Nanohashtag structures based on carbon nanotubes and molecular linkers

NASA Astrophysics Data System (ADS)

Frye, Connor W.; Rybolt, Thomas R.

2018-03-01

Molecular mechanics was used to study the noncovalent interactions between single-walled carbon nanotubes and molecular linkers. Groups of nanotubes have the tendency to form tight, parallel bundles (||||). Molecular linkers were introduced into our models to stabilize nanostructures with carbon nanotubes held in perpendicular orientations. Molecular mechanics makes it possible to estimate the strength of noncovalent interactions holding these structures together and to calculate the overall binding energy of the structures. A set of linkers were designed and built around a 1,3,5,7-cyclooctatetraene tether with two corannulene containing pincers that extend in opposite directions from the central cyclooctatetraene portion. Each pincer consists of a pairs of "arms." These molecular linkers were modified so that the "hand" portions of each pair of "arms" could close together to grab and hold two carbon nanotubes in a perpendicular arrangement. To illustrate the possibility of more complicated and open perpendicular CNTs structures, our primary goal was to create a model of a nanohashtag (#) CNT conformation that is more stable than any parallel CNT arrangements with bound linker molecules forming clumps of CNTs and linkers in non-hashtag arrangements. This goal was achieved using a molecular linker (C280H96) that utilizes van der Waals interactions to two perpendicular oriented CNTs. Hydrogen bonding was then added between linker molecules to augment the stability of the hashtag structure. In the hashtag structure with hydrogen bonding, four (5,5) CNTs of length 4.46 nm (18 rings) and four linkers (C276H92N8O8) stabilized the hashtag so that the average binding energy per pincer was 118 kcal/mol.

Cellulase Linkers Are Optimized Based on Domain Type and Function: Insights from Sequence Analysis, Biophysical Measurements, and Molecular Simulation

PubMed Central

Sammond, Deanne W.; Payne, Christina M.; Brunecky, Roman; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2012-01-01

Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions. PMID:23139804

Creating Hierarchical Pores by Controlled Linker Thermolysis in Multivariate Metal-Organic Frameworks.

PubMed

Feng, Liang; Yuan, Shuai; Zhang, Liang-Liang; Tan, Kui; Li, Jia-Luo; Kirchon, Angelo; Liu, Ling-Mei; Zhang, Peng; Han, Yu; Chabal, Yves J; Zhou, Hong-Cai

2018-02-14

Sufficient pore size, appropriate stability, and hierarchical porosity are three prerequisites for open frameworks designed for drug delivery, enzyme immobilization, and catalysis involving large molecules. Herein, we report a powerful and general strategy, linker thermolysis, to construct ultrastable hierarchically porous metal-organic frameworks (HP-MOFs) with tunable pore size distribution. Linker instability, usually an undesirable trait of MOFs, was exploited to create mesopores by generating crystal defects throughout a microporous MOF crystal via thermolysis. The crystallinity and stability of HP-MOFs remain after thermolabile linkers are selectively removed from multivariate metal-organic frameworks (MTV-MOFs) through a decarboxylation process. A domain-based linker spatial distribution was found to be critical for creating hierarchical pores inside MTV-MOFs. Furthermore, linker thermolysis promotes the formation of ultrasmall metal oxide nanoparticles immobilized in an open framework that exhibits high catalytic activity for Lewis acid-catalyzed reactions. Most importantly, this work provides fresh insights into the connection between linker apportionment and vacancy distribution, which may shed light on probing the disordered linker apportionment in multivariate systems, a long-standing challenge in the study of MTV-MOFs.

Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

PubMed Central

Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

2015-01-01

Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. PMID:25870416

Differential solvation of intrinsically disordered linkers drives the formation of spatially organized droplets in ternary systems of linear multivalent proteins

NASA Astrophysics Data System (ADS)

Harmon, Tyler S.; Holehouse, Alex S.; Pappu, Rohit V.

2018-04-01

Intracellular biomolecular condensates are membraneless organelles that encompass large numbers of multivalent protein and nucleic acid molecules. The bodies assemble via a combination of liquid–liquid phase separation and gelation. A majority of condensates included multiple components and show multilayered organization as opposed to being well-mixed unitary liquids. Here, we put forward a simple thermodynamic framework to describe the emergence of spatially organized droplets in multicomponent systems comprising of linear multivalent polymers also known as associative polymers. These polymers, which mimic proteins and/or RNA have the architecture of domains or motifs known as stickers that are interspersed by flexible spacers known as linkers. Using a minimalist numerical model for a four-component system, we have identified features of linear multivalent molecules that are necessary and sufficient for generating spatially organized droplets. We show that differences in sequence-specific effective solvation volumes of disordered linkers between interaction domains enable the formation of spatially organized droplets. Molecules with linkers that are preferentially solvated are driven to the interface with the bulk solvent, whereas molecules that have linkers with negligible effective solvation volumes form cores in the core–shell architectures that emerge in the minimalist four-component systems. Our modeling has relevance for understanding the physical determinants of spatially organized membraneless organelles.

Chromatic patchy particles: Effects of specific interactions on liquid structure

DOE PAGES

Vasilyev, Oleg A.; Tkachenko, Alexei V.; Klumov, Boris A.

2015-07-13

We study the structural and thermodynamic properties of patchy particle liquids, with a special focus on the role of “color,” i.e., specific interactions between individual patches. A possible experimental realization of such “chromatic” interactions is by decorating the particle patches with single-stranded DNA linkers. The complementarity of the linkers can promote selective bond formation between predetermined pairs of patches. By using MD simulations, we compare the local connectivity, the bond orientation order, and other structural properties of the aggregates formed by the “colored” and “colorless” systems. The analysis is done for spherical particles with two different patch arrangements (tetrahedral andmore » cubic). It is found that the aggregated (liquid) phase of the “colorless” patchy particles is better connected, denser and typically has stronger local order than the corresponding “colored” one. This, in turn, makes the colored liquid less stable thermodynamically. Specifically, we predict that in a typical case the chromatic interactions should increase the relative stability of the crystalline phase with respect to the disordered liquid, thus expanding its region in the phase diagram.« less

A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding

PubMed Central

Kostrhon, Sebastian; Kontaxis, Georg; Kaufmann, Tanja; Schirghuber, Erika; Kubicek, Stefan; Konrat, Robert

2017-01-01

N-terminal histone tails are subject to many posttranslational modifications that are recognized by and interact with designated reader domains in histone-binding proteins. BROMO domain adjacent to zinc finger 2B (BAZ2B) is a multidomain histone-binding protein that contains two histone reader modules, a plant homeodomain (PHD) and a bromodomain (BRD), linked by a largely disordered linker. Although previous studies have reported specificity of the PHD domain for the unmodified N terminus of histone H3 and of the BRD domain for H3 acetylated at Lys14 (H3K14ac), the exact mode of H3 binding by BAZ2B and its regulation are underexplored. Here, using isothermal titration calorimetry and NMR spectroscopy, we report that acidic residues in the BAZ2B PHD domain are essential for H3 binding and that BAZ2B PHD–BRD establishes a polyvalent interaction with H3K14ac. Furthermore, we provide evidence that the disordered interdomain linker modulates the histone-binding affinity by interacting with the PHD domain. In particular, lysine-rich stretches in the linker, which resemble the positively charged N terminus of histone H3, reduce the binding affinity of the PHD finger toward the histone substrate. Phosphorylation, acetylation, or poly(ADP-ribosyl)ation of the linker residues may therefore act as a cellular mechanism to transiently tune BAZ2B histone-binding affinity. Our findings further support the concept of interdomain linkers serving a dual role in substrate binding by appropriately positioning the adjacent domains and by electrostatically modulating substrate binding. Moreover, inhibition of histone binding by a histone-mimicking interdomain linker represents another example of regulation of protein–protein interactions by intramolecular mimicry. PMID:28864776

Influencing Antibody-Mediated Attenuation of Methamphetamine CNS Distribution through Vaccine Linker Design.

PubMed

Gooyit, Major; Miranda, Pedro O; Wenthur, Cody J; Ducime, Alex; Janda, Kim D

2017-03-15

Active vaccination examining a single hapten engendered with a series of peptidic linkers has resulted in the production of antimethamphetamine antibodies. Given the limited chemical complexity of methamphetamine, the structure of the linker species embedded within the hapten could have a substantial effect on the ultimate efficacy of the resulting vaccines. Herein, we investigate linker effects by generating a series of methamphetamine haptens that harbor a linker with varying amino acid identity, peptide length, and associated carrier protein. Independent changes in each of these parameters were found to result in alterations in both the quantity and quality of the antibodies induced by vaccination. Although it was found that the consequence of the linker design was also dependent on the identity of the carrier protein, we demonstrate overall that the inclusion of a short, structurally simple, amino acid linker benefits the efficacy of a methamphetamine vaccine in limiting brain penetration of the free drug.

The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains.

PubMed

English, Charles A; Sherman, Woody; Meng, Wenli; Gierasch, Lila M

2017-09-08

Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Entropic Elasticity in the Giant Muscle Protein Titin

NASA Astrophysics Data System (ADS)

Morgan, Ian; Saleh, Omar

Intrinsically disordered proteins (IDPs) are a large and functionally important class of proteins that lack a fixed three-dimensional structure. Instead, they adopt a conformational ensemble of states which facilitates their biological function as molecular linkers, springs, and switches. Due to their conformational flexibility, it can be difficult to study IDPs using typical experimental methods. To overcome this challenge, we use a high-resolution single-molecule magnetic stretching technique to quantify IDP flexibility. We apply this technique to the giant muscle protein titin, measuring its elastic response at low forces. We present results demonstrating that titin's native elastic response derives from the combined entropic elasticity of its ordered and disordered domains.

Effect of the SH3-SH2 domain linker sequence on the structure of Hck kinase.

PubMed

Meiselbach, Heike; Sticht, Heinrich

2011-08-01

The coordination of activity in biological systems requires the existence of different signal transduction pathways that interact with one another and must be precisely regulated. The Src-family tyrosine kinases, which are found in many signaling pathways, differ in their physiological function despite their high overall structural similarity. In this context, the differences in the SH3-SH2 domain linkers might play a role for differential regulation, but the structural consequences of linker sequence remain poorly understood. We have therefore performed comparative molecular dynamics simulations of wildtype Hck and of a mutant Hck in which the SH3-SH2 domain linker is replaced by the corresponding sequence from the homologous kinase Lck. These simulations reveal that linker replacement not only affects the orientation of the SH3 domain itself, but also leads to an alternative conformation of the activation segment in the Hck kinase domain. The sequence of the SH3-SH2 domain linker thus exerts a remote effect on the active site geometry and might therefore play a role in modulating the structure of the inactive kinase or in fine-tuning the activation process itself.

Chromatin Condensing Functions of the Linker Histone C-terminal Domain are mediated by Specific Amino Acid Composition and Intrinsic Protein Disorder†

PubMed Central

Lu, Xu; Hamkalo, Barbara; Parseghian, Missag H.; Hansen, Jeffrey C.

2009-01-01

Linker histones bind to the nucleosomes and linker DNA of chromatin fibers, causing changes in linker DNA structure and stabilization of higher order folded and oligomeric chromatin structures. Linker histones affect chromatin structure acting primarily through their ~100 residue C-terminal domain (CTD). We have previously shown that the ability of the linker histone H1° to alter chromatin structure was localized to two discontinuous 24-/25-residue CTD regions (Lu, X., and Hansen, J. C. (2004) J Biol Chem 279, 8701–8707). To determine the biochemical basis for these results, we have characterized chromatin model systems assembled with endogenous mouse somatic H1 isoforms, or recombinant H1° CTD mutants in which the primary sequence has been scrambled, the amino acid composition mutated, or the location of various CTD regions swapped. Our results indicate that specific amino acid composition plays a fundamental role in molecular recognition and function by the H1 CTD. Additionally, these experiments support a new molecular model for CTD function, and provide a biochemical basis for the redundancy observed in H1 isoform knockout experiments in vivo. PMID:19072710

The Role of Linkers in the Excited-State Dynamic Planarization Processes of Macrocyclic Oligothiophene 12-Mers.

PubMed

Kim, Woojae; Sung, Jooyoung; Park, Kyu Hyung; Shimizu, Hideyuki; Imamura, Mika; Han, Minwoo; Sim, Eunji; Iyoda, Masahiko; Kim, Dongho

2015-11-05

Linkers adjoining chromophores play an important role in modulating the structure of conjugated systems, which is bound up with their photophysical properties. However, to date, the focus of works dealing with linker effects was limited only to linear π-conjugated materials, and there have been no detailed studies on cyclic counterparts. Herein we report the linker effects on the dynamic planarization processes of π-conjugated macrocyclic oligothiophene 12-mers, where the different ratio between ethynylene and vinylene linkers was chosen to control the backbone rigidity. By analyzing transient fluorescence spectra, we demonstrate that the connecting linkers play a crucial role in the excited-state dynamics of cyclic conjugated systems. Faster dynamic planarization, longer exciton delocalization length, and higher degree of planarity were observed in vinylene inserted cyclic oligothiophenes. Molecular dynamics simulations and density functional theory calculations also stress the importance of the role of linkers in modulating the structure of cyclic oligothiophenes.

A Novel MS-Cleavable Azo Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS)

NASA Astrophysics Data System (ADS)

Iacobucci, Claudio; Hage, Christoph; Schäfer, Mathias; Sinz, Andrea

2017-10-01

The chemical cross-linking/mass spectrometry (MS) approach is a growing research field in structural proteomics that allows gaining insights into protein conformations. It relies on creating distance constraints between cross-linked amino acid side chains that can further be used to derive protein structures. Currently, the most urgent task for designing novel cross-linking principles is an unambiguous and automated assignment of the created cross-linked products. Here, we introduce the homobifunctional, amine-reactive, and water soluble cross-linker azobisimidoester (ABI) as a prototype of a novel class of cross-linkers. The ABI-linker possesses an innovative modular scaffold combining the benefits of collisional activation lability with open shell chemistry. This MS-cleavable cross-linker can be efficiently operated via free radical initiated peptide sequencing (FRIPS) in positive ionization mode. Our proof-of-principle study challenges the gas phase behavior of the ABI-linker for the three amino acids, lysine, leucine, and isoleucine, as well as the model peptide thymopentin. The isomeric amino acids leucine and isoleucine could be discriminated by their characteristic side chain fragments. Collisional activation experiments were conducted via positive electrospray ionization (ESI) on two Orbitrap mass spectrometers. The ABI-mediated formation of odd electron product ions in MS/MS and MS3 experiments was evaluated and compared with a previously described azo-based cross-linker. All cross-linked products were amenable to automated analysis by the MeroX software, underlining the future potential of the ABI-linker for structural proteomics studies. [Figure not available: see fulltext.

Binding of DNA-bending non-histone proteins destabilizes regular 30-nm chromatin structure

PubMed Central

Bajpai, Gaurav; Jain, Ishutesh; Inamdar, Mandar M.; Das, Dibyendu; Padinhateeri, Ranjith

2017-01-01

Why most of the in vivo experiments do not find the 30-nm chromatin fiber, well studied in vitro, is a puzzle. Two basic physical inputs that are crucial for understanding the structure of the 30-nm fiber are the stiffness of the linker DNA and the relative orientations of the DNA entering/exiting nucleosomes. Based on these inputs we simulate chromatin structure and show that the presence of non-histone proteins, which bind and locally bend linker DNA, destroys any regular higher order structures (e.g., zig-zag). Accounting for the bending geometry of proteins like nhp6 and HMG-B, our theory predicts phase-diagram for the chromatin structure as a function of DNA-bending non-histone protein density and mean linker DNA length. For a wide range of linker lengths, we show that as we vary one parameter, that is, the fraction of bent linker region due to non-histone proteins, the steady-state structure will show a transition from zig-zag to an irregular structure—a structure that is reminiscent of what is observed in experiments recently. Our theory can explain the recent in vivo observation of irregular chromatin having co-existence of finite fraction of the next-neighbor (i + 2) and neighbor (i + 1) nucleosome interactions. PMID:28135276

Structure of the Intermediate Filament-Binding Region of Desmoplakin

DOE PAGES

Kang, Hyunook; Weiss, Thomas M.; Bang, Injin; ...

2016-01-25

Here, desmoplakin (DP) is a cytoskeletal linker protein that connects the desmosomal cadherin/plakoglobin/plakophilin complex to intermediate filaments (IFs). The C-terminal region of DP (DPCT) mediates IF binding, and contains three plakin repeat domains (PRDs), termed PRD-A, PRD-B and PRD-C. Previous crystal structures of PRDs B and C revealed that each is formed by 4.5 copies of a plakin repeat (PR) and has a conserved positively charged groove on its surface. Although PRDs A and B are linked by just four amino acids, B and C are separated by a 154 residue flexible linker, which has hindered crystallographic analysis of themore » full DPCT. Here we present the crystal structure of a DPCT fragment spanning PRDs A and B, and elucidate the overall architecture of DPCT by small angle X-ray scattering (SAXS) analysis. The structure of PRD-A is similar to that of PRD-B, and the two domains are arranged in a quasi-linear arrangement, and separated by a 4 amino acid linker. Analysis of the B-C linker region using secondary structure prediction and the crystal structure of a homologous linker from the cytolinker periplakin suggests that the N-terminal ~100 amino acids of the linker form two PR-like motifs. SAXS analysis of DPCT indicates an elongated but non-linear shape with R g = 51.5 Å and D max = 178 Å. These data provide the first structural insights into an IF binding protein containing multiple PRDs and provide a foundation for studying the molecular basis of DP-IF interactions.« less

The length but not the sequence of peptide linker modules exerts the primary influence on the conformations of protein domains in cellulosome multi-enzyme complexes.

PubMed

Różycki, Bartosz; Cazade, Pierre-André; O'Mahony, Shane; Thompson, Damien; Cieplak, Marek

2017-08-16

Cellulosomes are large multi-protein catalysts produced by various anaerobic microorganisms to efficiently degrade plant cell-wall polysaccharides down into simple sugars. X-ray and physicochemical structural characterisations show that cellulosomes are composed of numerous protein domains that are connected by unstructured polypeptide segments, yet the properties and possible roles of these 'linker' peptides are largely unknown. We have performed coarse-grained and all-atom molecular dynamics computer simulations of a number of cellulosomal linkers of different lengths and compositions. Our data demonstrates that the effective stiffness of the linker peptides, as quantified by the equilibrium fluctuations in the end-to-end distances, depends primarily on the length of the linker and less so on the specific amino acid sequence. The presence of excluded volume - provided by the domains that are connected - dampens the motion of the linker residues and reduces the effective stiffness of the linkers. Simultaneously, the presence of the linkers alters the conformations of the protein domains that are connected. We demonstrate that short, stiff linkers induce significant rearrangements in the folded domains of the mini-cellulosome composed of endoglucanase Cel8A in complex with scaffoldin ScafT (Cel8A-ScafT) of Clostridium thermocellum as well as in a two-cohesin system derived from the scaffoldin ScaB of Acetivibrio cellulolyticus. We give experimentally testable predictions on structural changes in protein domains that depend on the length of linkers.

Dependence of the Linker Histone and Chromatin Condensation on the Nucleosome Environment.

PubMed

Perišić, Ognjen; Schlick, Tamar

2017-08-24

The linker histone (LH), an auxiliary protein that can bind to chromatin and interact with the linker DNA to form stem motifs, is a key element of chromatin compaction. By affecting the chromatin condensation level, it also plays an active role in gene expression. However, the presence and variable concentration of LH in chromatin fibers with different DNA linker lengths indicate that its folding and condensation are highly adaptable and dependent on the immediate nucleosome environment. Recent experimental studies revealed that the behavior of LH in mononucleosomes markedly differs from that in small nucleosome arrays, but the associated mechanism is unknown. Here we report a structural analysis of the behavior of LH in mononucleosomes and oligonucleosomes (2-6 nucleosomes) using mesoscale chromatin simulations. We show that the adapted stem configuration heavily depends on the strength of electrostatic interactions between LH and its parental DNA linkers, and that those interactions tend to be asymmetric in small oligonucleosome systems. Namely, LH in oligonucleosomes dominantly interacts with one DNA linker only, as opposed to mononucleosomes where LH has similar interactions with both linkers and forms a highly stable nucleosome stem. Although we show that the LH condensation depends sensitively on the electrostatic interactions with entering and exiting DNA linkers, other interactions, especially by nonparental cores and nonparental linkers, modulate the structural condensation by softening LH and thus making oligonucleosomes more flexible, in comparison to to mono- and dinucleosomes. We also find that the overall LH/chromatin interactions sensitively depend on the linker length because the linker length determines the maximal nucleosome stem length. For mononucleosomes with DNA linkers shorter than LH, LH condenses fully, while for DNA linkers comparable or longer than LH, the LH extension in mononucleosomes strongly follows the length of DNA linkers, unhampered by neighboring linker histones. Thus, LH is more condensed for mononucleosomes with short linkers, compared to oligonucleosomes, and its orientation is variable and highly environment-dependent. More generally, the work underscores the agility of LH whose folding dynamics critically controls genomic packaging and gene expression.

Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: structural and functional significance of the intermodular linker

PubMed Central

Petkun, Svetlana; Rozman Grinberg, Inna; Lamed, Raphael; Jindou, Sadanari; Burstein, Tal; Yaniv, Oren; Shoham, Yuval; Shimon, Linda J.W.; Frolow, Felix

2015-01-01

Non-cellulosomal processive endoglucanase 9I (Cel9I) from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b), separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences) restored 60–70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes. PMID:26401442

Structure of an Engineered β-Lactamase Maltose Binding Protein Fusion Protein: Insights into Heterotropic Allosteric Regulation

PubMed Central

Ke, Wei; Laurent, Abigail H.; Armstrong, Morgan D.; Chen, Yuchao; Smith, William E.; Liang, Jing; Wright, Chapman M.; Ostermeier, Marc; van den Akker, Focco

2012-01-01

Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn2+ at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn2+ concentration and determined its structure. The structure reveals that the MBP and TEM-1 domains are in close proximity connected via two linkers and a zinc ion bridging both domains. By bridging both TEM-1 and MBP, Zn2+ acts to “twist tie” the linkers thereby partially dislodging a linker between the two domains from its original catalytically productive position in TEM-1. This linker 1 contains residues normally part of the TEM-1 active site including the critical β3 and β4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn2+ site only slightly affected Zn2+ inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn2+ sites. Mutagenesis and molecular modeling of an intact TEM-1 domain near MBP within the RG13 framework indicated a close surface proximity of the two domains with maltose switching being critically dependent on MBP linker anchoring residues and linker length. Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive. Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via steric and/or linker juxtapositioning mechanisms. PMID:22720063

Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker.

