable with Kd = 1 x 10-8 M. The half-time for kinetic exchange of the 3HT_6M complex was determined to be approximately 100 min, from which we calculated association and dissociation rate constants ka = 5.11 x 103 M-1s-1 and kd = 5.11 x 10-5 s-1. RNA paranemic assembly of 3HT and 5HT complexes is blocked by single-base substitutions that disrupt individual intermolecular Watson-Crick basepairs and is restored by compensatory substitutions that restore those basepairs. The 3HT motif appears suitable for specific, programmable, and reversible tecto-RNA self-assembly for constructing artificial RNA molecular machines.

Role of the IXI/V motif in oligomer assembly and function of StHsp14.0, a small heat shock protein from the acidothermophilic archaeon, Sulfolobus tokodaii strain 7

Small heat shock proteins (sHsps) are one of the most ubiquitous molecular chaperones. They are grouped together based on a conserved domain, the alpha-crystallin domain. Generally, sHsps exist as oligomers of 9-40 subunits, and the oligomers undergo reversible temperature-dependent dissociation into smaller species as dimers, which interact with denaturing substrate proteins. Previous studies have shown that the C-terminal region, especially the consensus IXI/V motif, is responsible for oligomer assembly. In this study, we examined deletions or mutations in the C-terminal region on the oligomer assembly and function of StHsp14.0, an sHsp from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Mutated StHsp14.0 with C-terminal deletion or replacement of IIe residues in the IXI/V motif to Ala, Ser, or Phe residues could not form large oligomers and lost chaperone activity. StHsp14.0WKW, whose Ile residues in the IXI/V motif are changed to Trp, existed as an oligomer like that of the wild type. However, it dissociates to small oligomers and exhibits chaperone activity at relatively lowered temperature. Replacement of two Ile residues in the motif to relatively small residues, Ala or Ser, also resulted in the change of beta-sheet rich secondary structure and decrease of hydrophobicity. Interestingly, StHsp14.0 mutant with amino acid replacements to Phe kept almost the same secondary structure and relatively high hydrophobicity despite that it could not form an oligomeric structure. The results show that hydrophobicity and size of the amino acids in the IXI/V motif in the C-terminal region are responsible not only for assembly of the oligomer but also for the maintenance of beta-sheet rich secondary structure and hydrophobicity, which are important for the function of sHsp.

Comparative modeling of marsupial MHC class I molecules identifies structural polymorphisms affecting functional motifs

Major histocompatibility complex (MHC) class I molecules are transmembrane glycoproteins that present antigenic peptides to CD8+ T cells and are subsequently important for the initiation of an immune response. In this study novel MHC class I sequences from the tammar wallaby (Macropus eugenii) have been characterized. Analysis and comparative modeling of these and existing marsupial molecules reveals potential functional polymorphisms within peptide-binding grooves, MHC assembly motifs and the T cell receptor recognition interface. In addition, we show that a previously identified marsupial-specific insertion is within a region, which is known as a putative NK cell receptor (Ly49A) binding site in the mouse, suggesting that this site may be functionally active in marsupials. Further, the analysis highlighted differences in structural and sequence based grouping of marsupial MHC class I molecules.

Defining Self-Assembling Linear Oligo(dioxaborole)s

A route to self-assembling linear oligo(dioxaborole)s and detailed characterization using Fourier transform infrared and 1H NMR spectroscopies compared to small molecule analogues is described. The oligomer formed through the reaction between benzene-1,4-diboronic acid and 1,2,4,5-tetrahydroxybenzene is linked via covalent yet reversible cyclic boronate esters. Comparison of the spectral data for this oligomer compared to bis(dioxaborole) (diester) and anhydride containing model compounds forms a basis on which to derive information about bonding outcomes for larger polymeric systems from small molecule analogues. From this analysis, it is clear that caution must be exercised when evaluating the spectroscopic signatures related to certain boronate containing species because they are not always obvious and are highly dependent on molecular structure and environmental factors such as solvent. X-ray diffraction data along with 11B NMR and molecular modeling confirmed the planarity of the bis(dioxaborole)s and provided adequate information to propose a supramolecular π-stacking orientation for the oligomeric compounds. Careful interpretation of information obtained about small molecule boronate esters in the structural analysis of macromolecular structures creates a foundation for developing and analyzing self-assembling polymers and covalent organic frameworks on the basis of this assembly motif.

Peptide α-helices for synthetic nanostructures

Supramolecular structures arising from a broad range of chemical archetypes are of great technological promise. Defining such structures at the nanoscale is crucial to access principally new types of functional materials for applications in bionanotechnology. In this vein, biomolecular self-assembly has emerged as an efficient approach for building synthetic nanostructures from the bottom up. The approach predominantly employs the spontaneous folding of biopolymers to monodisperse three-dimensional shapes that assemble into hierarchically defined mesoscale composites. An immediate interest here is the extraction of reliable rules that link the chemistry of biopolymers to the mechanisms of their assembly. Once established these can be further harnessed in designing supramolecular objects de novo. Different biopolymer classes compile a rich repertoire of assembly motifs to facilitate the synthesis of otherwise inaccessible nanostructures. Among those are peptide alpha-helices, ubiquitous folding elements of natural protein assemblies. These are particularly appealing candidates for prescriptive supramolecular engineering, as their well-established and conservative design rules give unmatched predictability and rationale. Recent developments of self-assembling systems based on helical peptides, including fibrous systems, nanoscale linkers and reactors will be highlighted herein.

Perspectives on main‐chain hydrogen bonded supramolecular polymers

AbstractSupramolecular polymers are assembled from monomeric units held together by reversible non‐covalent interactions. These supramolecular materials display polymeric properties and may soon have important industrial applications. This mini review focuses on the advances in main‐chain supramolecular polymers whose assembly is guided primarily by hydrogen bonding interactions. The design constraints of these new systems discussed include assembly motifs, the strength and directionality of the non‐covalent interactions, association versus reversibility, and environmental effects on the degree of polymerization. Selected literature examples including Meijer's ureidopyrimidinone system are used to highlight the challenges and potential of these supramolecular polymeric materials. Copyright © 2007 Society of Chemical Industry