Coupling Motif Research Articles

Tuning the Diradical Character of Pentacene Derivatives via Non-Benzenoid Coupling Motifs.

The development of functional organic molecules requires structures of increasing size and complexity, which are typically obtained by the covalent coupling of smaller building blocks. Herein, with the aid of high-resolution scanning tunneling microscopy/spectroscopy and density functional theory, the coupling of a sterically demanded pentacene derivative on Au(111) into fused dimers connected by non-benzenoid rings was studied. The diradical character of the products was tuned according to the coupling section. In particular, the antiaromaticity of cyclobutadiene as the coupling motif and its position within the structure play a decisive role in shifting the natural orbital occupancies toward a stronger diradical electronic character. Understanding these structure-property relations is desirable not only for fundamental reasons but also for designing new complex and functional molecular structures.

An extended scale-free network evolution model based on star-like coupling motif embedding

As the only brand-new basic model that can study networks with scale change characteristics at present, the Barabási–Albert scale-free network evolution model (BA model) can well describe the entry phenomenon of a single subject. However, for many evolutionary networks in the real world, the entry of the subject is more realized in a team way. Therefore, the BA model’s assumption concerning one new node entering alone per unit of time can only be used as a particular case of a team entry. This deficiency makes it difficult for the BA model to analyze how the team entry mechanism affects the network’s performance and thus leads to the BA model’s certain limitations. To make up for this shortcoming, with the help of the motif’s concept, an extended BA model with a star-like coupling motif embedding (SCME-BA model) is constructed, where the motif’s scale obeys an arbitrary discrete probability distribution. The exact explicit expression of the network’s steady-state degree distribution of the SCME-BA model is first obtained by the Markov chain analytic method, and its numerical characteristics are explored. Then, the correctness of the analytic expression is verified by numerical simulation. The study results show that, compared with the BA model, the left tail of the SCME-BA model’s steady-state degree distribution can reflect the distribution law of the embedded motif scale well while the right one still has power-law attenuation characteristics whose power-law exponent is adjustable. Specifically, the power-law exponent is positively and negatively correlated with the expectation of the motif’s scale and the initial number of existing nodes connected to the new embedded motif, respectively. Moreover, the SCME-BA model can degenerate into the BA model when the motif’s scale is subject to the one-point distribution with parameter 1. Finally, the rationality of the SCME-BA model is verified through a case study.

An extended scale-free network evolution model based on global coupling motif embedding

The BA scale-free network evolution model assumes that one node enters at every unit time, which does not adequately reflect team entries that usually occur during the evolution of many practical networks, i.e. the phenomenon of motif embedment. Unfortunately, there are no specific studies on how the motif embedment mechanism affects the degree distribution of networks. In order to solve this problem, an extended scale-free network evolution model with global coupling motif embedment and with the motif size obeying an arbitrary discrete probability distribution (called the MEEBA model for short) is formulated with the help of the ‘motif’ concept. Using the Markov chain method, the accurate analytical expression of the network degree distribution of the MEEBA model is obtained and the correctness of the equation is verified through comparisons with numerical simulation results. The study results show that the right tail of the degree distribution of the MEEBA model still has a power-law behavior, while its left tail reflects the horse-head-like shapes of the degree distribution of many real networks. Furthermore, the power-law exponent and the horse-head shapes are both related to the distribution of the motif size. When the motif size follows a one-point distribution, the network degree distribution of the MEEBA model degenerates into a two-parameter Waring-like distribution and its horse-head shapes disappear. In particular, if the expectation of the one-point distribution is 1, the MEEBA model further degenerates to the BA model. Finally, the practicality and effectiveness of the MEEBA model are verified through a case study.

Molecular basis of chromatin remodeling by Rhp26, a yeast CSB ortholog

CSB/ERCC6 belongs to an orphan subfamily of SWI2/SNF2-related chromatin remodelers and plays crucial roles in gene expression, DNA damage repair, and the maintenance of genome integrity. The molecular basis of chromatin remodeling by Cockayne syndrome B protein (CSB) is not well understood. Here we investigate the molecular mechanism of chromatin remodeling by Rhp26, a Schizosaccharomyces pombe CSB ortholog. The molecular basis of chromatin remodeling and nucleosomal epitope recognition by Rhp26 is distinct from that of canonical chromatin remodelers, such as imitation switch protein (ISWI). We reveal that the remodeling activities are bidirectionally regulated by CSB-specific motifs: the N-terminal leucine-latch motif and the C-terminal coupling motif. Rhp26 remodeling activities depend mainly on H4 tails and to a lesser extent on H3 tails, but not on H2A and H2B tails. Rhp26 promotes the disruption of histone cores and the release of free DNA. Finally, we dissected the distinct contributions of two Rhp26 C-terminal regions to chromatin remodeling and DNA damage repair.