PubMed

Mo, Xuejun; Li, Qiushi; Yi Lui, Lena Wai; Zheng, Baixue; Kang, Chiang Huen; Nugraha, Bramasta; Yue, Zhilian; Jia, Rui Rui; Fu, Hong Xia; Choudhury, Deepak; Arooz, Talha; Yan, Jie; Lim, Chwee Teck; Shen, Shali; Hong Tan, Choon; Yu, Hanry

2010-10-01

Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications. 2010 Elsevier Ltd. All rights reserved.

Quantitative Characterization of Configurational Space Sampled by HIV-1 Nucleocapsid Using Solution NMR, X-ray Scattering and Protein Engineering.

PubMed

Deshmukh, Lalit; Schwieters, Charles D; Grishaev, Alexander; Clore, G Marius

2016-06-03

Nucleic-acid-related events in the HIV-1 replication cycle are mediated by nucleocapsid, a small protein comprising two zinc knuckles connected by a short flexible linker and flanked by disordered termini. Combining experimental NMR residual dipolar couplings, solution X-ray scattering and protein engineering with ensemble simulated annealing, we obtain a quantitative description of the configurational space sampled by the two zinc knuckles, the linker and disordered termini in the absence of nucleic acids. We first compute the conformational ensemble (with an optimal size of three members) of an engineered nucleocapsid construct lacking the N- and C-termini that satisfies the experimental restraints, and then validate this ensemble, as well as characterize the disordered termini, using the experimental data from the full-length nucleocapsid construct. The experimental and computational strategy is generally applicable to multidomain proteins. Differential flexibility within the linker results in asymmetric motion of the zinc knuckles which may explain their functionally distinct roles despite high sequence identity. One of the configurations (populated at a level of ≈40 %) closely resembles that observed in various ligand-bound forms, providing evidence for conformational selection and a mechanistic link between protein dynamics and function. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of linker regions and domain borders of the transcription activator protein NtrC from Escherichia coli by limited proteolysis, in-gel digestion, and mass spectrometry.

PubMed

Bantscheff, M; Weiss, V; Glocker, M O

1999-08-24

We have developed a mass spectrometry based method for the identification of linker regions and domain borders in multidomain proteins. This approach combines limited proteolysis and in-gel proteolytic digestions and was applied to the determination of linkers in the transcription factor NtrC from Escherichia coli. Limited proteolysis of NtrC with thermolysin and papain revealed that initial digestion yielded two major bands in SDS-PAGE that were identified by mass spectrometry as the R-domain and the still covalently linked OC-domains. Subsequent steps in limited proteolysis afforded further cleavage of the OC-fragment into the O- and the C-domain at accessible amino acid residues. Mass spectrometric identification of the tryptic/thermolytic peptides obtained after in-gel total proteolysis of the SDS-PAGE-separated domains determined the domain borders and showed that the protease accessible linker between R- and O-domain comprised amino acids Val-131 and Gln-132 within the "Q-linker" in agreement with papain and subtilisin digestion. The region between amino acid residues Thr-389 and Gln-396 marked the hitherto unknown linker sequence that connects the O- with the C-domain. High abundances of proline-, alanine-, serine-, and glutamic acid residues were found in this linker structure (PASE-linker) of related NtrC response regulator proteins. While R- and C-domains remained stable under the applied limited proteolysis conditions, the O-domain was further truncated yielding a core fragment that comprised the sequence from Ile-140 to Arg-320. ATPase activity was lost after separation of the R-domain from the OC-fragment. However, binding of OC- and C- fragments to specific DNA was observed by characteristic band-shifts in migration retardation assays, indicating intact tertiary structures of the C-domain. The outlined strategy proved to be highly efficient and afforded lead information of tertiary structural features necessary for protein design and engineering and for structure-function studies.

Small angle x-ray scattering of chromatin. Radius and mass per unit length depend on linker length.

PubMed Central

Williams, S P; Langmore, J P

1991-01-01

Analyses of low angle x-ray scattering from chromatin, isolated by identical procedures but from different species, indicate that fiber diameter and number of nucleosomes per unit length increase with the amount of nucleosome linker DNA. Experiments were conducted at physiological ionic strength to obtain parameters reflecting the structure most likely present in living cells. Guinier analyses were performed on scattering from solutions of soluble chromatin from Necturus maculosus erythrocytes (linker length 48 bp), chicken erythrocytes (linker length 64 bp), and Thyone briareus sperm (linker length 87 bp). The results were extrapolated to infinite dilution to eliminate interparticle contributions to the scattering. Cross-sectional radii of gyration were found to be 10.9 +/- 0.5, 12.1 +/- 0.4, and 15.9 +/- 0.5 nm for Necturus, chicken, and Thyone chromatin, respectively, which are consistent with fiber diameters of 30.8, 34.2, and 45.0 nm. Mass per unit lengths were found to be 6.9 +/- 0.5, 8.3 +/- 0.6, and 11.8 +/- 1.4 nucleosomes per 10 nm for Necturus, chicken, and Thyone chromatin, respectively. The geometrical consequences of the experimental mass per unit lengths and radii of gyration are consistent with a conserved interaction among nucleosomes. Cross-linking agents were found to have little effect on fiber external geometry, but significant effect on internal structure. The absolute values of fiber diameter and mass per unit length, and their dependencies upon linker length agree with the predictions of the double-helical crossed-linker model. A compilation of all published x-ray scattering data from the last decade indicates that the relationship between chromatin structure and linker length is consistent with data obtained by other investigators. Images FIGURE 1 PMID:2049522

Family-specific Kinesin Structures Reveal Neck-linker Length Based on Initiation of the Coiled-coil*

PubMed Central

Phillips, Rebecca K.; Peter, Logan G.; Gilbert, Susan P.

2016-01-01

Kinesin-1, -2, -5, and -7 generate processive hand-over-hand 8-nm steps to transport intracellular cargoes toward the microtubule plus end. This processive motility requires gating mechanisms to coordinate the mechanochemical cycles of the two motor heads to sustain the processive run. A key structural element believed to regulate the degree of processivity is the neck-linker, a short peptide of 12–18 residues, which connects the motor domain to its coiled-coil stalk. Although a shorter neck-linker has been correlated with longer run lengths, the structural data to support this hypothesis have been lacking. To test this hypothesis, seven kinesin structures were determined by x-ray crystallography. Each included the neck-linker motif, followed by helix α7 that constitutes the start of the coiled-coil stalk. In the majority of the structures, the neck-linker length differed from predictions because helix α7, which initiates the coiled-coil, started earlier in the sequence than predicted. A further examination of structures in the Protein Data Bank reveals that there is a great disparity between the predicted and observed starting residues. This suggests that an accurate prediction of the start of a coiled-coil is currently difficult to achieve. These results are significant because they now exclude simple comparisons between members of the kinesin superfamily and add a further layer of complexity when interpreting the results of mutagenesis or protein fusion. They also re-emphasize the need to consider factors beyond the kinesin neck-linker motif when attempting to understand how inter-head communication is tuned to achieve the degree of processivity required for cellular function. PMID:27462072

Thermal and chemical denaturation of the BRCT functional module of human 53BP1.

PubMed

Thanassoulas, Angelos; Nomikos, Michail; Theodoridou, Maria; Stavros, Philemon; Mastellos, Dimitris; Nounesis, George

2011-10-01

BRCTs are protein-docking modules involved in eukaryotic DNA repair. They are characterized by low sequence homology with generally well-conserved structure organization. In a considerable number of proteins, a pair of BRCT structural repeats occurs, connected with inter-BRCT linkers, variable in length, sequence and structure. Linkers may separate and control the relative position of BRCT domains as well as protect and stabilize the hydrophobic inter-BRCT interface region. Their vital role in protein function has been demonstrated by recent findings associating missense mutations in the inter-repeat linker region of the BRCT domain of BRCA1 (BRCA1-BRCT) to hereditary breast/ovarian cancer. The interaction of 53BP1 with the core domain of the p53 tumor suppressor involves the C-terminal BRCT repeat as well as the inert-BRCT linker of the tandem BRCT domain of 53BP1 (53BP1-BRCT). High-accuracy differential scanning calorimetry (DSC) and circular dichroism (CD) have been employed to characterize the heat-induced unfolding of 53BP1-BRCT domain. The calorimetric results provide evidence for unfolding to an intermediate, only partly unfolded state, which, based on the CD results, retains the secondary structural characteristics of the native protein. A direct comparison with the corresponding thermal processes for BRAC1-BRCT and BARD1-BRCT provides evidence that the observed behavior is analogous to BRCA1-BRCT even though the two domains differ substantially in the linker structure. Moreover, chemical denaturation experiments of the untagged 53BP1-BRCT and comparison with BRCA1 and BARD1 BRCTs show that no clear association can be drawn between the structural organization of the inter-BRCT linkers and the overall stability of the BRCT domains. Copyright © 2011 Elsevier B.V. All rights reserved.

Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability

NASA Astrophysics Data System (ADS)

Kong, Leopold; He, Linling; de Val, Natalia; Vora, Nemil; Morris, Charles D.; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Zhu, Jiang

2016-06-01

The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains.

Coiled-Coil Antagonism Regulates Activity of Venus Flytrap-Domain-Containing Sensor Kinases of the BvgS Family

PubMed Central

Lesne, Elodie; Dupré, Elian; Lensink, Marc F.; Locht, Camille

2018-01-01

ABSTRACT Bordetella pertussis controls the expression of its virulence regulon through the two-component system BvgAS. BvgS is a prototype for a family of multidomain sensor kinases. In BvgS, helical linkers connect periplasmic Venus flytrap (VFT) perception domains to a cytoplasmic Per-Arnt-Sim (PAS) domain and the PAS domain to the dimerization/histidine phosphotransfer (DHp) domain of the kinase. The two linkers can adopt coiled-coil structures but cannot do so simultaneously. The first linker forms a coiled coil in the kinase mode and the second in the phosphatase mode, with the other linker in both cases showing an increase in dynamic behavior. The intervening PAS domain changes its quaternary structure between the two modes. In BvgS homologues without a PAS domain, a helical “X” linker directly connects the VFT and DHp domains. Here, we used BvgS as a platform to characterize regulation in members of the PAS-less subfamily. BvgS chimeras of homologues with natural X linkers displayed various regulation phenotypes. We identified two distinct coiled-coil registers in the N- and C-terminal portions of the X linkers. Stable coil formation in the C-terminal moiety determines the phosphatase mode, similarly to BvgS; in contrast, coil formation in the N-terminal moiety along the other register leads to the kinase mode. Thus, antagonism between two registers in the VFT-DHp linker forms the basis for activity regulation in the absence of the PAS domain. The N and C moieties of the X linker play roles similar to those played by the two independent linkers in sensor kinases with a PAS domain, providing a unified mechanism of regulation for the entire family. PMID:29487240

Micromachined silicon acoustic delay line with 3D-printed micro linkers and tapered input for improved structural stability and acoustic directivity

NASA Astrophysics Data System (ADS)

Cho, Y.; Kumar, A.; Xu, S.; Zou, J.

2016-10-01

Recent studies have shown that micromachined silicon acoustic delay lines can provide a promising solution to achieve real-time photoacoustic tomography without the need for complex transducer arrays and data acquisition electronics. To achieve deeper imaging depth and wider field of view, a longer delay time and therefore delay length are required. However, as the length of the delay line increases, it becomes more vulnerable to structural instability due to reduced mechanical stiffness. In this paper, we report the design, fabrication, and testing of a new silicon acoustic delay line enhanced with 3D printed polymer micro linker structures. First, mechanical deformation of the silicon acoustic delay line (with and without linker structures) under gravity was simulated by using finite element method. Second, the acoustic crosstalk and acoustic attenuation caused by the polymer micro linker structures were evaluated with both numerical simulation and ultrasound transmission testing. The result shows that the use of the polymer micro linker structures significantly improves the structural stability of the silicon acoustic delay lines without creating additional acoustic attenuation and crosstalk. In addition, the improvement of the acoustic acceptance angle of the silicon acoustic delay lines was also investigated to better suppress the reception of unwanted ultrasound signals outside of the imaging plane. These two improvements are expected to provide an effective solution to eliminate current limitations on the achievable acoustic delay time and out-of-plane imaging resolution of micromachined silicon acoustic delay line arrays.

Dynamic Conformations of Nucleosome Arrays in Solution from Small-Angle X-ray Scattering

NASA Astrophysics Data System (ADS)

Howell, Steven C.

Chromatin conformation and dynamics remains unsolved despite the critical role of the chromatin in fundamental genetic functions such as transcription, replication, and repair. At the molecular level, chromatin can be viewed as a linear array of nucleosomes, each consisting of 147 base pairs (bp) of double-stranded DNA (dsDNA) wrapped around a protein core and connected by 10 to 90 bp of linker dsDNA. Using small-angle X-ray scattering (SAXS), we investigated how the conformations of model nucleosome arrays in solution are modulated by ionic condition as well as the effect of linker histone proteins. To facilitate ensemble modeling of these SAXS measurements, we developed a simulation method that treats coarse-grained DNA as a Markov chain, then explores possible DNA conformations using Metropolis Monte Carlo (MC) sampling. This algorithm extends the functionality of SASSIE, a program used to model intrinsically disordered biological molecules, adding to the previous methods for simulating protein, carbohydrates, and single-stranded DNA. Our SAXS measurements of various nucleosome arrays together with the MC generated models provide valuable solution structure information identifying specific differences from the structure of crystallized arrays.

Rigidifying fluorescent linkers by metal-organic framework formation for fluorescence blue shift and quantum yield enhancement.

PubMed

Wei, Zhangwen; Gu, Zhi-Yuan; Arvapally, Ravi K; Chen, Ying-Pin; McDougald, Roy N; Ivy, Joshua F; Yakovenko, Andrey A; Feng, Dawei; Omary, Mohammad A; Zhou, Hong-Cai

2014-06-11

We demonstrate that rigidifying the structure of fluorescent linkers by structurally constraining them in metal-organic frameworks (MOFs) to control their conformation effectively tunes the fluorescence energy and enhances the quantum yield. Thus, a new tetraphenylethylene-based zirconium MOF exhibits a deep-blue fluorescent emission at 470 nm with a unity quantum yield (99.9 ± 0.5%) under Ar, representing ca. 3600 cm(-1) blue shift and doubled radiative decay efficiency vs the linker precursor. An anomalous increase in the fluorescence lifetime and relative intensity takes place upon heating the solid MOF from cryogenic to ambient temperatures. The origin of these unusual photoluminescence properties is attributed to twisted linker conformation, intramolecular hindrance, and framework rigidity.

Biosynthesis of glycosaminoglycans: associated disorders and biochemical tests.

PubMed

Sasarman, Florin; Maftei, Catalina; Campeau, Philippe M; Brunel-Guitton, Catherine; Mitchell, Grant A; Allard, Pierre

2016-03-01

Glycosaminoglycans (GAG) are long, unbranched heteropolymers with repeating disaccharide units that make up the carbohydrate moiety of proteoglycans. Six distinct classes of GAGs are recognized. Their synthesis follows one of three biosynthetic pathways, depending on the type of oligosaccharide linker they contain. Chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin sulfate contain a common tetrasaccharide linker that is O-linked to specific serine residues in core proteins. Keratan sulfate can contain three different linkers, either N-linked to asparagine or O-linked to serine/threonine residues in core proteins. Finally, hyaluronic acid does not contain a linker and is not covalently attached to a core protein. Most inborn errors of GAG biosynthesis are reported in small numbers of patients. To date, in 20 diseases, convincing evidence for pathogenicity has been presented for mutations in a total of 16 genes encoding glycosyltransferases, sulfotransferases, epimerases or transporters. GAG synthesis defects should be suspected in patients with a combination of characteristic clinical features in more than one connective tissue compartment: bone and cartilage (short long bones with or without scoliosis), ligaments (joint laxity/dislocations), and subepithelial (skin, sclerae). Some produce distinct clinical syndromes. The commonest laboratory tests used for this group of diseases are analysis of GAGs, enzyme assays, and molecular testing. In principle, GAG analysis has potential as a general first-line diagnostic test for GAG biosynthesis disorders.

The attachment of α -synuclein to a fiber: A coarse-grain approach

NASA Astrophysics Data System (ADS)

Ilie, Ioana M.; den Otter, Wouter K.; Briels, Wim J.

2017-03-01

We present simulations of the amyloidogenic core of α-synuclein, the protein causing Parkinson's disease, as a short chain of coarse-grain patchy particles. Each particle represents a sequence of about a dozen amino acids. The fluctuating secondary structure of this intrinsically disordered protein is modelled by dynamic variations of the shape and interaction characteristics of the patchy particles, ranging from spherical with weak isotropic attractions for the disordered state to spherocylindrical with strong directional interactions for a β-sheet. Flexible linkers between the particles enable sampling of the tertiary structure. This novel model is applied here to study the growth of an amyloid fibril, by calculating the free energy profile of a protein attaching to the end of a fibril. The simulation results suggest that the attaching protein readily becomes trapped in a mis-folded state, thereby inhibiting further growth of the fibril until the protein has readjusted to conform to the fibril structure, in line with experimental findings and previous simulations on small fragments of other proteins.

The influence of the structural orientation of amide linkers on the serum compatibility and lung transfection properties of cationic amphiphiles.

PubMed

Srujan, Marepally; Chandrashekhar, Voshavar; Reddy, Rakesh C; Prabhakar, Rairala; Sreedhar, Bojja; Chaudhuri, Arabinda

2011-08-01

Understanding the structural parameters of cationic amphiphiles which can influence gene transfer efficiencies of cationic amphiphiles continues to remain important for designing efficient liposomal gene delivery reagents. Previously we demonstrated the influence of structural orientation of the ester linker (widely used in covalently tethering the polar head and the non-polar tails) in modulating in vitro gene transfer efficiencies of cationic amphiphiles. However, our previously described cationic amphiphiles with ester linkers failed to deliver genes under in vivo conditions. Herein we report on the development of a highly serum compatible cationic amphiphile with circulation stable amide linker which shows remarkable selectivity in transfecting mouse lung. We also demonstrate that reversing structural orientation of the amide linker adversely affects both serum compatibility and the lung selective gene transfer property. Dynamic laser light scattering and atomic force microscopic studies revealed smaller average hydrodynamic sizes of the liposomes of transfection efficient lipid than those for the liposomes of transfection incompetent analog (148 ± 1 nm vs 214 ± 4 nm). Average surface potential of the liposomes of transfection competent amphiphiles were found to be significantly higher than that for the liposomes of transfection incompetent analog (10.7 ± 5.4 mV vs 2.8 ± 1.3 mV, respectively). Findings in fluorescence resonance energy transfer and dye entrapment experiments support lower rigidity and higher biomembrane fusogenicity of the liposomes of the transfection efficient amphiphiles. Importantly, cationic lipoplexes of the novel amide-linker based amphiphile exhibited higher mouse lung selective gene transfer properties than DOTAP, one of the widely used commercially available liposomal lung transfection kits. In summary, the present findings demonstrate for the first time that amide linker structural orientation profoundly influences the serum compatibility and lung transfection efficiencies of cationic amphiphiles. Copyright © 2011 Elsevier Ltd. All rights reserved.

Novel Concepts of MS-Cleavable Cross-linkers for Improved Peptide Structure Analysis

NASA Astrophysics Data System (ADS)

Hage, Christoph; Falvo, Francesco; Schäfer, Mathias; Sinz, Andrea

2017-10-01

The chemical cross-linking/mass spectrometry (MS) approach is gaining increasing importance as an alternative method for studying protein conformation and for deciphering protein interaction networks. This study is part of our ongoing efforts to develop innovative cross-linking principles for a facile and efficient assignment of cross-linked products. We evaluate two homobifunctional, amine-reactive, and MS-cleavable cross-linkers regarding their potential for automated analysis of cross-linked products. We introduce the bromine phenylurea (BrPU) linker that possesses a unique structure yielding a distinctive fragmentation pattern on collisional activation. Moreover, BrPU delivers the characteristic bromine isotope pattern and mass defect for all cross-linker-decorated fragments. We compare the fragmentation behavior of the BrPU linker with that of our previously described MS-cleavable TEMPO-Bz linker (which consists of a 2,2,6,6-tetramethylpiperidine-1-oxy moiety connected to a benzyl group) that was developed to perform free-radical-initiated peptide sequencing. Comparative collisional activation experiments (collision-induced dissociation and higher-energy collision-induced dissociation) with both cross-linkers were conducted in negative electrospray ionization mode with an Orbitrap Fusion mass spectrometer using five model peptides. As hypothesized in a previous study, the presence of a cross-linked N-terminal aspartic acid residue seems to be the prerequisite for the loss of an intact peptide from the cross-linked products. As the BrPU linker combines a characteristic mass shift with an isotope signature, it presents a more favorable combination for automated assignment of cross-linked products compared with the TEMPO-Bz linker. [Figure not available: see fulltext.

Crystal structure of the Src family kinase Hck SH3-SH2 linker regulatory region supports an SH3-dominant activation mechanism.

PubMed

Alvarado, John J; Betts, Laurie; Moroco, Jamie A; Smithgall, Thomas E; Yeh, Joanne I

2010-11-12

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a "conformational switch" that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that "fine-tune" their sensitivities to activation by SH3-based ligands.

Independent movement, dimerization and stability of tandem repeats of chicken brain alpha-spectrin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusunoki, H.; Minasov, G.; Macdonald, R.I.

Previous X-ray crystal structures have shown that linkers of five amino acid residues connecting pairs of chicken brain {alpha}-spectrin and human erythroid {beta}-spectrin repeats can undergo bending without losing their {alpha}-helical structure. To test whether bending at one linker can influence bending at an adjacent linker, the structures of two and three repeat fragments of chicken brain {alpha}-spectrin have been determined by X-ray crystallography. The structure of the three-repeat fragment clearly shows that bending at one linker can occur independently of bending at an adjacent linker. This observation increases the possible trajectories of modeled chains of spectrin repeats. Furthermore, themore » three-repeat molecule crystallized as an antiparallel dimer with a significantly smaller buried interfacial area than that of {alpha}-actinin, a spectrin-related molecule, but large enough and of a type indicating biological specificity. Comparison of the structures of the spectrin and {alpha}-actinin dimers supports weak association of the former, which could not be detected by analytical ultracentrifugation, versus strong association of the latter, which has been observed by others. To correlate features of the structure with solution properties and to test a previous model of stable spectrin and dystrophin repeats, the number of inter-helical interactions in each repeat of several spectrin structures were counted and compared to their thermal stabilities. Inter-helical interactions, but not all interactions, increased in parallel with measured thermal stabilities of each repeat and in agreement with the thermal stabilities of two and three repeats and also partial repeats of spectrin.« less

Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome.