Scaffold-Induced Diketopyrrolopyrrole Molecular Stacks in a Covalent Organic Framework.

In recent years, covalent organic frameworks (COFs) have attracted considerable attention due to their crystalline and porous nature, which positions them as intriguing candidates for diverse applications such as catalysis, sensing, or optoelectronics. The incorporation of dyes or semiconducting moieties into a rigid two-dimensional COF can offer emergent features such as enhanced light harvesting or charge transport. However, this approach can be challenging when dealing with dye molecules that exhibit a large aromatic backbone, since the steric demand of solubilizing side chains also needs to be integrated into the framework. Here, we report the successful synthesis of DPP2-HHTP-COF consisting of diketopyrrolopyrrole (DPP) diboronic acid and hexahydroxytriphenylene (HHTP) building blocks. The well-known boronate ester coupling motif guides the formation of a planar and rigid backbone and long-range molecular DPP stacks, resulting in a highly crystalline and porous material. DPP2-HHTP-COF exhibits excellent optical properties including strong absorption over the visible spectral range, broad emission into the NIR and a singlet lifetime of over 5 ns attributed to the formation of molecular stacks with J-type interactions between the DPP subcomponents in the COF. Electrical conductivity measurements of crystalline DPP2-HHTP-COF pellets revealed conductivity values of up to 10–6 S cm–1.

Allosteric Signaling Is Bidirectional in an Outer-Membrane Transport Protein

In BtuB, the Escherichia coli TonB-dependent transporter for vitamin B12, substrate binding to the extracellular surface unfolds a conserved energy coupling motif termed the Ton box into the periplasm. This transmembrane signaling event facilitates an interaction between BtuB and the inner-membrane protein TonB. In this study, continuous-wave and pulse electron paramagnetic resonance in a native outer-membrane preparation demonstrate that signaling also occurs from the periplasmic to the extracellular surface in BtuB. The binding of a TonB fragment to the periplasmic interface alters the configuration of the second extracellular loop and partially dissociates a spin-labeled substrate analog. Moreover, mutants in the periplasmic Ton box that are transport-defective alter the binding site for vitamin B12 in BtuB. This work demonstrates that the Ton box and the extracellular substrate binding site are allosterically coupled in BtuB, and that TonB binding may initiate a partial round of transport.

Site-Directed Spin Labeling Reveals Multiple Modes for Regulating Protein-Protein Interactions in Bacterial Outer Membrane Transport

Structural transitions in three outer-membrane bacterial transport proteins have been investigated using site-directed spin labeling and EPR spectroscopy. In the vitamin B12 transporter BtuB and the ferric citrate transporter FecA, EPR spectra reveal the existence of a substrate-dependent order-to-disorder transition in the energy coupling motif (Ton box), which is localized at the periplasmic surface of the transporter. In BtuB, the Ton box unfolds into the periplasm, and in FecA, an N-terminal transcriptional domain disengages from the Ton box. Both these events expose the Ton box and initiate interactions with the inner membrane protein TonB, which drives transport. However, this disorder transition is not observed in all transporters, and in the ferrichrome transporter FhuA the EPR spectra indicate that the Ton box is constitutively disordered. Distance measurements using double electron-electron resonance (DEER) indicate how the FhuA Ton box is regulated, and each transporter regulates interactions with the inner membrane protein TonB using a distinct molecular mechanism. This work indicates that a variety of mechanisms may used to regulate protein-protein interactions in a single transporter family.

Energy Transduction Between Membranes: Characterization of the Interaction Between TonB and its Cognate Transporters

In gram-negative bacteria, essential nutrients such as iron and cobalamins are too large to diffuse through porins in the outer membrane and therefore require an active transport mechanism for their uptake. Since there is no source of energy at the outer membrane, this is a complicated task that involves coupling of the outer membrane transporter to the protonmotive force across the cytoplasmic membrane via the proteins ExbB, ExbD and TonB. Presumably, ExbB and ExbD utilize this gradient to conformationally energize TonB, which spans the periplasm and associates with the transporters in the outer membrane to deliver the energy that is required for transport. In this study, we use fluorescence anisotropy to measure the affinity of a TonB fragment (lacking the transmembrane domain) for three of its cognate transporters--BtuB, FhuA, and FecA. Additionally, we determine the effect of substrates, Colicin E3, and transport-defective mutations to the coupling motif (Ton box) on the affinity of this interaction. The oligomeric state and structure of TonB in solution and bound to the transporters will also be discussed. These results have implications for the development of new antibiotics that interfere with the TonB-transporter interaction.