PubMed

Siegel, Dawn H; Ashton, Gabrielle H S; Penagos, Homero G; Lee, James V; Feiler, Heidi S; Wilhelmsen, Kirk C; South, Andrew P; Smith, Frances J D; Prescott, Alan R; Wessagowit, Vesarat; Oyama, Noritaka; Akiyama, Masashi; Al Aboud, Daifullah; Al Aboud, Khalid; Al Githami, Ahmad; Al Hawsawi, Khalid; Al Ismaily, Abla; Al-Suwaid, Raouf; Atherton, David J; Caputo, Ruggero; Fine, Jo-David; Frieden, Ilona J; Fuchs, Elaine; Haber, Richard M; Harada, Takashi; Kitajima, Yasuo; Mallory, Susan B; Ogawa, Hideoki; Sahin, Sedef; Shimizu, Hiroshi; Suga, Yasushi; Tadini, Gianluca; Tsuchiya, Kikuo; Wiebe, Colin B; Wojnarowska, Fenella; Zaghloul, Adel B; Hamada, Takahiro; Mallipeddi, Rajeev; Eady, Robin A J; McLean, W H Irwin; McGrath, John A; Epstein, Ervin H

2003-07-01

Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed "KIND1" [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.

Methyl 2-methyl-4-(oxiran-2-ylmeth-oxy)-2H-1,2-benzothia-zine-3-carboxyl-ate 1,1-dioxide.

PubMed

Ahmad, Matloob; Siddiqui, Hamid Latif; Zia-Ur-Rehman, Muhammad; Elsegood, Mark R J; Weaver, George W

2010-01-09

In the title compound, C(14)H(15)NO(6)S, the thia-zine ring adopts a distorted half-chair conformation. The structure displays several cooperative weak inter-molecular C-H⋯O hydrogen-bonding inter-actions, giving rise to a two-dimensional sheet packing motif. The CH(2) group in the meth-oxy linker to the oxirane ring, and the CH group in that ring, exhibit twofold positional disorder. The three-membered oxirane ring is twisted approximately perpendicular with respect to thia-zine ring (dihedral angle = 60/86° for the major/minor disorder components). 1,2-Benzothia-zines of this kind have a wide range of biological activities and are mainly used as medicines in the treatment of inflammation and rheumatoid arthritis.

Functional interactions between A' helices in the C-linker of open CNG channels.

PubMed

Hua, Li; Gordon, Sharona E

2005-03-01

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels that are activated by the direct binding of the cyclic nucleotides cAMP and cGMP. The region linking the last membrane-spanning region (S6) to the cyclic nucleotide binding domain in the COOH terminus, termed the C-linker, has been shown to play an important role in coupling cyclic nucleotide binding to opening of the pore. In this study, we explored the intersubunit proximity between the A' helices of the C-linker regions of CNGA1 in functional channels using site-specific cysteine substitution. We found that intersubunit disulfide bonds can be formed between the A' helices in open channels, and that inducing disulfide bonds in most of the studied constructs resulted in potentiation of channel activation. This suggests that the A' helices of the C-linker regions are in close proximity when the channel is in the open state. Our finding is not compatible with a homology model of the CNGA1 C-linker made from the recently published X-ray crystallographic structure of the hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channel COOH terminus, and leads us to suggest that the C-linker region depicted in the crystal structure may represent the structure of the closed state. The opening conformational change would then involve a movement of the A' helices from a position parallel to the axis of the membrane to one perpendicular to the axis of the membrane.

Composite materials with metal oxide attached to lead chalcogenide nanocrystal quantum dots with linkers

DOEpatents

Fuke, Nobuhiro; Koposov, Alexey Y; Sykora, Milan; Hoch, Laura

2014-12-16

Composite materials useful for devices such as photoelectrochemical solar cells include a substrate, a metal oxide film on the substrate, nanocrystalline quantum dots (NQDs) of lead sulfide, lead selenide, and lead telluride, and linkers that attach the NQDs to the metal oxide film. Suitable linkers preserve the 1s absorption peak of the NQDs. A suitable linker has a general structure A-B-C where A is a chemical group adapted for binding to a MO.sub.x and C is a chemical group adapted for binding to a NQD and B is a divalent, rigid, or semi-rigid organic spacer moiety. Other linkers that preserve the 1s absorption peak may also be used.

Construction of hierarchically porous metal–organic frameworks through linker labilization

DOE PAGES

Yuan, Shuai; Zou, Lanfang; Qin, Jun-Sheng; ...

2017-05-25

One major goal of metal–organic framework (MOF) research is the expansion of pore size and volume. Although many approaches have been attempted to increase the pore size of MOF materials, it is still a challenge to construct MOFs with precisely customized pore apertures for specific applications. W present a new method, namely linker labilization, to increase the MOF porosity and pore size, giving rise to hierarchical-pore architectures. Microporous MOFs with robust metal nodes and pro-labile linkers were initially synthesized. The mesopores were subsequently created as crystal defects through the splitting of a pro-labile-linker and the removal of the linker fragmentsmore » by acid treatment. We also demonstrate that linker labilization method can create controllable hierarchical porous structures in stable MOFs, which facilitates the diffusion and adsorption process of guest molecules to improve the performances of MOFs in adsorption and catalysis.« less

Construction of hierarchically porous metal-organic frameworks through linker labilization

NASA Astrophysics Data System (ADS)

Yuan, Shuai; Zou, Lanfang; Qin, Jun-Sheng; Li, Jialuo; Huang, Lan; Feng, Liang; Wang, Xuan; Bosch, Mathieu; Alsalme, Ali; Cagin, Tahir; Zhou, Hong-Cai

2017-05-01

A major goal of metal-organic framework (MOF) research is the expansion of pore size and volume. Although many approaches have been attempted to increase the pore size of MOF materials, it is still a challenge to construct MOFs with precisely customized pore apertures for specific applications. Herein, we present a new method, namely linker labilization, to increase the MOF porosity and pore size, giving rise to hierarchical-pore architectures. Microporous MOFs with robust metal nodes and pro-labile linkers were initially synthesized. The mesopores were subsequently created as crystal defects through the splitting of a pro-labile-linker and the removal of the linker fragments by acid treatment. We demonstrate that linker labilization method can create controllable hierarchical porous structures in stable MOFs, which facilitates the diffusion and adsorption process of guest molecules to improve the performances of MOFs in adsorption and catalysis.

The linker region of AraC protein.

PubMed Central

Eustance, R J; Schleif, R F

1996-01-01

AraC protein, a transcriptional regulator of the L-arabinose operon in Escherichia coli, is dimeric. Each monomer consists of a domain for DNA binding plus transcription activation and a domain for dimerization plus arabinose binding. These are connected to one another by a linker region of at least 5 amino acids. Here we have addressed the question of whether any of the amino acids in the linker region play active, specific, and crucial structural roles or whether these amino acids merely serve as passive spacers between the functional domains. We found that all but one of the linker amino acids can be changed to other amino acids individually and in small groups without substantially affecting the ability of AraC protein to activate transcription when arabinose is present. When, however, the entire linker region is replaced with linker sequences from other proteins, the functioning of AraC is impaired. PMID:8955380

Construction of hierarchically porous metal–organic frameworks through linker labilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Shuai; Zou, Lanfang; Qin, Jun-Sheng

One major goal of metal–organic framework (MOF) research is the expansion of pore size and volume. Although many approaches have been attempted to increase the pore size of MOF materials, it is still a challenge to construct MOFs with precisely customized pore apertures for specific applications. W present a new method, namely linker labilization, to increase the MOF porosity and pore size, giving rise to hierarchical-pore architectures. Microporous MOFs with robust metal nodes and pro-labile linkers were initially synthesized. The mesopores were subsequently created as crystal defects through the splitting of a pro-labile-linker and the removal of the linker fragmentsmore » by acid treatment. We also demonstrate that linker labilization method can create controllable hierarchical porous structures in stable MOFs, which facilitates the diffusion and adsorption process of guest molecules to improve the performances of MOFs in adsorption and catalysis.« less

Solution conformation of a cohesin module and its scaffoldin linker from a prototypical cellulosome.

PubMed

Galera-Prat, Albert; Pantoja-Uceda, David; Laurents, Douglas V; Carrión-Vázquez, Mariano

2018-04-15

Bacterial cellulases are drawing increased attention as a means to obtain plentiful chemical feedstocks and fuels from renewable lignocellulosic biomass sources. Certain bacteria deploy a large extracellular multi-protein complex, called the cellulosome, to degrade cellulose. Scaffoldin, a key non-catalytic cellulosome component, is a large protein containing a cellulose-specific carbohydrate-binding module and several cohesin modules which bind and organize the hydrolytic enzymes. Despite the importance of the structure and protein/protein interactions of the cohesin module in the cellulosome, its structure in solution has remained unknown to date. Here, we report the backbone 1 H, 13 C and 15 N NMR assignments of the Cohesin module 5 from the highly stable and active cellulosome from Clostridium thermocellum. These data reveal that this module adopts a tightly packed, well folded and rigid structure in solution. Furthermore, since in scaffoldin, the cohesin modules are connected by linkers we have also characterized the conformation of a representative linker segment using NMR spectroscopy. Analysis of its chemical shift values revealed that this linker is rather stiff and tends to adopt extended conformations. This suggests that the scaffoldin linkers act to minimize interactions between cohesin modules. These results pave the way towards solution studies on cohesin/dockerin's fascinating dual-binding mode. Copyright © 2018 Elsevier Inc. All rights reserved.

Neck linker length determines the degree of processivity in kinesin-1 and kinesin-2 motors.

PubMed

Shastry, Shankar; Hancock, William O

2010-05-25

Defining the mechanical and biochemical determinates of kinesin processivity is important for understanding how diverse kinesins are tuned for specific cellular functions. Because transmission of mechanical forces through the 14-18 amino acid neck linker domain underlies coordinated stepping, we investigated the role of neck linker length, charge, and structure in kinesin-1 and kinesin-2 motor behavior. For optimum comparison with kinesin-1, the KIF3A head and neck linker of kinesin-2 were fused to the kinesin-1 neck coil and rod. Extending the 14-residue kinesin-1 neck linker reduced processivity, and shortening the 17-residue kinesin-2 neck linker enhanced processivity. When a proline in the kinesin-2 neck linker was replaced, kinesin-1 and kinesin-2 run lengths scaled identically with neck linker length, despite moving at different speeds. In low-ionic-strength buffer, charge had a dominant effect on motor processivity, which resolves ongoing controversy regarding the effect of neck linker length on kinesin processivity. From stochastic simulations, the results are best explained by neck linker extension slowing strain-dependent detachment of the rear head along with diminishing strain-dependent inhibition of ATP binding. These results help delineate how interhead strain maximizes stepping and suggest that less processive kinesins are tuned to coordinate with other motors differently than the maximally processive kinesin-1. Copyright 2010 Elsevier Ltd. All rights reserved.

Liquid-Crystalline Elastomers with Gold Nanoparticle Cross-Linkers.

PubMed

Wójcik, Michał M; Wróbel, Jarosław; Jańczuk, Zuzanna Z; Mieczkowski, Józef; Górecka, Ewa; Choi, Joonmyung; Cho, Maenghyo; Pociecha, Damian

2017-07-03

Embedding nanoparticles in a responsive polymer matrix is a formidable way to fabricate hybrid materials with predesigned properties and prospective applications in actuators, mechanically tunable optical elements, and electroclinic films. However, achieving chemical compatibility between nanoparticles and organic matter is not trivial and often results in disordered structures. Herein, it is shown that using nanoparticles as exclusive cross-linkers in the preparation of liquid-crystalline polymers can yield long-range-ordered liquid-crystalline elastomers with high loadings of well-dispersed nanoparticles, as confirmed by small-angle XRD measurements. Moreover, the strategy of incorporating NPs as cross-linking units does not result in disruption of mechanical properties of the polymer, and this phenomenon was explained by the means of all-atom molecular dynamics simulations. Such materials can exhibit switchable behavior under thermal stimulus with stability spanning over multiple heating/cooling cycles. The presented strategy has proven to be a promising approach for the preparation of new types of hybrid liquid-crystalline elastomers that can be of value for future photonic applications. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel

PubMed Central

James, Zachary M.; Borst, Andrew J.; Haitin, Yoni; Frenz, Brandon; DiMaio, Frank; Zagotta, William N.; Veesler, David

2017-01-01

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae—which shares sequence similarity to eukaryotic CNG and HCN channels—in the presence of a saturating concentration of cAMP. A short S4–S5 linker connects nearby voltage-sensing and pore domains to produce a non–domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies. PMID:28396445

Pentasubstituted ferrocene and dirhodium(II) tetracarboxylate as building blocks for discrete fullerene-like and extended supramolecular structures.

PubMed

Tong, Lok H; Guénée, Laure; Williams, Alan F

2011-03-21

The synthesis of a penta(1-methylpyrazole)ferrocenyl phosphine oxide ligand (1) [Fe(C(5)(C(3)H(2)N(2)CH(3))(5))(C(5)H(4)PO(t-C(4)H(9))(2))] is reported together with its X-ray crystal structure. Its self-assembly behavior with a dirhodium(II) tetraoctanoate linker (2) [Rh(2)(O(2)CC(7)H(15))(4)] was investigated for construction of fullerene-like assemblies of composition [(ligand)(12)(linker)(30)]. Reaction between 1 and 2 in acetonitrile resulted in the formation of a light purple precipitate (3). Evidence for the ligand-to-linker ratio of 1:2.5 expected for a fullerene-like structure [Fe(C(5)(C(3)H(2)N(2)CH(3))(5))(C(5)H(4)PO(t-C(4)H(9))(2))](12)[Rh(2)(O(2)CC(7)H(15))(4)](30) was obtained from (1)H NMR and elemental analysis. IR and Raman studies confirmed the diaxially bound coordination environment of the dirhodium linker by comparing the stretching frequencies of the carboxylate group and the rhodium-rhodium bond with those in model compound (5), [Rh(2)(O(2)CC(7)H(15))(4)](C(3)H(3)N(2)CH(3))(2), the bis-adduct of linker 2 with 1-methylpyrazole. X-ray powder diffraction and molecular modeling studies provide additional support for the formation of a spherical molecule topologically identical to fullerene with a diameter of approximately 38 Å and a molecular formula of [(1)(12)(2)(30)]. Dissolution of 3 in tetrahydrofuran (THF) followed by layering with acetonitrile afforded purple crystals of [(1)(2)(2)](∞) (6) [Fe(C(5)(C(3)H(2)N(2)CH(3))(5))(C(5)H(4)PO(t-C(4)H(9))(2))][Rh(2)(O(2)CC(7)H(15))(4)](2) with a two-dimensional polymeric structure determined by X-ray crystallography. The dirhodium linkers link ferrocenyl units by coordination to the pyrazoles but only four of the five pyrazole moieties of the pentapyrazole ligand are coordinated. The ligand-to-linker ratio of 1:2 in 6 was confirmed by (1)H NMR spectroscopy and elemental analysis, while results from IR and Raman are in agreement with the diaxially coordinated environment of the linker observed in the solid state.

Human polyhomeotic homolog 3 (PHC3) sterile alpha motif (SAM) linker allows open-ended polymerization of PHC3 SAM.

PubMed

Robinson, Angela K; Leal, Belinda Z; Nanyes, David R; Kaur, Yogeet; Ilangovan, Udayar; Schirf, Virgil; Hinck, Andrew P; Demeler, Borries; Kim, Chongwoo A

2012-07-10

Sterile alpha motifs (SAMs) are frequently found in eukaryotic genomes. An intriguing property of many SAMs is their ability to self-associate, forming an open-ended polymer structure whose formation has been shown to be essential for the function of the protein. What remains largely unresolved is how polymerization is controlled. Previously, we had determined that the stretch of unstructured residues N-terminal to the SAM of a Drosophila protein called polyhomeotic (Ph), a member of the polycomb group (PcG) of gene silencers, plays a key role in controlling Ph SAM polymerization. Ph SAM with its native linker created shorter polymers compared to Ph SAM attached to either a random linker or no linker. Here, we show that the SAM linker for the human Ph ortholog, polyhomeotic homolog 3 (PHC3), also controls PHC3 SAM polymerization but does so in the opposite fashion. PHC3 SAM with its native linker allows longer polymers to form compared to when attached to a random linker. Attaching the PHC3 SAM linker to Ph SAM also resulted in extending Ph SAM polymerization. Moreover, in the context of full-length Ph protein, replacing the SAM linker with PHC3 SAM linker, intended to create longer polymers, resulted in greater repressive ability for the chimera compared to wild-type Ph. These findings show that polymeric SAM linkers evolved to modulate a wide dynamic range of SAM polymerization abilities and suggest that rationally manipulating the function of SAM containing proteins through controlling their SAM polymerization may be possible.

Accurate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity.

PubMed

Sindbert, Simon; Kalinin, Stanislav; Nguyen, Hien; Kienzler, Andrea; Clima, Lilia; Bannwarth, Willi; Appel, Bettina; Müller, Sabine; Seidel, Claus A M

2011-03-02

In Förster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A detailed analysis of possible orientation effects (κ(2) problem) indicates that, for short linkers and unknown local environments, additional κ(2)-related uncertainties are clearly outweighed by better defined dye positions.

Human linker histones: interplay between phosphorylation and O-β-GlcNAc to mediate chromatin structural modifications

PubMed Central

2011-01-01

Eukaryotic chromatin is a combination of DNA and histone proteins. It is established fact that epigenetic mechanisms are associated with DNA and histones. Initial studies emphasize on core histones association with DNA, however later studies prove the importance of linker histone H1 epigenetic. There are many types of linker histone H1 found in mammals. These subtypes are cell specific and their amount in different types of cells varies as the cell functions. Many types of post-translational modifications which occur on different residues in each subtype of linker histone H1 induce conformational changes and allow the different subtypes of linker histone H1 to interact with chromatin at different stages during cell cycle which results in the regulation of transcription and gene expression. Proposed O-glycosylation of linker histone H1 promotes condensation of chromatin while phosphorylation of linker histone H1 is known to activate transcription and gene regulation by decondensation of chromatin. Interplay between phosphorylation and O-β-GlcNAc modification on Ser and Thr residues in each subtype of linker histone H1 in Homo sapiens during cell cycle may result in diverse functional regulation of proteins. This in silico study describes the potential phosphorylation, o-glycosylation and their possible interplay sites on conserved Ser/Thr residues in various subtypes of linker histone H1 in Homo sapiens. PMID:21749719

Molecular dissection of the interaction between the SH3 domain and the SH2-Kinase Linker region in PTK6.

PubMed

Kim, Han Ie; Jung, Jinwon; Lee, Eun-Saem; Kim, Yong-Chul; Lee, Weontae; Lee, Seung-Taek

2007-11-03

PTK6 (also known as Brk) is an intracellular tyrosine kinase that contains SH3, SH2, and tyrosine kinase catalytic (Kinase) domains. The SH3 domain of PTK6 interacts with the N-terminal half of the linker (Linker) region between the SH2 and Kinase domains. Site-directed mutagenesis and surface plasmon resonance studies showed that a tryptophan residue (Trp44) in the SH3 domain and proline residues in the Linker region, in the order of Pro177, Pro175, and Pro179, contribute to the interaction. The three-dimensional modeled structure of the SH3-Linker complex was in agreement with the biochemical data. Disruption of the intramolecular interaction between the SH3 domain and the Linker region by mutation of Trp44, Pro175, Pro177, and Pro179 markedly increased the catalytic activity of PTK6 in HEK 293 cells. These results demonstrate that Trp44 in the SH3 domain and Pro177, Pro175, and Pro179 in the N-terminal half of the Linker region play important roles in the SH3-Linker interaction to maintain the protein in an inactive conformation along with the phosphorylated Tyr447-SH2 interaction.

Micromachined silicon acoustic delay line with improved structural stability and acoustic directivity for real-time photoacoustic tomography

NASA Astrophysics Data System (ADS)

Cho, Young; Kumar, Akhil; Xu, Song; Zou, Jun

2017-03-01

Recent studies have shown that micromachined silicon acoustic delay lines can provide a promising solution to achieve real-time photoacoustic tomography without the need for complex transducer arrays and data acquisition electronics. However, as its length increases to provide longer delay time, the delay line becomes more vulnerable to structural instability due to reduced mechanical stiffness. In addition, the small cross-section area of the delay line results in a large acoustic acceptance angle and therefore poor directivity. To address these two issues, this paper reports the design, fabrication, and testing of a new silicon acoustic delay line enhanced with 3D printed polymer micro linker structures. First, mechanical deformation of the silicon acoustic delay line (with and without linker structures) under gravity was simulated by using finite element method. Second, the acoustic crosstalk and acoustic attenuation caused by the polymer micro linker structures were evaluated with both numerical simulation and ultrasound transmission testing. The result shows that the use of the polymer micro linker structures significantly improves the structural stability of the silicon acoustic delay lines without creating additional acoustic attenuation and crosstalk. In addition, a new tapered design for the input terminal of the delay line was also investigate to improve its acoustic directivity by reducing the acoustic acceptance angle. These two improvements are expected to provide an effective solution to eliminate current limitations on the achievable acoustic delay time and out-of-plane imaging resolution of micromachined silicon acoustic delay line arrays.

Structure of the voltage-gated K⁺ channel Eag1 reveals an alternative voltage sensing mechanism.

PubMed

Whicher, Jonathan R; MacKinnon, Roderick

2016-08-12

Voltage-gated potassium (K(v)) channels are gated by the movement of the transmembrane voltage sensor, which is coupled, through the helical S4-S5 linker, to the potassium pore. We determined the single-particle cryo-electron microscopy structure of mammalian K(v)10.1, or Eag1, bound to the channel inhibitor calmodulin, at 3.78 angstrom resolution. Unlike previous K(v) structures, the S4-S5 linker of Eag1 is a five-residue loop and the transmembrane segments are not domain swapped, which suggest an alternative mechanism of voltage-dependent gating. Additionally, the structure and position of the S4-S5 linker allow calmodulin to bind to the intracellular domains and to close the potassium pore, independent of voltage-sensor position. The structure reveals an alternative gating mechanism for K(v) channels and provides a template to further understand the gating properties of Eag1 and related channels. Copyright © 2016, American Association for the Advancement of Science.

Structure-function analysis of Sua5 protein reveals novel functional motifs required for the biosynthesis of the universal t6A tRNA modification.

PubMed

Pichard-Kostuch, Adeline; Zhang, Wenhua; Liger, Dominique; Daugeron, Marie-Claire; Letoquart, Juliette; Li de la Sierra-Gallay, Ines; Forterre, Patrick; Collinet, Bruno; van Tilbeurgh, Herman; Basta, Tamara

2018-04-12

N6-threonyl-carbamoyl adenosine (t6A) is a universal tRNA modification found at position 37, next to the anticodon, in almost all tRNAs decoding ANN codons (where N = A, U, G or C). t6A stabilizes the codon-anticodon interaction and hence promotes translation fidelity. The first step of the biosynthesis of t6A, the production of threonyl-carbamoyl adenylate (TC-AMP), is catalyzed by the Sua5/TsaC family of enzymes. While TsaC is a single domain protein, Sua5 enzymes are composed of the TsaC-like domain, a linker and an extra domain called SUA5 of unknown function. In the present study, we report structure-function analysis of Pyrococcus abyssi Sua5 (Pa-Sua5). Crystallographic data revealed binding sites for bicarbonate substrate and pyrophosphate product. The linker of Pa-Sua5 forms a loop structure that folds into the active site gorge and closes it. Using structure-guided mutational analysis we established that the conserved sequence motifs in the linker and the domain-domain interface are essential for the function of Pa-Sua5. We propose that the linker participates actively in the biosynthesis of TC-AMP by binding to ATP/PPi and by stabilizing the N-carboxy-L-threonine intermediate. Hence, TsaC orthologs which lack such a linker and SUA5 domain use different mechanism for TC-AMP synthesis. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Prevention of nanoparticle coalescence under high-temperature annealing.

PubMed

Mizuno, Mikihisa; Sasaki, Yuichi; Yu, Andrew C C; Inoue, Makoto

2004-12-21

An effective method of employing 3-aminopropyldimethylethoxysilane linker molecules to stabilize 4.4 nm FePt nanoparticle monolayer films on a SiO2 substrate as well as to prevent coalescence of the particles under 800 degrees C annealing is reported. As-deposited FePt nanoparticle films in chemically disordered face-centered-cubic phase transform to mostly chemically ordered L1 0 structure after annealing, while the nanoparticles are free from serious coalescence. The method may fulfill the pressing need to prevent nanoparticle coalescence under high-temperature annealing for the development of FePt nanoparticle based products, such as ultrahigh-density magnetic recording media and novel memory devices.

Density functional theory with van der waals corrections study of the adsorption of alkyl, alkylthiol, alkoxyl, and amino-alkyl chains on the H:Si(111) surface.

PubMed

Arefi, Hadi H; Nolan, Michael; Fagas, Giorgos

2014-11-11

Surface modification of silicon with organic monolayers tethered to the surface by different linkers is an important process in realizing future miniaturized electronic and sensor devices. Understanding the roles played by the nature of the linking group and the chain length on the adsorption structures and stabilities of these assemblies is vital to advance this technology. This paper presents a density functional theory (DFT) study of the hydrogen passivated Si(111) surface modified with alkyl chains of the general formula H:Si-(CH2)n-CH2 and H:Si-X-(CH2)n-CH3, where X = NH, O, S and n = (0, 1, 3, 5, 7, 9, 11), at half coverage. For (X)-hexane and (X)-dodecane functionalization, we also examined various coverages up to full monolayer grafting in order to validate the result of half covered surface and the linker effect on the coverage. We find that it is necessary to take into account the van der Waals interaction between the alkyl chains. The strongest binding is for the oxygen linker, followed by S, N, and C, irrespective of chain length. The result revealed that the sequence of the stability is independent of coverage; however, linkers other than carbon can shift the optimum coverage considerably and allow further packing density. For all linkers apart from sulfur, structural properties, in particular, surface-linker-chain angles, saturate to a single value once n > 3. For sulfur, we identify three regimes, namely, n = 0-3, n = 5-7, and n = 9-11, each with its own characteristic adsorption structures. Where possible, our computational results are shown to be consistent with the available experimental data and show how the fundamental structural properties of modified Si surfaces can be controlled by the choice of linking group and chain length.

Crystal structure of the Agrobacterium virulence complex VirE1-VirE2 reveals a flexible protein that can accommodate different partners.

PubMed

Dym, Orly; Albeck, Shira; Unger, Tamar; Jacobovitch, Jossef; Branzburg, Anna; Michael, Yigal; Frenkiel-Krispin, Daphna; Wolf, Sharon Grayer; Elbaum, Michael

2008-08-12

Agrobacterium tumefaciens infects its plant hosts by a mechanism of horizontal gene transfer. This capability has led to its widespread use in artificial genetic transformation. In addition to DNA, the bacterium delivers an abundant ssDNA binding protein, VirE2, whose roles in the host include protection from cytoplasmic nucleases and adaptation for nuclear import. In Agrobacterium, VirE2 is bound to its acidic chaperone VirE1. When expressed in vitro in the absence of VirE1, VirE2 is prone to oligomerization and forms disordered filamentous aggregates. These filaments adopt an ordered solenoidal form in the presence of ssDNA, which was characterized previously by electron microscopy and three-dimensional image processing. VirE2 coexpressed in vitro with VirE1 forms a soluble heterodimer. VirE1 thus prevents VirE2 oligomerization and competes with its binding to ssDNA. We present here a crystal structure of VirE2 in complex with VirE1, showing that VirE2 is composed of two independent domains presenting a novel fold, joined by a flexible linker. Electrostatic interactions with VirE1 cement the two domains of VirE2 into a locked form. Comparison with the electron microscopy structure indicates that the VirE2 domains adopt different relative orientations. We suggest that the flexible linker between the domains enables VirE2 to accommodate its different binding partners.

The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding.

PubMed

Chen, Shugui; Brier, Sébastien; Smithgall, Thomas E; Engen, John R

2007-04-01

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.

Stabilization of RNA hairpins using non-nucleotide linkers and circularization.

PubMed

Kiliszek, Agnieszka; Blaszczyk, Leszek; Kierzek, Ryszard; Rypniewski, Wojciech

2017-06-02

An RNA hairpin is an essential structural element of RNA. Hairpins play crucial roles in gene expression and intermolecular recognition but are also involved in the pathogenesis of some congenital diseases. Structural studies of the hairpin motifs are impeded by their thermodynamic instability, as they tend to unfold to form duplexes, especially at high concentrations required for crystallography or nuclear magnetic resonance spectroscopy. We have elaborated techniques to stabilize the RNA hairpins by linking the free ends of the RNA strand at the base of the hairpin stem. One method involves stilbene diether or hexaethylene glycol linkers and circularization by T4 RNA ligase. Another method uses click chemistry to stitch the RNA ends with a triazole linker. Both techniques are efficient and easy to perform. They should be useful in making stable, biologically relevant RNA constructs for structural studies. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Application of linker technique to trap transiently interacting protein complexes for structural studies

PubMed Central

Reddy Chichili, Vishnu Priyanka; Kumar, Veerendra; Sivaraman, J.

2016-01-01

Protein-protein interactions are key events controlling several biological processes. We have developed and employed a method to trap transiently interacting protein complexes for structural studies using glycine-rich linkers to fuse interacting partners, one of which is unstructured. Initial steps involve isothermal titration calorimetry to identify the minimum binding region of the unstructured protein in its interaction with its stable binding partner. This is followed by computational analysis to identify the approximate site of the interaction and to design an appropriate linker length. Subsequently, fused constructs are generated and characterized using size exclusion chromatography and dynamic light scattering experiments. The structure of the chimeric protein is then solved by crystallization, and validated both in vitro and in vivo by substituting key interacting residues of the full length, unlinked proteins with alanine. This protocol offers the opportunity to study crucial and currently unattainable transient protein interactions involved in various biological processes. PMID:26985443

Pathogenic proline mutation in the linker between spectrin repeats: disease caused by spectrin unfolding

PubMed Central

Johnson, Colin P.; Gaetani, Massimiliano; Ortiz, Vanessa; Bhasin, Nishant; Harper, Sandy

2007-01-01

Pathogenic mutations in α and β spectrin result in a variety of syndromes, including hereditary elliptocytosis (HE), hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS). Although some mutations clearly lie at sites of interaction, such as the sites of spectrin α-βtetramer formation, a surprising number of HE-causing mutations have been identified within linker regions between distal spectrin repeats. Here we apply solution structural and single molecule methods to the folding and stability of recombinant proteins consisting of the first 5 spectrin repeats of α-spectrin, comparing normal spectrin with a pathogenic linker mutation, Q471P, between repeats R4 and R5. Results show that the linker mutation destabilizes a significant fraction of the 5-repeat construct at 37°C, whereas the WT remains fully folded well above body temperature. In WT protein, helical linkers propagate stability from one repeat to the next, but the mutation disrupts the stabilizing influence of adjacent repeats. The results suggest a molecular mechanism for the high frequency of disease caused by proline mutations in spectrin linkers. PMID:17192394

Methyl 2-methyl-4-(oxiran-2-ylmeth­oxy)-2H-1,2-benzothia­zine-3-carboxyl­ate 1,1-dioxide

PubMed Central

Ahmad, Matloob; Siddiqui, Hamid Latif; Zia-ur-Rehman, Muhammad; Elsegood, Mark R. J.; Weaver, George W.

2010-01-01

In the title compound, C14H15NO6S, the thia­zine ring adopts a distorted half-chair conformation. The structure displays several cooperative weak inter­molecular C—H⋯O hydrogen-bonding inter­actions, giving rise to a two-dimensional sheet packing motif. The CH2 group in the meth­oxy linker to the oxirane ring, and the CH group in that ring, exhibit twofold positional disorder. The three-membered oxirane ring is twisted approximately perpendicular with respect to thia­zine ring (dihedral angle = 60/86° for the major/minor disorder components). 1,2-Benzothia­zines of this kind have a wide range of biological activities and are mainly used as medicines in the treatment of inflammation and rheumatoid arthritis. PMID:21579762

Bis-Acridines as Lead Antiparasitic Agents: Structure-Activity Analysis of a Discrete Compound Library In Vitro▿

PubMed Central

Caffrey, Conor R.; Steverding, Dietmar; Swenerton, Ryan K.; Kelly, Ben; Walshe, Deirdre; Debnath, Anjan; Zhou, Yuan-Min; Doyle, Patricia S.; Fafarman, Aaron T.; Zorn, Julie A.; Land, Kirkwood M.; Beauchene, Jessica; Schreiber, Kimberly; Moll, Heidrun; Ponte-Sucre, Alicia; Schirmeister, Tanja; Saravanamuthu, Ahilan; Fairlamb, Alan H.; Cohen, Fred E.; McKerrow, James H.; Weisman, Jennifer L.; May, Barnaby C. H.

2007-01-01

Parasitic diseases are of enormous public health significance in developing countries—a situation compounded by the toxicity of and resistance to many current chemotherapeutics. We investigated a focused library of 18 structurally diverse bis-acridine compounds for in vitro bioactivity against seven protozoan and one helminth parasite species and compared the bioactivities and the cytotoxicities of these compounds toward various mammalian cell lines. Structure-activity relationships demonstrated the influence of both the bis-acridine linker structure and the terminal acridine heterocycle on potency and cytotoxicity. The bioactivity of polyamine-linked acridines required a minimum linker length of approximately 10 Å. Increasing linker length resulted in bioactivity against most parasites but also cytotoxicity toward mammalian cells. N alkylation, but less so N acylation, of the polyamine linker ameliorated cytotoxicity while retaining bioactivity with 50% effective concentration (EC50) values similar to or better than those measured for standard drugs. Substitution of the polyamine for either an alkyl or a polyether linker maintained bioactivity and further alleviated cytotoxicity. Polyamine-linked compounds in which the terminal acridine heterocycle had been replaced with an aza-acridine also maintained acceptable therapeutic indices. The most potent compounds recorded low- to mid-nanomolar EC50 values against Plasmodium falciparum and Trypanosoma brucei; otherwise, low-micromolar potencies were measured. Importantly, the bioactivity of the library was independent of P. falciparum resistance to chloroquine. Compound bioactivity was a function of neither the potential to bis-intercalate DNA nor the inhibition of trypanothione reductase, an important drug target in trypanosomatid parasites. Our approach illustrates the usefulness of screening focused compound libraries against multiple parasite targets. Some of the bis-acridines identified here may represent useful starting points for further lead optimization. PMID:17371810

Acetone-Linked Peptides: A Convergent Approach for Peptide Macrocyclization and Labeling.

PubMed

Assem, Naila; Ferreira, David J; Wolan, Dennis W; Dawson, Philip E

2015-07-20

Macrocyclization is a broadly applied approach for overcoming the intrinsically disordered nature of linear peptides. Herein, it is shown that dichloroacetone (DCA) enhances helical secondary structures when introduced between peptide nucleophiles, such as thiols, to yield an acetone-linked bridge (ACE). Aside from stabilizing helical structures, the ketone moiety embedded in the linker can be modified with diverse molecular tags by oxime ligation. Insights into the structure of the tether were obtained through co-crystallization of a constrained S-peptide in complex with RNAse S. The scope of the acetone-linked peptides was further explored through the generation of N-terminus to side chain macrocycles and a new approach for generating fused macrocycles (bicycles). Together, these studies suggest that acetone linking is generally applicable to peptide macrocycles with a specific utility in the synthesis of stabilized helices that incorporate functional tags. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Intra-molecular cross-linking of acidic residues for protein structure studies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kruppa, Gary Hermann; Young, Malin M.; Novak, Petr

2005-03-01

Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of themore » lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry, increasing the probability that the protein target of choice will yield sufficient distance constraints to develop a structural model.« less

When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture

PubMed Central

Barik, Sailen

2008-01-01

The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the α-helices and β-strands of proteins than within the more flexible linker regions (‘turns’ and ‘loops’) connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the α-helix and the β-strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures. PMID:15381847

When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture.

PubMed

Barik, Sailen

2004-09-01

The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the alpha-helices and beta-strands of proteins than within the more flexible linker regions ('turns' and 'loops') connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the alpha-helix and the beta-strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures.

Protein Structural Analysis via Mass Spectrometry-Based Proteomics

PubMed Central

Artigues, Antonio; Nadeau, Owen W.; Rimmer, Mary Ashley; Villar, Maria T.; Du, Xiuxia; Fenton, Aron W.; Carlson, Gerald M.

2017-01-01

Modern mass spectrometry (MS) technologies have provided a versatile platform that can be combined with a large number of techniques to analyze protein structure and dynamics. These techniques include the three detailed in this chapter: 1) hydrogen/deuterium exchange (HDX), 2) limited proteolysis, and 3) chemical crosslinking (CX). HDX relies on the change in mass of a protein upon its dilution into deuterated buffer, which results in varied deuterium content within its backbone amides. Structural information on surface exposed, flexible or disordered linker regions of proteins can be achieved through limited proteolysis, using a variety of proteases and only small extents of digestion. CX refers to the covalent coupling of distinct chemical species and has been used to analyze the structure, function and interactions of proteins by identifying crosslinking sites that are formed by small multi-functional reagents, termed crosslinkers. Each of these MS applications is capable of revealing structural information for proteins when used either with or without other typical high resolution techniques, including NMR and X-ray crystallography. PMID:27975228

Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase.

PubMed

Nadar, Shamraja S; Muley, Abhijeet B; Ladole, Mayur R; Joshi, Pranoti U

2016-03-01

Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase were prepared by precipitation and subsequent cross-linking. The non-toxic, biodegradable, biocompatible, renewable polysaccharide based macromolecular cross-linkers viz. agar, chitosan, dextran, and gum arabic were used as a substitute for traditional glutaraldehyde to augment activity recovery toward macromolecular substrate. Macromolecular cross-linkers were prepared by periodate mediated controlled oxidation of polysaccharides. The effects of precipitating agent, concentration and different cross-linkers on activity recovery of α-amylase CLEAs were investigated. α-Amylase aggregated with ammonium sulphate and cross-linked by dextran showed 91% activity recovery, whereas glutaraldehyde CLEAs (G-CLEAs) exhibited 42% activity recovery. M-CLEAs exhibited higher thermal stability in correlation with α-amylase and G-CLEAs. Moreover, dextran and chitosan M-CLEAs showed same affinity for starch hydrolysis as of free α-amylase. The changes in secondary structures revealed the enhancements in structural and conformational rigidity attributed by cross-linkers. Finally, after five consecutive cycles dextran M-CLEAs retained 1.25 times higher initial activity than G-CLEAs. Copyright © 2015 Elsevier B.V. All rights reserved.

Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex.

PubMed

Ihling, Christian; Schmidt, Andreas; Kalkhof, Stefan; Schulz, Daniela M; Stingl, Christoph; Mechtler, Karl; Haack, Michael; Beck-Sickinger, Annette G; Cooper, Dermot M F; Sinz, Andrea

2006-08-01

For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central alpha-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few days.

Structure and function of photosystem I–[FeFe] hydrogenase protein fusions: An all-atom molecular dynamics study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harris, Bradley J.; Cheng, Xiaolin; Frymier, Paul

2015-12-15

All-atom molecular dynamics (MD) simulation was used to study the solution dynamics and protein protein interactions of protein fusions of photosystem I (PSI) from Thermosynechococcus elongatus and an [FeFe]-hydrogenase (FeFe H 2ase) from Clostridium pasteurianum, a unique complex capable of photocatalytic hydrogen production. This study involved fusions of these two proteins via dithiol linkers of different length including decanedithiol, octanedithiol, and hexanedithiol, for which experimental data had previously been obtained. Evaluation of root-mean-squared deviations (RMSDs) relative to the respective crystal structures of PSI and the FeFe H 2ase shows that these fusion complexes approach stable equilibrium conformations during the MDmore » simulations. Investigating protein mobility via root-mean-squared fluctuations (RMSFs) reveals that tethering via the shortest hexanedithiol linker results in increased atomic fluctuations of both PSI and the hydrogenase in these fusion complexes. Furthermore, evaluation of the inter- and intraprotein electron transfer distances in these fusion complexes indicates that the structural changes in the FeFe H 2ase arising from ligation to PSI via the shortest hexanedithiol linker may hinder electron transport in the hydrogenase, thus providing a molecular level explanation for the observation that the medium-length octanedithiol linker gives the highest hydrogen production rate.« less

One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA.

PubMed

Mochizuki, Yuki; Biyani, Manish; Tsuji-Ueno, Sachika; Suzuki, Miho; Nishigaki, Koichi; Husimi, Yuzuru; Nemoto, Naoto

2011-09-12

A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.

Different states of synaptotagmin regulate evoked versus spontaneous release

PubMed Central

Bai, Hua; Xue, Renhao; Bao, Huan; Zhang, Leili; Yethiraj, Arun; Cui, Qiang; Chapman, Edwin R.

2016-01-01

The tandem C2-domains of synaptotagmin 1 (syt) function as Ca2+-binding modules that trigger exocytosis; in the absence of Ca2+, syt inhibits spontaneous release. Here, we used proline linkers to constrain and alter the relative orientation of these C2-domains. Short poly-proline helices have a period of three, so large changes in the relative disposition of the C2-domains result from changing the length of the poly-proline linker by a single residue. The length of the linker was varied one residue at a time, revealing a periodicity of three for the ability of the linker mutants to interact with anionic phospholipids and drive evoked synaptic transmission; syt efficiently drove exocytosis when its tandem C2-domains pointed in the same direction. Analysis of spontaneous release revealed a reciprocal relationship between the activation and clamping activities of the linker mutants. Hence, different structural states of syt underlie the control of distinct forms of synaptic transmission. PMID:27001899

Fabrication of zinc-dicarboxylate- and zinc-pyrazolate-carboxylate-framework thin films through vapour-solid deposition.

PubMed

Medishetty, Raghavender; Zhang, Zongji; Sadlo, Alexander; Cwik, Stefan; Peeters, Daniel; Henke, Sebastian; Mangayarkarasi, Nagarathinam; Devi, Anjana

2018-05-17

Fabrication of three-dimensional metal-organic framework (MOF) thin films has been investigated for the first time through the conversion of a ZnO layer via a pure vapour-solid deposition reaction at ambient pressure. The fabrication of MOF thin films with a dicarboxylate linker, (DMA)2[Zn3(bdc)4] (1) (bdc = 1,4-benzenedicarboxylate), and a carboxy-pyrazolate linker, [Zn4O(dmcapz)6] (2) (dmcapz = 3,5-dimethyl-4-carboxypyrazole), involves the deposition of the linker and/or the preparation of a composite film preliminarily and its subsequent conversion into a MOF film using closed cell thermal treatment. Furthermore, it was possible to isolate thin films with a MOF-5 isotype structure grown along the [110] direction, using a carboxy-pyrazolate linker. This was achieved just by the direct reaction of the ZnO film and the organic linker vapors, employing a simple route that demonstrates the feasibility of MOF thin film fabrication using inexpensive routes at ambient pressure.

The Impact of O-Glycan Chemistry on the Stability of Intrinsically Disordered Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beckham, Gregg T; Prates, Erica T; Crowley, Michael F

2018-03-02

Protein glycosylation is a diverse post-translational modification that serves myriad biological functions. O-linked glycans in particular vary widely in extent and chemistry in eukaryotes, with secreted proteins from fungi and yeast commonly exhibiting O-mannosylation in intrinsically disordered regions of proteins, likely for proteolysis protection, among other functions. However, it is not well understood why mannose is often the preferred glycan, and more generally, if the neighboring protein sequence and glycan have coevolved to protect against proteolysis in glycosylated intrinsically disordered proteins (IDPs). Here, we synthesized variants of a model IDP, specifically a natively O-mannosylated linker from a fungal enzyme, withmore » a-O-linked mannose, glucose, and galactose moieties, along with a non-glycosylated linker. Upon exposure to thermolysin, O-mannosylation, by far, provides the highest extent of proteolysis protection. To explain this observation, extensive molecular dynamics simulations were conducted, revealing that the axial configuration of the C2-hydroxyl group (2-OH) of a-mannose adjacent to the glycan-peptide bond strongly influences the conformational features of the linker. Specifically, a-mannose restricts the torsions of the IDP main chain more than other glycans whose equatorial 2-OH groups exhibit interactions that favor perpendicular glycan-protein backbone orientation. We suggest that IDP stiffening due to O-mannosylation impairs protease action, with contributions from protein-glycan interactions, protein flexibility, and protein stability. Our results further imply that resistance to proteolysis is an important driving force for evolutionary selection of a-mannose in eukaryotic IDPs, and more broadly, that glycan motifs for proteolysis protection likely coevolve with the protein sequence to which they attach.« less

Crystal structure of the Mus81-Eme1 complex.

PubMed

Chang, Jeong Ho; Kim, Jeong Joo; Choi, Jung Min; Lee, Jung Hoon; Cho, Yunje

2008-04-15

The Mus81-Eme1 complex is a structure-specific endonuclease that plays an important role in rescuing stalled replication forks and resolving the meiotic recombination intermediates in eukaryotes. We have determined the crystal structure of the Mus81-Eme1 complex. Both Mus81 and Eme1 consist of a central nuclease domain, two repeats of the helix-hairpin-helix (HhH) motif at their C-terminal region, and a linker helix. While each domain structure resembles archaeal XPF homologs, the overall structure is significantly different from those due to the structure of a linker helix. We show that a flexible intradomain linker that formed with 36 residues in the nuclease domain of Eme1 is essential for the recognition of DNA. We identified several basic residues lining the outer surface of the active site cleft of Mus81 that are involved in the interaction with a flexible arm of a nicked Holliday junction (HJ). These interactions might contribute to the optimal positioning of the opposite junction across the nick into the catalytic site, which provided the basis for the "nick and counternick" mechanism of Mus81-Eme1 and for the nicked HJ to be the favored in vitro substrate of this enzyme.