Poly(2-hydroxyethyl methacrylate) for Enzyme Immobilization: Impact on Activity and Stability of Horseradish Peroxidase

On the basis of their versatile structure and chemistry as well as tunable mechanical properties, polymer brushes are well-suited as supports for enzyme immobilization. However, a robust surface design is hindered by an inadequate understanding of the impact on activity from the coupling motif and enzyme distribution within the brush. Herein, horseradish peroxidase C (HRP C, 44 kDa), chosen as a model enzyme, was immobilized covalently through its lysine residues on a N-hydroxysuccinimidyl carbonate-activated poly(2-hydroxyethyl methacrylate) (PHEMA) brush grafted chemically onto a flat impenetrable surface. Up to a monolayer coverage of HRP C is achieved, where most of the HRP C resides at or near the brush-air interface. Molecular modeling shows that lysines 232 and 241 are the most probable binding sites, leading to an orientation of the immobilized HRP C that does not block the active pocket of the enzyme. Michaelis-Menten kinetics of the immobilized HRP C indicated little change in the K(m) (Michaelis constant) but a large decrease in the V(max) (maximum substrate conversion rate) and a correspondingly large decrease in the k(cat) (overall catalytic rate). This indicates a loss in the percentage of active enzymes. Given the relatively ideal geometry of the HRPC-PHEMA brush, the loss of activity is most likely due to structural changes in the enzyme arising from either secondary constraints imposed by the connectivity of the N-hydroxysuccinimidyl carbonate linking moiety or nonspecific interactions between HRP C and DSC-PHEMA. Therefore, a general enzyme-brush coupling motif must optimize reactive group density to balance binding with neutrality of surroundings.

Conformational Exchange in a Membrane Transport Protein Is Altered in Protein Crystals

Successful macromolecular crystallography requires solution conditions that may alter the conformational sampling of a macromolecule. Here, site-directed spin labeling is used to examine a conformational equilibrium within BtuB, the Escherichia coli outer membrane transporter for vitamin B12. Electron paramagnetic resonance (EPR) spectra from a spin label placed within the N-terminal energy coupling motif (Ton box) of BtuB indicate that this segment is in equilibrium between folded and unfolded forms. In bilayers, substrate binding shifts this equilibrium toward the unfolded form; however, EPR spectra from this same spin-labeled mutant indicate that this unfolding transition is blocked in protein crystals. Moreover, crystal structures of this spin-labeled mutant are consistent with the EPR result. When the free energy difference between substates is estimated from the EPR spectra, the crystal environment is found to alter this energy by 3 kcal/mol when compared to the bilayer state. Approximately half of this energy change is due to solutes or osmolytes in the crystallization buffer, and the remainder is contributed by the crystal lattice. These data provide a quantitative measure of how a conformational equilibrium in BtuB is modified in the crystal environment, and suggest that more-compact, less-hydrated substates will be favored in protein crystals.

Rational Design of Two-Dimensional Nanoscale Networks by Electrostatic Interactions at Surfaces

The self-assembly of aromatic carboxylic acids and cesium adatoms on a Cu(100) surface at room temperature has been investigated by scanning tunneling microscopy and X-ray photoelectron spectroscopy. The highly ordered molecular nanostructures are comprised of a central ionic coupling motif between the anionic carboxylate moieties and Cs cations that generate distinctive chiral arrangements of the network structures. The primary electrostatic interaction results in highly flexible bond lengths and geometries. The adsorbate-substrate coupling is found to be important for the determination of the structures. With the use of rod-like carboxylic linker molecules, the dimension of the porous networks can be tuned through the variation of the aromatic backbone length.

Osmolytes modulate conformational exchange in solvent‐exposed regions of membrane proteins

Site-directed spin labeling (SDSL) was used to investigate local structure and conformational exchange in two bacterial outer-membrane TonB-dependent transporters, BtuB and FecA. Protecting osmolytes, such as polyethylene glycols (PEGs) are known to modulate a substrate-dependent conformational equilibrium in the energy coupling motif (Ton box) of BtuB. Here, we demonstrate that a segment that is N-terminal to the Ton box in BtuB, is in conformational exchange between ordered and disordered states with or without substrate. Protecting osmolytes shift this equilibrium to favor the more ordered, folded state. However, a segment of BtuB that is C-terminal to the Ton box that is not solvent exposed is insensitive to PEGs. Protecting osmolytes also modulate a conformational equilibrium in the Ton box of FecA, with larger molecular weight PEGs producing the largest shifts in the conformational free energy. These data indicate that solvent-exposed regions of these transporters undergo conformational exchange and that regions of these transporters that are involved in protein-protein interactions sample multiple conformational substates. The sensitivity to solute provides an explanation for differences seen between two high-resolution structures of BtuB, which each likely represent one conformation from a subset of states that are normally sampled by the protein. This work also illustrates how SDSL and osmolytes may be used to characterize and quantitate conformational equilibria in membrane proteins.