Bisquaternary pyridinium oximes: Comparison of in-vitro reactivation potency of compounds bearing aliphatic linkers and heteroaromatic linkers for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase

PubMed Central

Bharate, Sandip B.; Guo, Lilu; Reeves, Tony E.; Cerasoli, Douglas M.; Thompson, Charles M.

2009-01-01

Oxime reactivators are the drugs of choice for the post-treatment of OP (organophosphorus) intoxication and used widely for mechanistic and kinetic studies of OP-inhibited cholinesterases. The purpose of the present study was to evaluate new oxime compounds to reactivate acetylcholinesterase (AChE) inhibited by the OP paraoxon. Several new bisquaternary pyridinium oximes with heterocyclic linkers along with some known bisquaternary pyridinium oximes bearing aliphatic linkers were synthesized and evaluated for their in vitro reactivation potency against paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE). Results herein indicate that most of the compounds are better reactivators of EeAChE than of rHuAChE. The reactivation potency of two different classes of compounds with varying linker chains was compared and observed that the structure of the connecting chain is an important factor for the activity of the reactivators. At a higher concentration (10−3 M), compounds bearing aliphatic linker showed better reactivation than compounds with heterocyclic linkers. Interestingly, oximes with a heterocyclic linker inhibited AChE at higher concentration (10−3 M), whereas their ability to reactivate was increased at lower concentrations (10−4 M and 10−5 M). Compounds bearing either a thiophene linker 26, 46 or a furan linker 31 showed 59%, 49% and 52% reactivation of EeAChE, respectively, at 10−5 M. These compounds showed 14%, 6% and 15% reactivation of rHuAChE at 10−4 M. Amongst newly synthesized analogs with heterocyclic linkers (26–35 and 45–46), compound 31, bearing furan linker chain, was found to be the most effective reactivator with a kr 0.042 min−1, which is better than obidoxime (3) for paraoxon-inhibited EeAChE. Compound 31 showed a kr 0.0041 min−1 that is near equal to pralidoxime (1) for paraoxon-inhibited rHuAChE. PMID:20005727

Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis*S⃞

PubMed Central

Guhaniyogi, Jayita; Sohar, Istvan; Das, Kalyan; Stock, Ann M.; Lobel, Peter

2009-01-01

Late infantile neuronal ceroid lipofuscinosis is a fatal childhood neurological disorder caused by a deficiency in the lysosomal protease tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian member of the S53 family of serine proteases, a group characterized by a subtilisin-like fold, a Ser-Glu-Asp catalytic triad, and an acidic pH optimum. TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is proteolytically processed into the active enzyme after exposure to low pH in vitro or targeting to the lysosome in vivo. In this study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing four Asn-linked N-acetylglucosamines that is indistinguishable from fully glycosylated TPP1 in terms of autocatalytic processing of the proform and enzymatic properties of the mature protease. The crystal structure of deglycosylated pro-TPP1 was determined at 1.85 Å resolution. A large 151-residue C-shaped prodomain makes extensive contacts as it wraps around the surface of the catalytic domain with the two domains connected by a 24-residue flexible linker that passes through the substrate-binding groove. The proenzyme structure reveals suboptimal catalytic triad geometry with its propiece linker partially blocking the substrate-binding site, which together serve to prevent premature activation of the protease. Finally, we have identified numerous processing intermediates and propose a structural model that explains the pathway for TPP1 activation in vitro. These data provide new insights into TPP1 function and represent a valuable resource for constructing improved TPP1 variants for treatment of late infantile neuronal ceroid lipofuscinosis. PMID:19038967

Mesoscale Modeling of Chromatin Folding

NASA Astrophysics Data System (ADS)

Schlick, Tamar

2009-03-01

Eukaryotic chromatin is the fundamental protein/nucleic acid unit that stores the genetic material. Understanding how chromatin fibers fold and unfold in physiological conditions is important for interpreting fundamental biological processes like DNA replication and transcription regulation. Using a mesoscopic model of oligonucleosome chains and tailored sampling protocols, we elucidate the energetics of oligonucleosome folding/unfolding and the role of each histone tail, linker histones, and divalent ions in regulating chromatin structure. The resulting compact topologies reconcile features of the zigzag model with straight linker DNAs with the solenoid model with bent linker DNAs for optimal fiber organization and reveal dynamic and energetic aspects involved.

The length of glycine-rich linker in DNA-binding domain is critical for optimal functioning of quorum-sensing master regulatory protein HapR.

PubMed

Singh, Naorem Santa; Kachhap, Sangita; Singh, Richa; Mishra, Rahul Chandra; Singh, Balvinder; Raychaudhuri, Saumya

2014-12-01

HapR is a quorum-sensing master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of this wild-type protein. While unraveling the underlying cause of functional inertness of a natural variant (HapRV2), the significance of a conserved glycine residue at position 39 in a glycine-rich linker in DNA-binding domain comes into light. This work aims at investigating how the length of glycine-rich linker (R(33)GIGRGG(39)) bridging helices α1 and α2 modulates the functionality of HapR. In pursuit of our interest, glycine residues were inserted after terminal glycine (G39) of the linker in a sequential manner. To evaluate functionality, all the glycine linker variants were subjected to a battery of performance tests under various conditions. Combined in vitro and in vivo results clearly demonstrated a gradual functional impairment of HapR linker variants coupled with increasing length of glycine-rich linker and finally, linker variant harboring four glycine residues resulted in a functionally compromised protein with significant loss of communication with cognate DNAs. Molecular dynamics studies of modeled HapR linker variants in complex with cognate promoter region show that residues namely Ser50, Thr53 and Asn56 are involved in varying degree of interactions with different nucleotides of HapR-DNA complex. The diminished functionality between variants and DNA appears to result from reduced or no interactions between Phe55 and nucleotides of cognate DNA as observed during simulations.

Lipid-based nanotubes as functional architectures with embedded fluorescence and recognition capabilities.

PubMed

John, George; Mason, Megan; Ajayan, Pulickel M; Dordick, Jonathan S

2004-11-24

A limited combinatorial strategy was used to synthesize a small library of soft lipid-based materials ranging from structurally unordered fibers to highly uniform nanotubes. The latter nanotubes are comprised of a bilayer structure with interdigitated alkyl chains associated through hydrophobic interactions. These tubes contain accessible 2,6-diaminopyridine linkers that can interact with thymidine and related nucleosides through multipoint hydrogen bonding, thereby quenching the intrinsic fluorescence of the aromatic linker. These results are the first example of a systematic strategy to design functional lipid nanotubes with precise structural and functional features.

A Folding Zone in the Ribosomal Exit Tunnel for Kv1.3 Helix Formation

PubMed Central

Tu, LiWei; Deutsch, Carol

2010-01-01

SUMMARY Although it is now clear that protein secondary structure can be acquired early, while the nascent peptide resides within the ribosomal exit tunnel, the principles governing folding of native polytopic proteins have not yet been elucidated. We now report an extensive investigation of native Kv1.3, a voltage-gated K+ channel, including transmembrane and linker segments synthesized in sequence. These native segments form helices vectorially (N- to C-terminus) only in a permissive vestibule located in the last 20Å of the tunnel. Native linker sequences similarly fold in this vestibule. Finally, secondary structure acquired in the ribosome is retained in the translocon. These findings emerge from accessibility studies of a diversity of native transmembrane and linker sequences and may therefore be applicable to protein biogenesis in general. PMID:20060838

Novel regulation of Smad3 oligomerization and DNA binding by its linker domain.

PubMed

Vasilaki, Eleftheria; Siderakis, Manos; Papakosta, Paraskevi; Skourti-Stathaki, Konstantina; Mavridou, Sofia; Kardassis, Dimitris

2009-09-08

Smad proteins are key effectors of the transforming growth factor beta (TGFbeta) signaling pathway in mammalian cells. Smads are composed of two highly structured and conserved domains called Mad homology 1 (MH1) and 2 (MH2), which are linked together by a nonconserved linker region. The recent identification of phosphorylation sites and binding sites for ubiquitin ligases in the linker regions of TGFbeta and bone morphogenetic protein (BMP) receptor-regulated Smads suggested that the linker may contribute to the regulation of Smad function by facilitating cross-talks with other signaling pathways. In the present study, we have generated and characterized novel Smad3 mutants bearing individual substitutions of conserved and nonconserved amino acid residues within a previously described transcriptionally active linker fragment. Our analysis showed that the conserved linker amino acids glutamine 222 and proline 229 play important roles in Smad functions such as homo- and hetero-oligomerization, nuclear accumulation in response to TGFbeta stimulation, and DNA binding. Furthermore, a Smad3 mutant bearing a substitution of the nonconserved amino acid asparagine 218 to alanine displayed enhanced transactivation potential relative to wild type Smad3. Finally, Smad3 P229A inhibited TGFbeta signaling when overexpressed in mammalian cells. In conclusion, our data are in line with previous studies supporting an important regulatory role of the linker region of Smads in their function as key transducers of TGFbeta signaling.

Equilibrium & Nonequilibrium Fluctuation Effects in Biopolymer Networks

NASA Astrophysics Data System (ADS)

Kachan, Devin Michael

Fluctuation-induced interactions are an important organizing principle in a variety of soft matter systems. In this dissertation, I explore the role of both thermal and active fluctuations within cross-linked polymer networks. The systems I study are in large part inspired by the amazing physics found within the cytoskeleton of eukaryotic cells. I first predict and verify the existence of a thermal Casimir force between cross-linkers bound to a semi-flexible polymer. The calculation is complicated by the appearance of second order derivatives in the bending Hamiltonian for such polymers, which requires a careful evaluation of the the path integral formulation of the partition function in order to arrive at the physically correct continuum limit and properly address ultraviolet divergences. I find that cross linkers interact along a filament with an attractive logarithmic potential proportional to thermal energy. The proportionality constant depends on whether and how the cross linkers constrain the relative angle between the two filaments to which they are bound. The interaction has important implications for the synthesis of biopolymer bundles within cells. I model the cross-linkers as existing in two phases: bound to the bundle and free in solution. When the cross-linkers are bound, they behave as a one-dimensional gas of particles interacting with the Casimir force, while the free phase is a simple ideal gas. Demanding equilibrium between the two phases, I find a discontinuous transition between a sparsely and a densely bound bundle. This discontinuous condensation transition induced by the long-ranged nature of the Casimir interaction allows for a similarly abrupt structural transition in semiflexible filament networks between a low cross linker density isotropic phase and a higher cross link density bundle network. This work is supported by the results of finite element Brownian dynamics simulations of semiflexible filaments and transient cross-linkers. I speculate that cells take advantage of this equilibrium effect by tuning near the transition point, where small changes in free cross-linker density will affect large structural rearrangements between free filament networks and networks of bundles. Cells are naturally found far from equilibrium, where the active influx of energy from ATP consumption controls the dynamics. Motor proteins actively generate forces within biopolymer networks, and one may ask how these differ from the random stresses characteristic of equilibrium fluctuations. Besides the trivial observation that the magnitude is independent of temperature, I find that the processive nature of the motors creates a temporally correlated, or colored, noise spectrum. I model the network with a nonlinear scalar elastic theory in the presence of active driving, and study the long distance and large scale properties of the system with renormalization group techniques. I find that there is a new critical point associated with diverging correlation time, and that the colored noise produces novel frequency dependence in the renormalized transport coefficients. Finally, I study marginally elastic solids which have vanishing shear modulus due to the presence of soft modes, modes with zero deformation cost. Although network coordination is a useful metric for determining the mechanical response of random spring networks in mechanical equilibrium, it is insufficient for describing networks under external stress. In particular, under-constrained networks which are fluid-like at zero load will dynamically stiffen at a critical strain, as observed in numerical simulations and experimentally in many biopolymer networks. Drawing upon analogies to the stress induced unjamming of emulsions, I develop a kinetic theory to explain the rigidity transition in spring and filament networks. Describing the dynamic evolution of non-affine deformation via a simple mechanistic picture, I recover the emergent nonlinear strain-stiffening behavior and compare this behavior to the yield stress flow seen in soft glassy fluids. I extend this theory to account for coordination number inhomogeneities and predict a breakdown of universal scaling near the critical point at sufficiently high disorder, and discuss the utility for this type of model in describing biopolymer networks.

Directing the breathing behavior of pillared-layered metal-organic frameworks via a systematic library of functionalized linkers bearing flexible substituents.

PubMed

Henke, Sebastian; Schneemann, Andreas; Wütscher, Annika; Fischer, Roland A

2012-06-06

Flexible metal-organic frameworks (MOFs), also referred to as soft porous crystals (SPCs), show reversible structural transitions dependent on the nature and quantity of adsorbed guest molecules. In recent studies it has been reported that covalent functionalization of the organic linker can influence or even integrate framework flexibility ("breathing") in MOFs. However, rational fine-tuning of such responsive properties is very desirable but challenging as well. Here we present a powerful approach for the targeted manipulation of responsiveness and framework flexibility of an important family of pillared-layered MOFs based on the parent structure [Zn(2)(bdc)(2)(dabco)](n) (bdc = 1,4-benzenedicarboxylate; dabco = 1,4-diazabicyclo[2.2.2]octane). A library of functionalized bdc-type linkers (fu-bdc), which bear additional dangling side groups at different positions of the benzene core (alkoxy groups of varying chain length with diverse functionalities and polarity), was generated. Synthesis of the materials [Zn(2)(fu-bdc)(2)(dabco)](n) yields the respective collection of highly responsive MOFs. The parent MOF is only weakly flexible; however, the substituted frameworks of [Zn(2)(fu-bdc)(2)(dabco)](n) contract drastically upon guest removal and expand again upon adsorption of DMF (N,N-dimethylformamide), EtOH, or CO(2), etc., while N(2) is hardly adsorbed and does not open the narrow-pored form. These "breathing" dynamics are attributed to the dangling side chains that act as immobilized "guests", which interact with mobile guest molecules as well as with themselves and with the framework backbone. The structural details of the guest-free, contracted form and the gas sorption behavior (phase transition pressure, hysteresis loop) are highly dependent on the nature of the substituent at the linker and can therefore be adjusted using our approach. Combining our library of functionalized linkers with the concept of mixed-component MOFs (solid solutions) offers very rich additional dimensions of tailoring the structural dynamics and responsiveness. Implementation of two differently functionalized linkers in varying ratios yields multicomponent single-phased [Zn(2)(fu-bdc')(2x)(fu-bdc″)(2-2x)(dabco)](n) MOFs (0 < x < 1) of increased inherent complexity, which feature a non-linear dependence of their gas sorption properties on the applied ratio of components. Hence, the responsive behavior of such pillared-layered MOFs can be extensively tuned via an intelligent combination of functionalized linkers.

The length of the linker between the epidermal growth factor-like domains in factor VIIa is critical for a productive interaction with tissue factor.

PubMed

Persson, Egon; Madsen, Jesper J; Olsen, Ole H

2014-12-01

Formation of the factor VIIa (FVIIa)-tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure-based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)-like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ-carboxyglutamic acid (Gla)-containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly-Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF-mediated stabilization of the active conformation of FVIIa and abrogated TF-induced activity enhancement. In addition, FVIIa variants with short linkers associated 80-fold slower with soluble TF (sTF) as compared with wild-type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca(2+)-free FVIIa; stepwise truncation resulting in gradually higher activity with des(83-86)-FVIIa reaching the level of Gla-domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo-FVIIa activity. © 2014 The Protein Society.

A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore.

PubMed

Tomczak, Adam P; Fernández-Trillo, Jorge; Bharill, Shashank; Papp, Ferenc; Panyi, Gyorgy; Stühmer, Walter; Isacoff, Ehud Y; Pardo, Luis A

2017-05-01

Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4-S5 linker). However, our recent work on channels disrupted in the S4-S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of K V 10.1 revealed that the S4-S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use "split" channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in K V 10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4-S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4-S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism. © 2017 Tomczak et al.

A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore

PubMed Central

Fernández-Trillo, Jorge; Bharill, Shashank; Panyi, Gyorgy; Stühmer, Walter; Isacoff, Ehud Y.

2017-01-01

Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4–S5 linker). However, our recent work on channels disrupted in the S4–S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of KV10.1 revealed that the S4–S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use “split” channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in KV10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4–S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4–S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism. PMID:28360219

Improving TCO-Conjugated Antibody Reactivity for Bioorthogonal Pretargeting

NASA Astrophysics Data System (ADS)

Chu, Tina Tingyi

Cancer remains a major cause of death because of its unpredictable progression. Utilizing bioorthogonal chemistry between trans-cyclooctene (TCO) and tetrazine to target imaging agents to tumors in two subsequent steps offers a more versatile platform for molecular imaging. This is accomplished by pretargeting TCO-modified primary antibody to cell surface biomarkers, followed by delivery of tetrazine-modified imaging probes. In previous work, it has been established that TCO-tetrazine chemistry can be applied to in vivo imaging, resulting in precise tumor detection. However, most TCO modifications on an antibody are not reactive because they are buried within hydrophobic domains. To expose and improve the reactivity, Rahim et al. incorporated a polyethylene glycol (PEG) linker through a two-step reaction with DBCO-azide, which successfully maintained 100% TCO functionality. In this project, various types of linkers were studied to improve the reactivity in a single step. Three primary types of linkers were studied: hydrophilic PEG chains, hydrophobic short linkers, and amphiphilic linkers. Our results show that PEG chain alone can only maintain 40% TCO reactivity. Unexpectedly, a short alkyl chain (valeric acid) provided superior results, with 60% TCO reactivity. Lengthening the alkyl chain did not improve results further. Finally, an amphiphilic linker containing valeric acid and PEG performed worse than either linker type alone, at ˜30% functionality. We conclude that our previous 100% functional TCO result obtained with the two-step coupling may have stemmed from generation of the DBCO/azide cycloaddition product. Future work will explore factors such as rigidity of linker structure, polarity, or charges.

Structure of calmodulin complexed with an olfactory CNG channel fragment and role of the central linker: residual dipolar couplings to evaluate calmodulin binding modes outside the kinase family.

PubMed

Contessa, Gian Marco; Orsale, Maria; Melino, Sonia; Torre, Vincent; Paci, Maurizio; Desideri, Alessandro; Cicero, Daniel O

2005-03-01

The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs.

Detection of protease activity by fluorescent protein FRET sensors: from computer simulation to live cells

NASA Astrophysics Data System (ADS)

Goryashchenko, Alexander S.; Khrenova, Maria G.; Savitsky, Alexander P.

2018-04-01

Förster resonance energy transfer (FRET) sensors are widely used for the detection of protease activity in vitro and in vivo. Usually they consist of a FRET pair connected with a polypeptide linker containing a specific cleavage site for the relevant protease. Use of the fluorescent proteins as components of the FRET pair allows genetic encoding of such sensors and solves the problem of their delivery into live cells and animals. There are several ways to improve the properties of such sensors, mainly to increase FRET efficiency and therefore the dynamic range. One of the ways to achieve this is to use a non-fluorescent chromoprotein as an acceptor. Molecular dynamic simulations may assist in the construction of linker structures connecting donor and acceptor molecules. Estimation of the orientation factor κ 2 can be obtained by methods based on quantum theory and combined quantum mechanics/molecular mechanics approaches. The linker can be structured by hydrophobic interactions, bringing it into a closed conformation that shortens the distance between donor and acceptor and, consequently, increases FRET efficiency. We analyzed the effects of different linker structures on the detection of caspase-3 activity using a non-fluorescent acceptor. Also we have constructed the Tb3+- TagRFP sensor in which a complex of the terbium ion and terbium-binding peptide is used as a donor. This allowed us to use the unique property of lanthanide ions—fluorescence lifetime up to milliseconds—to perform measurements with time delay and exclude the nanosecond-order fluorescence. Using our systems as a starting point, by changing the recognition site in the linker it is possible to perform imaging of different protease activity in vitro or in vivo.

Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116

NASA Astrophysics Data System (ADS)

Zhang, Yuan; Palla, Mirkó; Sun, Andrew; Liao, Jung-Chi

2013-09-01

DEAD-box RNA helicases are ATP-dependent proteins implicated in nearly all aspects of RNA metabolism. The yeast DEAD-box helicase Mss116 is unique in its functions of splicing group I and group II introns and activating mRNA translation, but the structural understanding of why it performs these unique functions remains unclear. Here we used sequence analysis and molecular dynamics simulation to identify residues in the flexible linker specific for yeast Mss116, potentially associated with its unique functions. We first identified residues that are 100% conserved in Mss116 of different species of the Saccharomycetaceae family. The amino acids of these conserved residues were then compared with the amino acids of the corresponding residue positions of other RNA helicases to identify residues that have distinct amino acids from other DEAD-box proteins. Four residues in the flexible linker, i.e. N334, E335, P336 and H339, are conserved and Mss116-specific. Molecular dynamics simulation was conducted for the wild-type Mss116 structure and mutant models to examine mutational effects of the linker on the conformational equilibrium. Relatively short MD simulation runs (within 20 ns) were enough for us to observe mutational effects, suggesting serious structural perturbations by these mutations. The mutation of E335 depletes the interactions between E335 and K95 in domain 1. The interactions between N334/P336 and N496/I497 of domain 2 are also abolished by mutation. Our results suggest that tight interactions between the Mss116-specific flexible linker and the two RecA-like domains may be mechanically required to crimp RNA for the unique RNA processes of yeast Mss116.

Metal-organic framework assembled from erbium and a tetrapodal polyphosphonic acid organic linker.

PubMed

Mendes, Ricardo F; Firmino, Ana D G; Tomé, João P C; Almeida Paz, Filipe A

2018-06-01

A three-dimensional metal-organic framework (MOF), poly[[μ 6 -5'-pentahydrogen [1,1'-biphenyl]-3,3',5,5'-tetrayltetrakis(phosphonato)]erbium(III)] 2.5-hydrate], formulated as [Er(C 12 H 11 O 12 P 4 )]·2.5H 2 O or [Er(H 5 btp)]·2.5H 2 O (I) and isotypical with a Y 3+ -based MOF reported previously by our research group [Firmino et al. (2017b). Inorg. Chem. 56, 1193-1208], was constructed based solely on Er 3+ and on the polyphosphonic organic linker [1,1'-biphenyl]-3,3',5,5'-tetrakis(phosphonic acid) (H 8 btp). The present work describes our efforts to introduce lanthanide cations into the flexible network, demonstrating that, on the one hand, the compound can be obtained using three distinct experimental methods, i.e. hydro(solvo)thermal (Hy), microwave-assisted (MW) and one-pot (Op), and, on the other hand, that crystallite size can be approximately fine-tuned according to the method employed. MOF I contains hexacoordinated Er 3+ cations which are distributed in a zigzag inorganic chain running parallel to the [100] direction of the unit cell. The chains are, in turn, bridged by the anionic organic linker to form a three-dimensional 6,6-connected binodal network. This connectivity leads to the existence of one-dimensional channels (also running parallel to the [100] direction) filled with disordered and partially occupied water molecules of crystalization which are engaged in O-H...O hydrogen-bonding interactions with the [Er(H 5 btp)] framework. Additional weak π-π interactions [intercentroid distance = 3.957 (7) Å] exist between aromatic rings, which help to maintain the structural integrity of the network.

Location of Dual Sites in E. coli FtsZ Important for Degradation by ClpXP; One at the C-Terminus and One in the Disordered Linker

PubMed Central

Camberg, Jodi L.; Viola, Marissa G.; Rea, Leslie; Hoskins, Joel R.; Wickner, Sue

2014-01-01

ClpXP is a two-component ATP-dependent protease that unfolds and degrades proteins bearing specific recognition signals. One substrate degraded by Escherichia coli ClpXP is FtsZ, an essential cell division protein. FtsZ forms polymers that assemble into a large ring-like structure, termed the Z-ring, during cell division at the site of constriction. The FtsZ monomer is composed of an N-terminal polymerization domain, an unstructured linker region and a C-terminal conserved region. To better understand substrate selection by ClpXP, we engineered FtsZ mutant proteins containing amino acid substitutions or deletions near the FtsZ C-terminus. We identified two discrete regions of FtsZ important for degradation of both FtsZ monomers and polymers by ClpXP in vitro. One region is located 30 residues away from the C-terminus in the unstructured linker region that connects the polymerization domain to the C-terminal region. The other region is near the FtsZ C-terminus and partially overlaps the recognition sites for several other FtsZ-interacting proteins, including MinC, ZipA and FtsA. Mutation of either region caused the protein to be more stable and mutation of both caused an additive effect, suggesting that both regions are important. We also observed that in vitro MinC inhibits degradation of FtsZ by ClpXP, suggesting that some of the same residues in the C-terminal site that are important for degradation by ClpXP are important for binding MinC. PMID:24722340

From force-fields to photons: MD simulations of dye-labeled nucleic acids and Monte Carlo modeling of FRET

NASA Astrophysics Data System (ADS)

Milas, Peker; Gamari, Ben; Parrot, Louis; Buckman, Richard; Goldner, Lori

2011-11-01

Fluorescence resonance energy transfer (FRET) is a powerful experimental technique for understanding the structural fluctuations and transformations of RNA, DNA and proteins. Molecular dynamics (MD) simulations provide a window into the nature of these fluctuations on a faster time scale inaccessible to experiment. We use Monte Carlo methods to model and compare FRET data from dye-labeled RNA with what might be predicted from the MD simulation. With a few notable exceptions, the contribution of fluorophore and linker dynamics to these FRET measurements has not been investigated. We include the dynamics of the ground state dyes and linkers along with an explicit water solvent in our study of a 16mer double-stranded RNA. Cyanine dyes are attached at either the 3' or 5' ends with a three carbon linker, providing a basis for contrasting the dynamics of similar but not identical molecular structures.

A protein interaction mechanism for suppressing the mechanosensitive Piezo channels.

PubMed

Zhang, Tingxin; Chi, Shaopeng; Jiang, Fan; Zhao, Qiancheng; Xiao, Bailong

2017-11-27

Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca 2+ ATPase (SERCA), including the widely expressed SERCA2, as Piezo interacting proteins. SERCA2 strategically suppresses Piezo1 via acting on a 14-residue-constituted intracellular linker connecting the pore-module and mechanotransduction-module. Mutating the linker impairs mechanogating and SERCA2-mediated modulation of Piezo1. Furthermore, the synthetic linker-peptide disrupts the modulatory effects of SERCA2, demonstrating the key role of the linker in mechanogating and regulation. Importantly, the SERCA2-mediated regulation affects Piezo1-dependent migration of endothelial cells. Collectively, we identify SERCA-mediated regulation of Piezos and the functional significance of the linker, providing important insights into the mechanogating and regulation mechanisms of Piezo channels.

Replacement of the double bond of antitubulin chalcones with triazoles and tetrazoles: Synthesis and biological evaluation.

PubMed

Mesenzani, Ornella; Massarotti, Alberto; Giustiniano, Mariateresa; Pirali, Tracey; Bevilacqua, Valentina; Caldarelli, Antonio; Canonico, Pierluigi; Sorba, Giovanni; Novellino, Ettore; Genazzani, Armando A; Tron, Gian Cesare

2011-01-15

In the chalcone scaffold, it is thought that the double bond is an important structural linker but it is likely not essential for the interaction with tubulin. Yet, it may be a potential site of metabolic degradation and interaction with biological nucleophiles. In this letter, we have replaced this olefinic portion of chalcones with two metabolically stable and chemically inert heterocyclic rings, namely triazole or tetrazole. Yet, our biologic data suggest that, unlike in other antitubulinic structures, the olephinic ring might not be merely a structural linker. Copyright © 2010 Elsevier Ltd. All rights reserved.

Theoretical Investigation of Charge Transfer in Metal Organic Frameworks for Electrochemical Device Applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patwardhan, Sameer; Schatz, George C.

For electrochemical device applications metal organic frameworks (MOFs) must exhibit suitable conduction properties. To this end, we have performed computational studies of intermolecular charge transfer in MOFs consisting of hexa-ZrIV nodes and tetratopic carboxylate linkers. This includes an examination of the electronic structure of linkers that are derived from tetraphenyl benzene 1, tetraphenyl pyrene 2, and tetraphenyl porphyrin 3 molecules. These results are used to determine charge transfer propensities in MOFs, within the framework of Marcus theory, including an analysis of the key parameters (charge transfer integral t, reorganization energy λ, and free energy change ΔG0) and evaluation of figuresmore » of merit for charge transfer based on the chemical structures of the linkers. This qualitative analysis indicates that delocalization of the HOMO/LUMO on terminal substituents increases t and decreases λ, while weaker binding to counterions decreases ΔG0, leading to better charge transfer propensity. Subsequently, we study hole transfer in the linker 2 containing MOFs, NU-901 and NU-1000, in detail and describe mechanisms (hopping and superexchange) that may be operative under different electrochemical conditions. Comparisons with experiment are provided where available. On the basis of the redox and catalytic activity of nodes and linkers, we propose three possible schemes for constructing electrochemical devices for catalysis. We believe that the results of this study will lay the foundation for future experimental work on this topic.« less

Mechanical Coupling via the Membrane Fusion SNARE Protein Syntaxin 1A: A Molecular Dynamics Study

PubMed Central

Knecht, Volker; Grubmüller, Helmut

2003-01-01

SNARE trans complexes between membranes likely promote membrane fusion. For the t-SNARE syntaxin 1A involved in synaptic transmission, the secondary structure and bending stiffness of the five-residue juxtamembrane linker is assumed to determine the required mechanical energy transfer from the cytosolic core complex to the membrane. These properties have here been studied by molecular dynamics and annealing simulations for the wild-type and a C-terminal-prolongated mutant within a neutral and an acidic bilayer, suggesting linker stiffnesses above 1.7 but below 50 × 10−3 kcal mol−1 deg−2. The transmembrane helix was found to be tilted by 15° and tightly anchored within the membrane with a stiffness of 4–5 kcal mol−1 Å−2. The linker turned out to be marginally helical and strongly influenced by its lipid environment. Charged lipids increased the helicity and H3 helix tilt stiffness. For the wild type, the linker was seen embedded deeply within the polar region of the bilayer, whereas the prolongation shifted the linker outward. This reduced its helicity and increased its average tilt, thereby presumably reducing fusion efficiency. Our results suggest that partially unstructured linkers provide considerable mechanical coupling; the energy transduced cooperatively by the linkers in a native fusion event is thus estimated to be 3–8 kcal/mol, implying a two-to-five orders of magnitude fusion rate increase. PMID:12609859

Electrostatic Interactions Govern "Odd/Even" Effects in Water-Induced Gemini Surfactant Self-Assembly.

PubMed

Mantha, Sriteja; McDaniel, Jesse G; Perroni, Dominic V; Mahanthappa, Mahesh K; Yethiraj, Arun

2017-01-26

Gemini surfactants comprise two single-tailed surfactants connected by a linker at or near the hydrophilic headgroup. They display a variety of water-concentration-dependent lyotropic liquid crystal morphologies that are sensitive to surfactant molecular structure and the nature of the headgroups and counterions. Recently, an interesting dependence of the aqueous-phase behavior on the length of the linker has been discovered; odd-numbered linker length surfactants exhibit characteristically different phase diagrams than even-numbered linker surfactants. In this work, we investigate this "odd/even effect" using computer simulations, focusing on experimentally studied gemini dicarboxylates with Na + counterions, seven nonterminal carbon atoms in the tails, and either three, four, five, or six carbon atoms in the linker (denoted Na-73, Na-74, Na-75, and Na-76, respectively). We find that the relative electrostatic repulsion between headgroups in the different morphologies is correlated with the qualitative features of the experimental phase diagrams, predicting destabilization of hexagonal phases as the cylinders pack close together at low water content. Significant differences in the relative headgroup orientations of Na-74 and Na-76 compared to those of Na-73 and Na-75 surfactants lead to differences in linker-linker packing and long-range headgroup-headgroup electrostatic repulsion, which affects the delicate electrostatic balance between the hexagonal and gyroid phases. Much of the fundamental insight presented in this work is enabled by the ability to computationally construct and analyze metastable phases that are not observable in experiments.

Synthesis and Characterization of Metal-Organic Frameworks (MOFs) That Are Difficult to Access De Novo

NASA Astrophysics Data System (ADS)

Karagiaridi, Olga

Metal-organic frameworks (MOFs) are a class of intriguing hybrid materials, comprised of metal-based nodes joined by organic linkers into a crystalline, porous, three-dimensional lattice. Their signature properties (well-defined surfaces, tailorability and ultra-high porosity) render them promising candidates for many applications, including, but not limited to, gas storage, gas separation, catalysis and sensing. One of the greatest challenges associated with MOF synthesis lies in the fact that obtaining a desired MOF structure that is tailored to perform a specific application is often not trivial. Traditional synthetic pathways termed "de novo synthesis" (typically one-pot reactions between the MOF structural building blocks under solvothermal conditions) often give rise to side products that do not possess the desired structure. To circumvent this problem, we have studied in depth two powerful MOF synthetic techniques -- solvent-assisted linker exchange (SALE) and transmetalation. These are heterogeneous reactions of parent MOF crystals with concentrated solutions of organic linkers and inorganic metal salts, respectively, that lead to the replacement of the linkers or metal nodes within the parent MOFs by the desired components, while the overall framework topology is preserved. The projects described in this dissertation have aimed to apply these techniques to transform simple (unfunctionalized) and easy to synthesize representative materials from various MOF systems to structurally and functionally interesting daughter products. Examples include synthesis of MOFs that are energetically "unfavorable", extension of MOF cages by longer linker incorporation, functionalization of MOF pores and endowment of MOFs with permanent and persistent porosity. Through these projects, we have been able to formulate a set of rules that can be applied to predict the successful outcome of SALE. Since the allure of MOFs lies in their applications, expanding the range of accessible MOF structures translates into potentially solving more relevant problems - especially those related to alternative energy and sustainability, which urgently need to be addressed given our current energy demands. We hope that SALE and transmetalation can provide alternative routes towards the synthesis of many new and exciting MOFs that can provide creative solutions to many of the problems that we face.

An in vitro and in vivo investigation of bivalent ligands that display preferential binding and functional activity for different melanocortin receptor homodimers

PubMed Central

Lensing, Cody J.; Freeman, Katie T.; Schnell, Sathya M.; Adank, Danielle N.; Speth, Robert C.; Haskell-Luevano, Carrie

2017-01-01

Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 (CJL-1-140) possessed a functional profile that was unique from its monovalent counterparts providing evidence of the discrete effects of bivalent ligands. Lead compound 7 (CJL-1-87) significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects. PMID:26959173

Autoinhibition of ankyrin-B/G membrane target bindings by intrinsically disordered segments from the tail regions

PubMed Central

Wang, Chao; Wei, Zhiyi

2017-01-01

Ankyrins together with their spectrin partners are the master organizers of micron-scale membrane domains in diverse tissues. The 24 ankyrin (ANK) repeats of ankyrins bind to numerous membrane proteins, linking them to spectrin-based cytoskeletons at specific membrane microdomains. The accessibility of the target binding groove of ANK repeats must be regulated to achieve spatially defined functions of ankyrins/target complexes in different tissues, though little is known in this regard. Here we systemically investigated the autoinhibition mechanism of ankyrin-B/G by combined biochemical, biophysical and structural biology approaches. We discovered that the entire ANK repeats are inhibited by combinatorial and quasi-independent bindings of multiple disordered segments located in the ankyrin-B/G linkers and tails, suggesting a mechanistic basis for differential regulations of membrane target bindings by ankyrins. In addition to elucidating the autoinhibition mechanisms of ankyrins, our study may also shed light on regulations on target bindings by other long repeat-containing proteins. PMID:28841137

Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes.

PubMed

Lössl, Philip; Sinz, Andrea

2016-01-01

During the last 15 years, the combination of chemical cross-linking and high-resolution mass spectrometry (MS) has matured into an alternative approach for analyzing 3D-structures of proteins and protein complexes. Using the distance constraints imposed by the cross-links, models of the protein or protein complex under investigation can be created. The majority of cross-linking studies are currently conducted with homobifunctional amine-reactive cross-linkers. We extend this "traditional" cross-linking/MS strategy by adding complementary photo-cross-linking data. For this, the diazirine-containing unnatural amino acids photo-leucine and photo-methionine are incorporated into the proteins and cross-link formation is induced by UV-A irradiation. The advantage of the photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can be targeted, which is advantageous for investigating membrane proteins. We consider the strategy of combining cross-linkers with orthogonal reactivities and distances to be ideally suited for maximizing the amount of structural information that can be gained from a cross-linking experiment.

Development of Bioorthogonally Degradable Linkers and Polymers Using alpha-Azidoethers

NASA Astrophysics Data System (ADS)

Rajagopalan, Chandrasekhar Ramasubramanian

Degradable polymers have gained a lot of attention in recent years for applications in biotechnology and medicine. External control over polymer degradation can be obtained by incorporating functional groups that cleave in the presence of triggers that would normally be absent in biological environments, i.e. are bioorthogonal. This thesis explores the use of chemically cleavable alpha-azidoethers as a new method to obtain external control over the degradation behavior of polymers. My first goal is to illustrate the potential of alpha-azidoethers toward developing cleavable linkers. We have studied the relationship between alpha-azidoether structure and hydrolytic stability, to prepare linkers that withstand background hydrolytic cleavage until they are exposed to the cleaving trigger. The cleavage kinetics of the alpha-azidoether functional group was quantified. In addition to the conventionally used tris(2-carboxyethyl)phosphine (TCEP), dihydrolipoic acid (DHLA), a previously unexplored, biocompatible reducing agent, was also evaluated as a cleaving trigger. Based on these results, we have proposed design rules for utilizing alpha-azidoethers as cleavable linkers in applications that require bioorthogonal control over linker cleavage. Secondly, the alpha-azidoether cleavable linker chemistry was implemented into the development of polymeric materials. Two different types of polymers were developed. Polyamides incorporating alpha-azidoethers along the backbone were synthesized, and their physical properties and chemically triggered degradation behavior were characterized. The degradation timescale of these polymers can be tuned simply by manipulating the concentration of the externally applied chemical trigger. The alpha-azidoether functional group was then utilized to develop a unique triggered-release polymeric adhesive for potential applications in dental adhesive formulations. A methacrylamide-phosphonate adhesive monomer incorporating an alpha-azidoether group was designed and synthesized. The monomer was polymerized to adhere polymer-composite substrates. Adhesion strength was quantified, and on-demand release of bonded substrates was demonstrated using DHLA as a trigger. The results presented here shed some light on the scope, advantages and drawbacks of utilizing alpha-azidoethers to develop new types of cleavable linkers and degradable polymers. In principle, the triggered degradation method described here could be incorporated into polymers with different chemical structures, to develop a variety of materials that offer an external control over degradation.

An atomistic view of Hsp70 allosteric crosstalk: from the nucleotide to the substrate binding domain and back

PubMed Central

Chiappori, Federica; Merelli, Ivan; Milanesi, Luciano; Colombo, Giorgio; Morra, Giulia

2016-01-01

The Hsp70 is an allosterically regulated family of molecular chaperones. They consist of two structural domains, NBD and SBD, connected by a flexible linker. ATP hydrolysis at the NBD modulates substrate recognition at the SBD, while peptide binding at the SBD enhances ATP hydrolysis. In this study we apply Molecular Dynamics (MD) to elucidate the molecular determinants underlying the allosteric communication from the NBD to the SBD and back. We observe that local structural and dynamical modulation can be coupled to large-scale rearrangements, and that different combinations of ligands at NBD and SBD differently affect the SBD domain mobility. Substituting ADP with ATP in the NBD induces specific structural changes involving the linker and the two NBD lobes. Also, a SBD-bound peptide drives the linker docking by increasing the local dynamical coordination of its C-terminal end: a partially docked DnaK structure is achieved by combining ATP in the NBD and peptide in the SBD. We propose that the MD-based analysis of the inter domain dynamics and structure modulation could be used as a tool to computationally predict the allosteric behaviour and functional response of Hsp70 upon introducing mutations or binding small molecules, with potential applications for drug discovery. PMID:27025773

Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM4- siderophore analogues of varied linker length

NASA Astrophysics Data System (ADS)

Wilde, Ellis J.; Hughes, Adam; Blagova, Elena V.; Moroz, Olga V.; Thomas, Ross P.; Turkenburg, Johan P.; Raines, Daniel J.; Duhme-Klair, Anne-Kathrin; Wilson, Keith S.

2017-04-01

Bacteria use siderophores to mediate the transport of essential Fe(III) into the cell. In Campylobacter jejuni the periplasmic binding protein CeuE, an integral part of the Fe(III) transport system, has adapted to bind tetradentate siderophores using a His and a Tyr side chain to complete the Fe(III) coordination. A series of tetradentate siderophore mimics was synthesized in which the length of the linker between the two iron-binding catecholamide units was increased from four carbon atoms (4-LICAM4-) to five, six and eight (5-, 6-, 8-LICAM4-, respectively). Co-crystal structures with CeuE showed that the inter-planar angles between the iron-binding catecholamide units in the 5-, 6- and 8-LICAM4- structures are very similar (111°, 110° and 110°) and allow for an optimum fit into the binding pocket of CeuE, the inter-planar angle in the structure of 4-LICAM4- is significantly smaller (97°) due to restrictions imposed by the shorter linker. Accordingly, the protein-binding affinity was found to be slightly higher for 5- compared to 4-LICAM4- but decreases for 6- and 8-LICAM4-. The optimum linker length of five matches that present in natural siderophores such as enterobactin and azotochelin. Site-directed mutagenesis was used to investigate the relative importance of the Fe(III)-coordinating residues H227 and Y288.

Synthesis, structure and in vitro cytostatic activity of ferrocene-Cinchona hybrids.

PubMed

Kocsis, László; Szabó, Ildikó; Bősze, Szilvia; Jernei, Tamás; Hudecz, Ferenc; Csámpai, Antal

2016-02-01

Exploring copper(I)- and ruthenium(II)-catalyzed azide-alkyne cycloadditions and a Sonogashira protocol, novel cytostatic ferrocene-cinchona hybrids were synthetized displaying significant in vitro activity on HepG-2 and HT-29 cells. Preliminary SAR studies disclosed that compounds incorporating linkers with 1,2,3-triazole and chalchone residues can be considered as promising lead structures. According to the best of our knowledge this is the first letter on the incorporation of ferrocene nucleus in the reputed cinchona family via triazole and chalcone linkers with established pharmaceutical profile. Copyright © 2015 Elsevier Ltd. All rights reserved.

Adenoviral vector tethering to metal surfaces via hydrolysable cross-linkers for the modulation of vector release and transduction

PubMed Central

Fishbein, Ilia; Forbes, Scott P.; Chorny, Michael; Connolly, Jeanne M.; Adamo, Richard F.; Corrales, Ricardo; Alferiev, Ivan S.; Levy, Robert J.

2013-01-01

The use of arterial stents and other medical implants as a delivery platform for surface immobilized gene vectors allows for safe and efficient localized expression of therapeutic transgenes. In this study we investigate the use of hydrolysable cross-linkers with distinct kinetics of hydrolysis for delivery of gene vectors from polyallylamine bisphosphonate-modified metal surfaces. Three cross-linkers with the estimated t1/2 of ester bonds hydrolysis of 5, 12 and 50 days demonstrated a cumulative 20%, 39% and 45% vector release, respectively, after 30 days exposure to physiological buffer at 37°C. Transgene expression in endothelial and smooth muscles cells transduced with substrate immobilized adenovirus resulted in significantly different expression profiles for each individual cross-linker. Furthermore, immobilization of adenoviral vectors effectively extended their transduction effectiveness beyond the initial phase of release. Transgene expression driven by adenovirus-tethered stents in rat carotid arteries demonstrated that a faster rate of cross-linker hydrolysis resulted in higher expression levels at day 1, which declined by day 8 after stent implantation, while inversely, slower hydrolysis was associated with increased arterial expression at day 8 in comparison with day 1. In conclusion, adjustable release of transduction-competent adenoviral vectors from metallic surfaces can be achieved, both in vitro and in vivo, through surface immobilization of adenoviral vectors using hydrolysable cross-linkers with structure-specific release kinetics. PMID:23777912

Facile self-assembly and stabilization of metal oxide nanoparticles.

PubMed

Charbonneau, Cecile; Holliman, Peter J; Davies, Matthew L; Watson, Trystan M; Worsley, David A

2015-03-15

This paper describes a facile method of self-assembling different metal oxide nanoparticles into nanostructured materials via di-carboxylate linkers (oxalic acid) using TiO2 as an example. In this method, the di-carboxylate linkers react with surface hydroxyls on metal oxide nanoparticles forming covalent, ester-like bonds, which enable the binding of two metal oxide particles, one at either end of the linker and facilitates efficient self-assembly of one group of metal oxide nanoparticles homogeneously distributed onto the surface of another group. The oxalate linkers can then be removed by thermal decomposition. This approach is shown to be effective using differently-sized TiO2 nanoparticles, namely in-house synthesized 3-5nm anatase nanocrystals and Degussa P25 titania particles (mean 21nm particle size). Our data show that the application of a high temperature heat treatment (450°C for 30min), conventionally applied to achieve a stable porous structure by thermal decomposition of the linker molecules and by inducing inter-particle necking, damages the surface area of the nanostructured material. However, here we show that sintering at 300°C for 30min or by flash near infrared radiation sintering for 12s efficiently decomposes the oxalate linkers and stabilizes the nanostructure of the material whilst maintaining its high surface area. Copyright © 2013 Elsevier Inc. All rights reserved.