An aromatic coupling motif for two-dimensional supramolecular architectures

We show, using scanning tunneling microscopy, how a coupling motif based on self-complementary helical aromatic units is able to drive the formation of a chiral porous supramolecular network and chains based on lateral aromatic interactions in two dimensions.

Molecular Basis for Substrate-dependent Transmembrane Signaling in an Outer-membrane Transporter

Transmembrane signaling events that propagate through receptors and transporters have critical roles in cellular function and regulation. In the Escherichia coli vitamin B 12 transporter, BtuB, substrate binding to the extracellular surface of the protein triggers the unfolding of an energy coupling motif at the periplasmic surface. Here, the molecular interactions mediating this substrate-dependent transmembrane signaling event were investigated in a novel way by combining a two mutant cycle analysis with site-directed spin labeling (SDSL). SDSL was used to monitor the unfolding and conformational equilibrium of the energy-coupling motif, and a thermodynamic two-mutant cycle analysis was used to estimate pair-wise interaction free energies for a pair of charged residues (D316 and R14) within the protein interior. The data indicate that D316 and R14 are critical to this structural transition. Substrate binding is shown to reduce the interaction free energy between these residues, thereby triggering the unfolding of the energy coupling motif of this membrane transporter. The result indicates that SDSL when used in combination with a mutant cycle analysis provides an approach to examine the molecular interactions mediating signaling events in membrane proteins.

An ATP Hydrolysis Sensor in the DNA Packaging Motor from Bacteriophage T4 Suggests an Inchworm-Type Translocation Mechanism

Tailed bacteriophages and large eukaryotic viruses employ powerful molecular motors to translocate dsDNA into a preassembled capsid shell. The phage T4 motor is composed of a dodecameric portal and small and large terminase subunits assembled at the special head–tail connector vertex of the prohead. The motor pumps DNA through the portal channel, utilizing ATP hydrolysis energy provided by an ATPase present in the large terminase subunit. We report that the ATPase motors of terminases, helicases, translocating restriction enzymes, and protein translocases possess a common coupling motif (C-motif). Mutations in the phage T4 terminase C-motif lead to loss of stimulated ATPase and DNA translocation activities. Surprisingly, the mutants can catalyze at least one ATP hydrolysis event but are unable to turn over and reset the motor. This is the first report of a catalytic block in translocating ATPase motor after ATP hydrolysis occurred. We suggest that the C-motif is an ATP hydrolysis sensor, linking product release to mechanical motion. A novel terminase-driven mechanism is proposed for translocation of dsDNA in viruses.

Diverse Functional Motifs within the Three Intracellular Loops of the CGRP1 Receptor

The CGRP(1) receptor exists as a heterodimeric complex between a single-pass transmembrane accessory protein (RAMP1) and a family B G-protein-coupled receptor (GPCR) called the calcitonin receptor-like receptor (CLR). This study investigated the structural motifs found in the intracellular loops (ICLs) of this receptor. Molecular modeling was used to predict active and inactive conformations of each ICL. Conserved residues were altered to alanine by site-directed mutagenesis. cAMP accumulation, cell-surface expression, agonist affinity, and CGRP-stimulated receptor internalization were characterized. Within ICL1, L147 and particularly R151 were important for coupling to G(s). R151 may interact directly with the G-protein, accessing it following conformational changes involving ICL2 and ICL3. At the proximal end of ICL3, I290 and L294, probably lying on the same face of an alpha helix, formed a G-protein coupling motif. The largest effects on coupling were observed with I290A; additionally, it reduced CGRP affinity and impaired internalization. I290 may interact with TM6 to stabilize the conformation of ICL3, but it could also interact directly with Gs. R314, at the distal end of ICL3, impaired G-protein coupling and to a lesser extent reduced CGRP affinity; it may stabilize the TM6-ICL3 junction by interacting with the polar headgroups of membrane phospholipids. Y215 and L214 in ICL2 are required for cell-surface expression; they form a microdomain with H216 which has the same function. This study reveals similarities between the activation of CLR and other GPCRs in the role of TM6 and ICL3 but shows that other conserved motifs differ in their function.