Electrostatic Interactions Govern “Odd/Even” Effects in Water-Induced Gemini Surfactant Self-Assembly

DOE PAGES

Mantha, Sriteja; McDaniel, Jesse G.; Perroni, Dominic V.; ...

2016-12-27

Gemini surfactants comprise two single-tailed surfactants connected by a linker at or near the hydrophilic headgroup. They display a variety of water concentration-dependent lyotropic liquid crystal (LLC) morphologies that are sensitive to surfactant molecular structure, and na- ture of the headgroups and counterions. Recently, an interesting dependence of the aqueous phase behavior on the length of the linker has been discovered; odd-numbered linker length surfactants exhibit characteristically different phase diagrams than even-numbered linker sur- factants. In this work, we investigate this “odd/even effect” using computer simulations, focusing on experimentally studied gemini dicarboxylates with Na + counterions, 7 non-terminal carbon atomsmore » in the tails, and either 3, 4, 5, or 6 carbon atoms in the linker (denoted Na-73, Na-74, Na-75, and Na-76 respectively). We find that the relative electrostatic repulsion be- tween headgroups in the different morphologies is correlated with qualitative features of the experimental phase diagrams, predicting destabilization of hexagonal phases as the cylinders pack close together at low water content. Significant differences in the relative headgroup ori- entations of Na-74 and Na-76 compared to Na-73 and Na-75 surfactants lead to differences in linker-linker packing, and long-range headgroup/headgroup electrostatic repulsion, which affects the delicate electrostatic balance between hexagonal and gyroid phases. Finally, much of the fundamental insight presented in this work is enabled by the ability to computationally construct and analyze metastable phases that are not observable in experiments.« less

2-(Maleimidomethyl)-1,3-Dioxanes (MD): a Serum-Stable Self-hydrolysable Hydrophilic Alternative to Classical Maleimide Conjugation

NASA Astrophysics Data System (ADS)

Dovgan, Igor; Kolodych, Sergii; Koniev, Oleksandr; Wagner, Alain

2016-08-01

The vast majority of antibody-drug conjugates (ADC) are prepared through amine-to-thiol conjugation. To date, N-Succinimidyl-4-(maleimidomethyl) cyclohexanecarboxylate (SMCC) has been one of the most frequently applied reagents for the preparation of ADC and other functional conjugates. However, SMCC-based conjugates suffer from limited stability in blood circulation and from a hydrophobic character of the linker, which may give rise to major pharmacokinetic implications. To address this issue, we have developed a heterobifunctional analogue of a SMCC reagent, i.e., sodium 4-(maleimidomethyl)-1,3-dioxane-5-carbonyl)oxy)-2,3,5,6- tetrafluorobenzenesulfonate (MDTF) for amine-to-thiol conjugation. By replacing the cyclohexyl ring in the SMCC structure with the 1,3-dioxane, we increased the hydrophilicity of the linker. A FRET probe based on MD linker was prepared and showed superior stability compared to the MCC linker in human plasma, as well as in a variety of aqueous buffers. A detailed investigation demonstrated an accelerated succinimide ring opening for MD linker, resulting in stabilized conjugates. Finally, the MDTF reagent was applied for the preparation of serum stable antibody-dye conjugate.

A structured interdomain linker directs self-polymerization of human uromodulin

PubMed Central

Bokhove, Marcel; Nishimura, Kaoru; Brunati, Martina; Han, Ling; de Sanctis, Daniele; Rampoldi, Luca

2016-01-01

Uromodulin (UMOD)/Tamm–Horsfall protein, the most abundant human urinary protein, plays a key role in chronic kidney diseases and is a promising therapeutic target for hypertension. Via its bipartite zona pellucida module (ZP-N/ZP-C), UMOD forms extracellular filaments that regulate kidney electrolyte balance and innate immunity, as well as protect against renal stones. Moreover, salt-dependent aggregation of UMOD filaments in the urine generates a soluble molecular net that captures uropathogenic bacteria and facilitates their clearance. Despite the functional importance of its homopolymers, no structural information is available on UMOD and how it self-assembles into filaments. Here, we report the crystal structures of polymerization regions of human UMOD and mouse ZP2, an essential sperm receptor protein that is structurally related to UMOD but forms heteropolymers. The structure of UMOD reveals that an extensive hydrophobic interface mediates ZP-N domain homodimerization. This arrangement is required for filament formation and is directed by an ordered ZP-N/ZP-C linker that is not observed in ZP2 but is conserved in the sequence of deafness/Crohn’s disease-associated homopolymeric glycoproteins α-tectorin (TECTA) and glycoprotein 2 (GP2). Our data provide an example of how interdomain linker plasticity can modulate the function of structurally similar multidomain proteins. Moreover, the architecture of UMOD rationalizes numerous pathogenic mutations in both UMOD and TECTA genes. PMID:26811476

Unraveling Structure-Property Relationships in Polymer Blends for Intelligent Materials Design

NASA Astrophysics Data System (ADS)

Irwin, Matthew Tyler

Block polymers provide an accessible route to structured, composite materials by combining two or more components with disparate mechanical, chemical, and electrical properties into a single bulk material with nanoscale domains. However, the characteristic lengthscale of these systems is limited, and the choice of components is restricted to those that are able to undergo microstructural ordering at accessible temperatures. This thesis details routes to overcoming these limitations through the addition of a lithium salt, a blend of homopolymers, or both. Chapter 2 describes a study wherein complex sphere phases such as the Frank-Kasper sigma phase can be observed in otherwise disordered asymmetric block polymers through the addition of a lithium salt. Chapter 3 discusses the development and characterization of a ternary polymer blend of an AB diblock copolymer and A and B homopolymers doped with a lithium salt. Detailed characterization showed that doping blends that are otherwise disordered with lithium salt induced microstructural ordering and largely recovers the phase behavior of traditional ternary polymer blends. A systematic study of the ionic conductivity of the blends at a fixed salt concentration demonstrates that, at a given composition, disordered, yet highly structured blends consistently exhibit better conductivity than polycrystalline morphologies with long range order. Chapter 4 extends the methodology of Chapter 3 and details a systematic study of the effects of cross-linker concentration on the performance of polymer electrolyte membranes produced via polymerization-induced microphase separation that exhibit a highly structured, globally disordered microstructure. Finally, Chapter 5 details efforts to develop a water filtration membrane using a polyethylene template derived from a polymeric bicontinuous microemulsion. Throughout all of this work, the goal is to better understand structure-property relationships at the molecular level in order to ultimately inform design criteria for materials where simultaneous control over morphology and mechanical, chemical, or electrical properties is important.

Unusual and highly tunable missing-linker defects in zirconium metal-organic framework UiO-66 and their important effects on gas adsorption.

PubMed

Wu, Hui; Chua, Yong Shen; Krungleviciute, Vaiva; Tyagi, Madhusudan; Chen, Ping; Yildirim, Taner; Zhou, Wei

2013-07-17

UiO-66 is a highly important prototypical zirconium metal-organic framework (MOF) compound because of its excellent stabilities not typically found in common porous MOFs. In its perfect crystal structure, each Zr metal center is fully coordinated by 12 organic linkers to form a highly connected framework. Using high-resolution neutron power diffraction technique, we found the first direct structural evidence showing that real UiO-66 material contains significant amount of missing-linker defects, an unusual phenomenon for MOFs. The concentration of the missing-linker defects is surprisingly high, ∼10% in our sample, effectively reducing the framework connection from 12 to ∼11. We show that by varying the concentration of the acetic acid modulator and the synthesis time, the linker vacancies can be tuned systematically, leading to dramatically enhanced porosity. We obtained samples with pore volumes ranging from 0.44 to 1.0 cm(3)/g and Brunauer-Emmett-Teller surface areas ranging from 1000 to 1600 m(2)/g, the largest values of which are ∼150% and ∼60% higher than the theoretical values of defect-free UiO-66 crystal, respectively. The linker vacancies also have profound effects on the gas adsorption behaviors of UiO-66, in particular CO2. Finally, comparing the gas adsorption of hydroxylated and dehydroxylated UiO-66, we found that the former performs systematically better than the latter (particularly for CO2) suggesting the beneficial effect of the -OH groups. This finding is of great importance because hydroxylated UiO-66 is the practically more relevant, non-air-sensitive form of this MOF. The preferred gas adsorption on the metal center was confirmed by neutron diffraction measurements, and the gas binding strength enhancement by the -OH group was further supported by our first-principles calculations.

Design of an Active Ultrastable Single-chain Insulin Analog

PubMed Central

Hua, Qing-xin; Nakagawa, Satoe H.; Jia, Wenhua; Huang, Kun; Phillips, Nelson B.; Hu, Shi-quan; Weiss, Michael A.

2008-01-01

Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (<3 residues) prevent induced fit upon receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 ± 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (ΔΔGu = 0.7 ± 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 ± 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts ValA3 at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 α-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world. PMID:18332129

The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson,J.; Ryan, Z.; Salisbury, J.

2006-01-01

Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered {alpha}-helical linker. A stretch of the amino-terminalmore » domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an {alpha}-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin.« less

A dual affinity-tag strategy for the expression and purification of human linker histone H1.4 in Escherichia coli.

PubMed

Ryan, Daniel P; Tremethick, David J

2016-04-01

Linker histones are an abundant and critical component of the eukaryotic chromatin landscape. They play key roles in regulating the higher order structure of chromatin and many genetic processes. Higher eukaryotes possess a number of different linker histone subtypes and new data are consistently emerging that indicate these subtypes are functionally distinct. We were interested in studying one of the most abundant human linker histone subtypes, H1.4. We have produced recombinant full-length H1.4 in Escherichia coli. An N-terminal Glutathione-S-Transferase tag was used to promote soluble expression and was combined with a C-terminal hexahistidine tag to facilitate a simple non-denaturing two-step affinity chromatography procedure that results in highly pure full-length H1.4. The purified H1.4 was shown to be functional via in vitro chromatin assembly experiments and remains active after extended storage at -80 °C. Copyright © 2015 Elsevier Inc. All rights reserved.

Labeling of DOTA-conjugated HPMA-based polymers with trivalent metallic radionuclides for molecular imaging.

PubMed

Eppard, Elisabeth; de la Fuente, Ana; Mohr, Nicole; Allmeroth, Mareli; Zentel, Rudolf; Miederer, Matthias; Pektor, Stefanie; Rösch, Frank

2018-02-27

In this work, the in vitro and in vivo stabilities and the pharmacology of HPMA-made homopolymers were studied by means of radiometal-labeled derivatives. Aiming to identify the fewer amount and the optimal DOTA-linker structure that provides quantitative labeling yields, diverse DOTA-linker systems were conjugated in different amounts to HPMA homopolymers to coordinate trivalent radiometals Me(III)* = gallium-68, scandium-44, and lutetium-177. Short linkers and as low as 1.6% DOTA were enough to obtain labeling yields > 90%. Alkoxy linkers generally exhibited lower labeling yields than alkane analogues despite of similar chain length and DOTA incorporation rate. High stability of the radiolabel in all examined solutions was observed for all conjugates. Labeling with scandium-44 allowed for in vivo PET imaging and ex vivo measurements of organ distribution for up to 24 h. This study confirms the principle applicability of DOTA-HPMA conjugates for labeling with different trivalent metallic radionuclides allowing for diagnosis and therapy.

Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach.

PubMed

Anumala, Upendra Rao; Waaler, Jo; Nkizinkiko, Yves; Ignatev, Alexander; Lazarow, Katina; Lindemann, Peter; Olsen, Petter Angell; Murthy, Sudarshan; Obaji, Ezeogo; Majouga, Alexander G; Leonov, Sergey; von Kries, Jens Peter; Lehtiö, Lari; Krauss, Stefan; Nazaré, Marc

2017-12-28

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC 50 values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.

Postsynthetic Tuning of Metal-Organic Frameworks for Targeted Applications.

PubMed

Islamoglu, Timur; Goswami, Subhadip; Li, Zhanyong; Howarth, Ashlee J; Farha, Omar K; Hupp, Joseph T

2017-04-18

Metal-organic frameworks (MOFs) are periodic, hybrid, atomically well-defined porous materials that typically form by self-assembly and consist of inorganic nodes (metal ions or clusters) and multitopic organic linkers. MOFs as a whole offer many intriguing properties, including ultrahigh porosity, tunable chemical functionality, and low density. These properties point to numerous potential applications, including gas storage, chemical separations, catalysis, light harvesting, and chemical sensing, to name a few. Reticular chemistry, or the linking of molecular building blocks into predetermined network structures, has been employed to synthesize thousands of MOFs. Given the vast library of candidate nodes and linkers, the number of potentially synthetically accessible MOFs is enormous. Nevertheless, a powerful complementary approach to obtain specific structures with desired chemical functionality is to modify known MOFs after synthesis. This approach is particularly useful when incorporation of particular chemical functionalities via direct synthesis is challenging or impossible. The challenges may stem from limited stability or solubility of precursors, unwanted secondary reactivity of precursors, or incompatibility of functional groups with the conditions needed for direct synthesis. MOFs can be postsynthetically modified by replacing the metal nodes and/or organic linkers or via functionalization of the metal nodes and/or organic linkers. Here we describe some of our efforts toward the development and application of postsynthetic strategies for imparting desired chemical functionalities in MOFs of known topology. The techniques include methods for functionalizing MOF nodes, i.e., solvent-assisted ligand incorporation (SALI) and atomic layer deposition in MOFs (AIM) as well as a method to replace structural linkers, termed solvent-assisted linker exchange (SALE), also known as postsynthethic exchange (PSE). For each functionalization strategy, we first describe its chemical basis along with the requirements for its successful implementation. We then present a small number of examples, with an emphasis on those that (a) convey the underlying concepts and/or (b) lead to functional structures (e.g., catalysts) that would be difficult or impossible to access via direct routes. The examples, however, are only illustrative, and a significant body of work exists from both our lab and others, especially for the SALE/PSE strategy. We refer readers to the papers cited and to the references therein. More exciting, in our view, will be new examples and new applications of the functionalization strategies-especially applications made possible by creatively combining the strategies. Underexplored (again, in our view) are implementations that impart electrical conductivity, enable increasingly selective chemical sensing, or facilitate cascade catalysis. It will be interesting to see where these strategies and others take this compelling field over the next few years.

Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers.

PubMed

Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2014-01-01

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.

Dynamics of Linker Residues Modulate the Nucleic Acid Binding Properties of the HIV-1 Nucleocapsid Protein Zinc Fingers

PubMed Central

Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2014-01-01

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity. PMID:25029439

The structurally disordered paramyxovirus nucleocapsid protein tail domain is a regulator of the mRNA transcription gradient.

PubMed

Cox, Robert M; Krumm, Stefanie A; Thakkar, Vidhi D; Sohn, Maximilian; Plemper, Richard K

2017-02-01

The paramyxovirus RNA-dependent RNA-polymerase (RdRp) complex loads onto the nucleocapsid protein (N)-encapsidated viral N:RNA genome for RNA synthesis. Binding of the RdRp of measles virus (MeV), a paramyxovirus archetype, is mediated through interaction with a molecular recognition element (MoRE) located near the end of the carboxyl-terminal Ntail domain. The structurally disordered central Ntail section is thought to add positional flexibility to MoRE, but the functional importance of this Ntail region for RNA polymerization is unclear. To address this question, we dissected functional elements of Ntail by relocating MoRE into the RNA-encapsidating Ncore domain. Linker-scanning mutagenesis identified a microdomain in Ncore that tolerates insertions. MoRE relocated to Ncore supported efficient interaction with N, MoRE-deficient Ntails had a dominant-negative effect on bioactivity that was alleviated by insertion of MoRE into Ncore, and recombinant MeV encoding N with relocated MoRE grew efficiently and remained capable of mRNA editing. MoRE in Ncore also restored viability of a recombinant lacking the disordered central Ntail section, but this recombinant was temperature-sensitive, with reduced RdRp loading efficiency and a flattened transcription gradient. These results demonstrate that virus replication requires high-affinity RdRp binding sites in N:RNA, but productive RdRp binding is independent of positional flexibility of MoRE and cis-acting elements in Ntail. Rather, the disordered central Ntail section independent of the presence of MoRE in Ntail steepens the paramyxovirus transcription gradient by promoting RdRp loading and preventing the formation of nonproductive polycistronic viral mRNAs. Disordered Ntails may have evolved as a regulatory element to adjust paramyxovirus gene expression.

Two Novel Phycoerythrin-Associated Linker Proteins in the Marine Cyanobacterium Synechococcus sp. Strain WH8102

PubMed Central

Six, Christophe; Thomas, Jean-Claude; Thion, Laurent; Lemoine, Yves; Zal, Frank; Partensky, Frédéric

2005-01-01

The recent availability of the whole genome of Synechococcus sp. strain WH8102 allows us to have a global view of the complex structure of the phycobilisomes of this marine picocyanobacterium. Genomic analyses revealed several new characteristics of these phycobilisomes, consisting of an allophycocyanin core and rods made of one type of phycocyanin and two types of phycoerythrins (I and II). Although the allophycocyanin appears to be similar to that found commonly in freshwater cyanobacteria, the phycocyanin is simpler since it possesses only one complete set of α and β subunits and two rod-core linkers (CpcG1 and CpcG2). It is therefore probably made of a single hexameric disk per rod. In contrast, we have found two novel putative phycoerythrin-associated linker polypeptides that appear to be specific for marine Synechococcus spp. The first one (SYNW2000) is unusually long (548 residues) and apparently results from the fusion of a paralog of MpeC, a phycoerythrin II linker, and of CpeD, a phycoerythrin-I linker. The second one (SYNW1989) has a more classical size (300 residues) and is also an MpeC paralog. A biochemical analysis revealed that, like MpeC, these two novel linkers were both chromophorylated with phycourobilin. Our data suggest that they are both associated (partly or totally) with phycoerythrin II, and we propose to name SYNW2000 and SYNW1989 MpeD and MpeE, respectively. We further show that acclimation of phycobilisomes to high light leads to a dramatic reduction of MpeC, whereas the two novel linkers are not significantly affected. Models for the organization of the rods are proposed. PMID:15716439

Two novel phycoerythrin-associated linker proteins in the marine cyanobacterium Synechococcus sp. strain WH8102.

PubMed

Six, Christophe; Thomas, Jean-Claude; Thion, Laurent; Lemoine, Yves; Zal, Frank; Partensky, Frédéric

2005-03-01

The recent availability of the whole genome of Synechococcus sp. strain WH8102 allows us to have a global view of the complex structure of the phycobilisomes of this marine picocyanobacterium. Genomic analyses revealed several new characteristics of these phycobilisomes, consisting of an allophycocyanin core and rods made of one type of phycocyanin and two types of phycoerythrins (I and II). Although the allophycocyanin appears to be similar to that found commonly in freshwater cyanobacteria, the phycocyanin is simpler since it possesses only one complete set of alpha and beta subunits and two rod-core linkers (CpcG1 and CpcG2). It is therefore probably made of a single hexameric disk per rod. In contrast, we have found two novel putative phycoerythrin-associated linker polypeptides that appear to be specific for marine Synechococcus spp. The first one (SYNW2000) is unusually long (548 residues) and apparently results from the fusion of a paralog of MpeC, a phycoerythrin II linker, and of CpeD, a phycoerythrin-I linker. The second one (SYNW1989) has a more classical size (300 residues) and is also an MpeC paralog. A biochemical analysis revealed that, like MpeC, these two novel linkers were both chromophorylated with phycourobilin. Our data suggest that they are both associated (partly or totally) with phycoerythrin II, and we propose to name SYNW2000 and SYNW1989 MpeD and MpeE, respectively. We further show that acclimation of phycobilisomes to high light leads to a dramatic reduction of MpeC, whereas the two novel linkers are not significantly affected. Models for the organization of the rods are proposed.

Cell biology of Smad2/3 linker region phosphorylation in vascular smooth muscle.

PubMed

Rezaei, Hossein B; Kamato, Danielle; Ansari, Ghazaleh; Osman, Narin; Little, Peter J

2012-08-01

The transforming growth factor (TGF)-β superfamily of ligands regulates a diverse set of cellular functions. Transforming growth factor-β induces its biological effects through Type I and Type II transmembrane receptors that have serine/threonine kinase activities and weak tyrosine kinase activity. In vascular smooth muscle, TGF-β binds to the TGF-β Type II receptor (TβRII) at the cell surface, recruiting the Type I receptor (TβRI) to form a heterocomplex. Consequently, after phosphorylation and activation of TβRI, the transcription factors receptor activated (R-) Smad2 and Smad3 are recruited and activated through phosphorylation of C terminal residues. Overall, Smad2/3 and co-Smad4 have similar structures consisting of three regions an N-terminal MH1 domain, a C-terminal MH2 domain and a central linker region. Phosphorylation of the Smad linker region appears to have an important role in the regulation of Smad activity and function. The mitogen-activated protein kinase (MAPK) family, CDK2, CDK4 and calcium-calmodulin dependent kinase are the main kinases that phosphorylate sites in the linker region. The role of the linker region includes enabling the formation of Smad homo-oligomers and provision of phosphorylation sites for MAPK and other kinases. In some instances, linker region phosphorylation regulates the inhibition of the nuclear translocation of Smads. In the present review, we describe TGF-β signalling through Smad2/3 and the importance of the linker region in the regulation and expression of genes induced by TGF-β superfamily ligands in the context of vascular smooth muscle. © 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.

The S4–S5 Linker Acts as a Signal Integrator for hERG K+ Channel Activation and Deactivation Gating

PubMed Central

Ng, Chai Ann; Perry, Matthew D.; Tan, Peter S.; Hill, Adam P.; Kuchel, Philip W.; Vandenberg, Jamie I.

2012-01-01

Human ether-à-go-go-related gene (hERG) K+ channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4–S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4–S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4–S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4–S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4–S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4–S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel. PMID:22359612

Kinetic Methods for Understanding Linker Exchange in Metal-Organic Frameworks

NASA Astrophysics Data System (ADS)

Morabito, Joseph V.

Exchange reactions have enabled a new level of control in the rational, stepwise preparation of metal-organic framework (MOF) materials. However, their full potential is limited by a lack of understanding of the molecular mechanisms by which they occur. This dissertation describes our efforts to understand this important class of reactions in two parts. The first reports our use of a linker exchange process to encapsulate guest molecules larger than the limiting pore aperture of the MOF. The concept is demonstrated, along with evidence for guest encapsulation and its relation to a dissociative linker exchange process. The second part describes our development of the first quantitative kinetic method for studying MOF linker exchange reactions and our application of this method to understand the solvent dependence of the reaction of ZIF-8 with imidazole. This project involved the collection of the largest set of rate data available on any MOF linker exchange reaction. The combination of this dataset with small molecule encapsulation experiments allowed us to formulate a mechanistic model that could account for all the observed kinetic and structural data. By comparison with the kinetic behavior of complexes in solution, we were able to fit the kinetic behavior of ZIF-8 into the broader family of coordination compounds. Aside from the specific use that our kinetic data may have in predicting the reactivity of ZIF linker exchange, we hope that the conceptual bridges made between MOFs and related metal?organic compounds can help reveal underlying patterns in behavior and advance the field.