Substrate-Dependent Unfolding of the Energy Coupling Motif of a Membrane Transport Protein Determined by Double Electron-Electron Resonance

BtuB is a TonB-dependent transport protein that binds and carries vitamin B(12) across the outer membrane of Gram negative bacteria such as Escherichia coli. Previous work has demonstrated that the Ton box, a highly conserved segment near the N-terminus of the protein, undergoes an order-to-disorder transition upon the binding of substrate. Here, we incorporate pairs of nitroxide spin labels into membrane reconstituted BtuB and utilize a four-pulse double electron-electron resonance (DEER) experiment to measure distances between the Ton box and the periplasmic surface of the transporter with and without substrate. During reconstitution, the labeled membrane protein was diluted with wild-type protein, which significantly reduced the intermolecular electron spin-spin relaxation rate and increased the DEER signal-to-noise ratio. In the absence of substrate, each spin pair gives rise to a single distribution of distances that is consistent with the crystal structure obtained for BtuB; however, distances that are much longer are found in the presence of substrate, and the data are consistent with the existence of an equilibrium between folded and unfolded states of the Ton box. From these distances, a model for the position of the Ton box was constructed, and it indicates that the N-terminal end of the Ton box extends approximately 20 to 30 A into the periplasm upon the addition of substrate. We propose that this substrate-induced extension provides the signal that initiates interactions between BtuB and the inner membrane protein TonB.

Density Functional Theory Analysis of Carboxylate-Bridged Diiron Units in Two-Dimensional Metal−Organic Grids

Carboxylate-bridged diiron units have been realized recently in two dimensions via metal-directed self-assembly of simple organic linkers. They represent notably a coupling motif stabilizing a series of nanoporous metal-organic coordination networks and are reminiscent of catalytically active sites in metalloproteins. To rationalize their atomic structure, chemical bonding, and electronic properties, we performed density functional theory calculations for exemplary Fe-terephthalate grids assembled on the Cu(100) surface. The obtained atomistic description and understanding is decisive for the considerate use of two-dimensional metal-organic coordination networks as templates, guest systems, or catalysts.

Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter.

BtuB is a TonB-dependent outer-membrane transporter for vitamin B12 (or cyanocobalamin, CN-Cbl) in Escherichia coli. The binding of CN-Cbl is believed to promote an unfolding or undocking of the Ton box, the conserved N-terminal energy coupling motif at the periplasmic surface of the transporter. This structural change may facilitate the interaction of BtuB with the inner membrane protein TonB. In this work, the effect of the receptor binding fragment of colicin E3 (E3R) on the conformation of the Ton box was examined with site-directed spin labeling. Addition of E3R reverses the undocking of the Ton box that is promoted by CN-Cbl, consistent with a competitive binding between the substrate and the colicin fragment. EPR spectroscopy indicates that the Ton box is in a two-state equilibrium between docked and undocked conformations. In the absence of substrate, the docked conformation is the predominant state; however, the equilibrium can be shifted to the undocked state by the addition of detergents or site-specific proline substitutions. Even when the undocking is induced by detergents or by certain proline mutations, E3R binding shifts the equilibrium to the docked conformation. Thus, two competitive extracellular ligands, CN-Cbl and ER3, transduce opposite conformations of the N-terminal Ton box. Substrate binding stabilizes an undocked conformation, whereas E3R binding stabilizes a docked conformation of the Ton box.

Molecular cloning and structural analysis of the porcine homologue to CD97 antigen

CD97 is a member of a novel subfamily of leukocyte proteins that are characterized by the presence of tandemly repeated extracellular epidermal growth factor (EGF)-like domains and a seven-span transmembrane region, known as EGF–TM7. We here report the cloning of cDNA encoding the pig homologue of CD97. A pig CD97 specific probe was generated by PCR amplification of pig leukocyte cDNA, using primers based on consensus regions among the known sequences of mouse and human CD97. Screening of a pig aorta smooth muscle cDNA library identified one clone containing an open reading frame (ORF) that encoded an 18 amino acid putative signal peptide, a 141 amino acid sequence consisting of three EGF domains, a mucin-like spacer region of 276 amino acid, containing a G-protein coupling motif of 52 amino acids, followed by a 250 amino acid region containing seven membrane spanning domains and a 47 amino acid cytoplasmic tail. The amino acid sequence of the clone was 75, 67 and 59% homologous to cattle, human and mouse CD97 antigen, respectively. Therefore, it was termed pig CD97. Pig CD97 antigen shares many structural features with human, cattle and mouse CD97. RT-PCR analysis of cDNA from different pig cells and tissues showed that CD97 was highly expressed in leukocytes and lymph node cells. This is the first report describing the identification of a member of the EGF–TM7 family in the pig.