Supramolecular Packing Controls H 2 Photocatalysis in Chromophore Amphiphile Hydrogels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weingarten, Adam S.; Kazantsev, Roman V.; Palmer, Liam C.

2015-11-21

Light harvesting supramolecular assemblies are potentially useful structures as components of solar-to-fuel conversion materials. The development of these functional constructs requires an understanding of optimal packing modes for chromophores. We investigated here assembly in water and the photocatalytic function of perylene monoimide chromophore amphiphiles with different alkyl linker lengths separating their hydrophobic core and the hydrophilic carboxylate headgroup. We found that these chromophore amphiphiles (CAs) self-assemble into charged nanostructures of increasing aspect ratio as the linker length is increased. The addition of salt to screen the charged nanostructures induced the formation of hydrogels and led to internal crystallization within somemore » of the nanostructures. For linker lengths up to seven methylenes, the CAs were found to pack into 2D crystalline unit cells within ribbon-shaped nanostructures, whereas the nine methylene CAs assembled into long nanofibers without crystalline molecular packing. At the same time, the different molecular packing arrangements after charge screening led to different absorbance spectra, despite the identical electronic properties of all PMI amphiphiles. While the crystalline CAs formed electronically coupled H-aggregates, only CAs with intermediate linker lengths showed evidence of high intermolecular orbital overlap. Photocatalytic hydrogen production using a nickel-based catalyst was observed in all hydrogels, with the highest turnovers observed for CA gels having intermediate linker lengths. We conclude that the improved photocatalytic performance of the hydrogels formed by supramolecular assemblies of the intermediate linker CA molecules likely arises from improved exciton splitting efficiencies due to their higher orbital overlap.« less

Pivotal role of extended linker 2 in the activation of Gα by G protein-coupled receptor.

PubMed

Huang, Jianyun; Sun, Yutong; Zhang, J Jillian; Huang, Xin-Yun

2015-01-02

G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gαs mutant proteins (from a screen of 66 Gαs mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gαs. This extended Linker 2 is the target site of a natural product inhibitor of Gq. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β2/β3 loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A mechanism for histone chaperoning activity of nucleoplasmin: thermodynamic and structural models.

PubMed

Taneva, Stefka G; Bañuelos, Sonia; Falces, Jorge; Arregi, Igor; Muga, Arturo; Konarev, Petr V; Svergun, Dmitri I; Velázquez-Campoy, Adrián; Urbaneja, María A

2009-10-23

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different "affinity windows" for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.

Assembly of bipolar microtubule structures by passive cross-linkers and molecular motors

NASA Astrophysics Data System (ADS)

Johann, D.; Goswami, D.; Kruse, K.

2016-06-01

During cell division, sister chromatids are segregated by the mitotic spindle, a bipolar assembly of interdigitating antiparallel polar filaments called microtubules. The spindle contains the midzone, a stable region of overlapping antiparallel microtubules, that is essential for maintaining bipolarity. Although a lot is known about the molecular players involved, the mechanism underlying midzone formation and maintenance is still poorly understood. We study the interaction of polar filaments that are cross-linked by molecular motors moving directionally and by passive cross-linkers diffusing along microtubules. Using a particle-based stochastic model, we find that the interplay of motors and passive cross-linkers can generate a stable finite overlap between a pair of antiparallel polar filaments. We develop a mean-field theory to study this mechanism in detail and investigate the influence of steric interactions between motors and passive cross-linkers on the overlap dynamics. In the presence of interspecies steric interactions, passive cross-linkers mimic the behavior of molecular motors and stable finite overlaps are generated even for non-cross-linking motors. Finally, we develop a mean-field theory for a bundle of aligned polar filaments and show that they can self-organize into a spindlelike pattern. Our work suggests possible ways as to how cells can generate spindle midzones and control their extensions.

Molecular dynamics-based model of VEGF-A and its heparin interactions.

PubMed

Uciechowska-Kaczmarzyk, Urszula; Babik, Sándor; Zsila, Ferenc; Bojarski, Krzysztof Kamil; Beke-Somfai, Tamás; Samsonov, Sergey A

2018-06-01

We present a computational model of the Vascular Endothelial Growth Factor (VEGF), an important regulator of blood vessels formation, which function is affected by its heparin interactions. Although structures of a receptor binding (RBD) and a heparin binding domain (HBD) of VEGF are known, there are structural data neither on the 12 amino acids interdomain linker nor on its complexes with heparin. We apply molecular docking and molecular dynamics techniques combined with circular dichroism spectroscopy to model the full structure of the dimeric VEGF and to propose putative molecular mechanisms underlying the function of VEGF/VEGF receptors/heparin system. We show that both the conformational flexibility of the linker and the formation of HBD-heparin-HBD sandwich-like structures regulate the mutual disposition of HBDs and so affect the VEGF-mediated signalling. Copyright © 2018 Elsevier Inc. All rights reserved.

Solid-phase organic synthesis of difluoroalkyl entities using a novel fluorinating cleavage strategy: part 1. Linker development: scope and limitations.

PubMed

Wiehn, Matthias S; Lindell, Stephen D; Bräse, Stefan

2009-01-01

An efficient method to synthesize gem-difluorinated compounds on solid supports is described. The strategy is based on the design of a novel sulfur linker system that enables, to the best of our knowledge for the first time, the release of target structures from the resin under simultaneous fluorination. Starting from an immobilized dithiol, coupling with an excess of aldehyde or ketone furnished dithianes. These can be further functionalized prior to release from the resin using our newly developed fluorinating cleavage conditions. Amide forming reactions, palladium-catalyzed reactions (Heck, Suzuki, and Sonogashira couplings), reductions, alkylations, and olefinations were successfully explored on the linker. The difluorinated target substances were obtained in modest to excellent yields and in high purities.

Visual Detection of Human Antibodies Using Sugar Chain-Immobilized Fluorescent Nanoparticles: Application as a Point of Care Diagnostic Tool for Guillain-Barré Syndrome.

PubMed

Shinchi, Hiroyuki; Yuki, Nobuhiro; Ishida, Hideharu; Hirata, Koichi; Wakao, Masahiro; Suda, Yasuo

2015-01-01

Sugar chain binding antibodies have gained substantial attention as biomarkers due to their crucial roles in various disorders. In this study, we developed simple and quick detection method of anti-sugar chain antibodies in sera using our previously developed sugar chain-immobilized fluorescent nanoparticles (SFNPs) for the point-of-care diagnostics. Sugar chain structure on SFNPs was modified with the sugar moieties of the GM1 ganglioside via our original linker molecule to detect anti-GM1 antibodies. The structures and densities of the sugar moieties immobilized on the nanoparticles were evaluated in detail using lectins and sera containing anti-GM1 antibodies from patients with Guillain-Barré syndrome, a neurological disorder, as an example of disease involving anti-sugar chain antibodies. When optimized SFNPs were added to sera from patients with Guillain-Barré syndrome, fluorescent aggregates were able to visually detect under UV light in three hours. The sensitivity of the detection method was equivalent to that of the current ELISA method used for the diagnosis of Guillain-Barré syndrome. These results suggest that our method using SFNPs is suitable for the point-of-care diagnostics of diseases involving anti-sugar chain antibodies.

Optimizing the relaxivity of GdIII complexes appended to InP/ZnS quantum dots by linker tuning.

PubMed

Stasiuk, Graeme J; Tamang, Sudarsan; Imbert, Daniel; Gateau, Christelle; Reiss, Peter; Fries, Pascal; Mazzanti, Marinella

2013-06-21

Three bimodal MRI/optical nanosized contrast agents with high per-nanoparticle relaxivity (up to 2523 mM(-1) s(-1) at 35 MHz and 932 mM(-1) s(-1) at 200 MHz) have been prepared connecting up to 115 tris-aqua Gd(III) complexes to fluorescent non-toxic InP/ZnS quantum dots. The structure of the linker has an important effect on the relaxivity of the final multimeric contrast agent.

Structure, function, and tethering of DNA-binding domains in σ 54 transcriptional activators

DOE PAGES

Vidangos, Natasha; Maris, Ann E.; Young, Anisa; ...

2013-07-02

In this paper, we compare the structure, activity, and linkage of DNA-binding domains (DBDs) from σ 54 transcriptional activators and discuss how the properties of the DBDs and the linker to the neighboring domain are affected by the overall properties and requirements of the full proteins. These transcriptional activators bind upstream of specific promoters that utilize σ 54-polymerase. Upon receiving a signal the activators assemble into hexamers, which then, through adenosine triphosphate (ATP) hydrolysis, drive a conformational change in polymerase that enables transcription initiation. We present structures of the DBDs of activators nitrogen regulatory protein C 1 (NtrC1) and Nif-likemore » homolog 2 (Nlh2) from the thermophile Aquifex aeolicus. The structures of these domains and their relationship to other parts of the activators are discussed. These structures are compared with previously determined structures of the DBDs of NtrC4, NtrC, ZraR, and factor for inversion stimulation. The N-terminal linkers that connect the DBDs to the central domains in NtrC1 and Nlh2 were studied and found to be unstructured. Additionally, a crystal structure of full-length NtrC1 was solved, but density of the DBDs was extremely weak, further indicating that the linker between ATPase and DBDs functions as a flexible tether. Flexible linking of ATPase and DBDs is likely necessary to allow assembly of the active hexameric ATPase ring. Finally, the comparison of this set of activators also shows clearly that strong dimerization of the DBD only occurs when other domains do not dimerize strongly.« less

Electronic Structure Calculations of Hydrogen Storage in Lithium-Decorated Metal-Graphyne Framework.

PubMed

Kumar, Sandeep; Dhilip Kumar, Thogluva Janardhanan

2017-08-30

Porous metal-graphyne framework (MGF) made up of graphyne linker decorated with lithium has been investigated for hydrogen storage. Applying density functional theory spin-polarized generalized gradient approximation with the Perdew-Burke-Ernzerhof functional containing Grimme's diffusion parameter with double numeric polarization basis set, the structural stability, and physicochemical properties have been analyzed. Each linker binds two Li atoms over the surface of the graphyne linker forming MGF-Li 8 by Dewar coordination. On saturation with hydrogen, each Li atom physisorbs three H 2 molecules resulting in MGF-Li 8 -H 24 . H 2 and Li interact by charge polarization mechanism leading to elongation in average H-H bond length indicating physisorption. Sorption energy decreases gradually from ≈0.4 to 0.20 eV on H 2 loading. Molecular dynamics simulations and computed sorption energy range indicate the high reversibility of H 2 in the MGF-Li 8 framework with the hydrogen storage capacity of 6.4 wt %. The calculated thermodynamic practical hydrogen storage at room temperature makes the Li-decorated MGF system a promising hydrogen storage material.

Self-Assembly of Emulsion Droplets into Polymer Chains

NASA Astrophysics Data System (ADS)

Bargteil, Dylan; McMullen, Angus; Brujic, Jasna

We experimentally investigate `beads-on-a-string' models of polymers using the spontaneous assembly of emulsion droplets into linear chains. Droplets functionalized with surface-mobile DNA allow for programmable 'monomers' through which we can influence the three-dimensional structure of the assembled 'polymer'. Such model polymers can be used to study conformational changes of polypeptides and the principles governing protein folding. In our system, we find that droplets bind via complementary DNA strands that are recruited into adhesion patches. Recruitment is driven by the DNA hybridization energy, and is limited by the energy cost of surface deformation and the entropy loss of the mobile linkers, yielding adhesion patches of a characteristic size with a given number of linkers. By tuning the initial surface coverage of linkers, we control valency between the droplets to create linear or branched polymer chains. We additionally control the flexibility of the model polymers by varying the salt concentration and study their dynamics between extended and collapsed states. This system opens the possibility of programming stable three-dimensional structures, such as those found within folded proteins.

A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

PubMed Central

Umasankar, Perunthottathu K; Ma, Li; Thieman, James R; Jha, Anupma; Doray, Balraj; Watkins, Simon C; Traub, Linton M

2014-01-01

Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization. DOI: http://dx.doi.org/10.7554/eLife.04137.001 PMID:25303365

Diketopyrrolopyrrole Polymers with Thienyl and Thiazolyl Linkers for Application in Field-Effect Transistors and Polymer Solar Cells.

PubMed

Yu, Yaping; Wu, Yang; Zhang, Andong; Li, Cheng; Tang, Zheng; Ma, Wei; Wu, Yonggang; Li, Weiwei

2016-11-09

Conjugated polymers consisting of diketopyrrolopyrrole (DPP) units have been successfully applied in field-effect transistors (FETs) and polymer solar cells (PSCs), while most of the DPP polymers were designed as symmetric structures containing identical aromatic linkers. In this manuscript, we design a new asymmetric DPP polymer with varied aromatic linkers in the backbone for application in FETs and PSCs. The designation provides the chance to finely adjust the energy levels of conjugated polymers so as to influence the device performance. The asymmetric polymer exhibits highly crystalline properties, high hole mobilities of 3.05 cm 2 V -1 s -1 in FETs, and a high efficiency of 5.9% in PSCs with spectra response from 300 to 850 nm. Morphology investigation demonstrates that the asymmetric polymer has a large crystal domain in blended thin films, indicating that the solar cell performance can be further enhanced by optimizing the microphase separation. The study reveals that the asymmetric design via adjusting the aromatic linkers in DPP polymers is a useful route toward flexible electronic devices.

Synthesis and structure-activity relationships of varied ether linker analogues of the antitubercular drug (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5h-imidazo[2,1-b][1,3]oxazine (PA-824).

PubMed

Thompson, Andrew M; Sutherland, Hamish S; Palmer, Brian D; Kmentova, Iveta; Blaser, Adrian; Franzblau, Scott G; Wan, Baojie; Wang, Yuehong; Ma, Zhenkun; Denny, William A

2011-10-13

New analogues of antitubercular drug PA-824 were synthesized, featuring alternative side chain ether linkers of varying size and flexibility, seeking drug candidates with enhanced metabolic stability and high efficacy. Both α-methyl substitution and removal of the benzylic methylene were broadly tolerated in vitro, with a biaryl example of the latter class exhibiting an 8-fold better efficacy than the parent drug in a mouse model of acute Mycobacterium tuberculosis infection and negligible fragmentation to an alcohol metabolite in liver microsomes. Extended linkers (notably propenyloxy, propynyloxy, and pentynyloxy) provided greater potencies against replicating M. tb (monoaryl analogues), with propynyl ethers being most effective under anaerobic (nonreplicating) conditions (mono/biaryl analogues). For benzyloxybenzyl and biaryl derivatives, aerobic activity was maximal with the original (OCH(2)) linker. One propynyloxy-linked compound displayed an 89-fold higher efficacy than the parent drug in the acute model, and it was slightly superior to antitubercular drug OPC-67683 in a chronic infection model.

Polyimide Aerogels Using Triisocyanate as Cross-linker.

PubMed

Nguyen, Baochau N; Meador, Mary Ann B; Scheiman, Daniel; McCorkle, Linda

2017-08-16

A family of polyimide (PI)-based aerogels is produced using Desmodur N3300A, an inexpensive triisocyanate, as the cross-linker. The aerogels are prepared by cross-linking amine end-capped polyimide oligomers with the triisocyanate. The polyimide oligomers are formulated using 2,2'-dimethylbenzidine, 4,4'-oxydianiline, or mixtures of both diamines, combined with 3,3',4,4'-biphenyltetracarboxylic dianhydride, and are chemically imidized at room temperature. Depending on the backbone chemistry, chain length, and polymer concentration, density of the aerogels ranged from 0.06 to 0.14 g/cm 3 and Brunauer-Emmett-Teller surface areas ranged from 350 to 600 m 2 /g. Compressive moduli of these aerogels were as high as 225 MPa, which are comparable to, or higher than, those previously reported prepared with similar backbone structures but with other cross-linkers. Because of their lower cost and commercial availability as cross-linker, the aerogels may have further potential as insulation for building and construction, clothing, sporting goods, and automotive applications, although lower-temperature stability may limit their use in some aerospace applications.

Structural characterization by NMR of a double phosphorylated chimeric peptide vaccine for treatment of Alzheimer's disease.

PubMed

Ramírez-Gualito, Karla; Richter, Monique; Matzapetakis, Manolis; Singer, David; Berger, Stefan

2013-04-26

Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer's disease (AD) and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau₂₂₉₋₂₃₇[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B₂₄₁₋₂₅₅ originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a β-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible.

Supramolecular Packing Controls H 2 Photocatalysis in Chromophore Amphiphile Hydrogels

DOE PAGES

Weingarten, Adam S.; Kazantsev, Roman V.; Palmer, Liam C.; ...

2015-11-21

Light harvesting supramolecular assemblies are potentially useful structures as components of solar-to-fuel conversion materials. The development of these functional constructs requires an understanding of optimal packing modes for chromophores. Here, we investigated assembly in water and the photocatalytic function of perylene monoimide chromophore amphiphiles with different alkyl linker lengths separating their hydrophobic core and the hydrophilic carboxylate headgroup. We found that these chromophore amphiphiles (CAs) self-assemble into charged nanostructures of increasing aspect ratio as the linker length is increased. The addition of salt to screen the charged nanostructures induced the formation of hydrogels and led to internal crystallization within somemore » of the nanostructures. For linker lengths up to seven methylenes, the CAs were found to pack into 2D crystalline unit cells within ribbon-shaped nanostructures, whereas the nine methylene CAs assembled into long nanofibers without crystalline molecular packing. At the same time, the different molecular packing arrangements after charge screening led to different absorbance spectra, despite the identical electronic properties of all PMI amphiphiles. While the crystalline CAs formed electronically coupled H-aggregates, only CAs with intermediate linker lengths showed evidence of high intermolecular orbital overlap. Photocatalytic hydrogen production using a nickel-based catalyst was observed in all hydrogels, with the highest turnovers observed for CA gels having intermediate linker lengths. Lastly, we conclude that the improved photocatalytic performance of the hydrogels formed by supramolecular assemblies of the intermediate linker CA molecules likely arises from improved exciton splitting efficiencies due to their higher orbital overlap.« less

Virtual Screening and X-ray Crystallography for Human Kallikrein 6 Inhibitors with an Amidinothiophene P1 Group.

PubMed

Liang, Guyan; Chen, Xin; Aldous, Suzanne; Pu, Su-Fen; Mehdi, Shujaath; Powers, Elaine; Giovanni, Andrew; Kongsamut, Sathapana; Xia, Tianhui; Zhang, Ying; Wang, Rachel; Gao, Zhongli; Merriman, Gregory; McLean, Larry R; Morize, Isabelle

2012-02-09

A series of compounds with an amidinothiophene P1 group and a pyrrolidinone-sulphonamide scaffold linker was identified as potent inhibitors of human kallikrein 6 by structure-based virtual screening based on the union accessible binding space of serine proteases. As the first series of potent nonmechanism-based hK6 inhibitors, they may be used as tool compounds for target validation. An X-ray structure of a representative compound complexed with hK6, resolved at a resolution of 1.88 Å, revealed that the amidinothiophene moiety bound in the S1 pocket and the pyrrolidinone-sulphonamide linker projected the aromatic tail into the S' pocket.

Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative.

PubMed

Scuotto, Maria; Rivieccio, Elisa; Varone, Alessia; Corda, Daniela; Bucci, Mariarosaria; Vellecco, Valentina; Cirino, Giuseppe; Virgilio, Antonella; Esposito, Veronica; Galeone, Aldo; Borbone, Nicola; Varra, Michela; Mayol, Luciano

2015-09-18

Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13. © Crown copyright 2015.

Site specific replacements of a single loop nucleoside with a dibenzyl linker may switch the activity of TBA from anticoagulant to antiproliferative

PubMed Central

Scuotto, Maria; Rivieccio, Elisa; Varone, Alessia; Corda, Daniela; Bucci, Mariarosaria; Vellecco, Valentina; Cirino, Giuseppe; Virgilio, Antonella; Esposito, Veronica; Galeone, Aldo; Borbone, Nicola; Varra, Michela; Mayol, Luciano

2015-01-01

Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13. PMID:26250112

Structural Basis for Activation of ZAP-70 by Phosphorylation of the SH2-Kinase Linker

PubMed Central

Yan, Qingrong; Barros, Tiago; Visperas, Patrick R.; Deindl, Sebastian; Kadlecek, Theresa A.; Weiss, Arthur

2013-01-01

Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only ∼5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 ∼100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck. PMID:23530057

Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker.

PubMed

Yan, Qingrong; Barros, Tiago; Visperas, Patrick R; Deindl, Sebastian; Kadlecek, Theresa A; Weiss, Arthur; Kuriyan, John

2013-06-01

Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only ∼5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 ∼100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck.

Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

DOE PAGES

Lee, Ho-Hsien; Cherni, Irene; Yu, HongQi; ...

2014-08-20

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli . The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to amore » resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.« less

Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Ho-Hsien; Cherni, Irene; Yu, HongQi

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli . The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to amore » resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.« less

Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meshcheryakov, Vladimir A.; Kitao, Akio; Core Research for Evolutionary Science and Technology, Tokyo 113-0032

2013-05-01

Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhB{sub C}). Here, themore » crystal structures of FlhB{sub C} from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhB{sub C} structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhB{sub C} leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhB{sub C} and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhB{sub C} molecule.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Talin, Albert Alec; Jones, Reese E.; Spataru, Dan Catalin

Metal organic frameworks (MOFs) are extended, nanoporous crystalline compounds consisting of metal ions interconnected by organic ligands. Their synthetic versatility suggest a disruptive class of opto - electronic materials with a high degree of electrical tunability and without the property - degrading disorder of organic conductors. In this project we determined the factors controlling charge and energy transport in MOFs and evaluated their potential for thermoelectric energy conversion. Two strategies for a chieving electronic conductivity in MOFs were explored: 1) using redox active 'guest' molecules introduced into the pores to dope the framework via charge - transfer coupling (Guest@MOF), 2)more » metal organic graphene analogs (MOGs) with dispersive band structur es arising from strong electronic overlap between the MOG metal ions and its coordinating linker groups. Inkjet deposition methods were developed to facilitate integration of the guest@MOF and MOG materials into practical devices.« less

Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Vaidya; E Velazquez-Delgado; G Abbruzzese

2011-12-31

Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60's and 130's helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergomore » a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.« less

Discovery of aliphatic-chain hydroxamates containing indole derivatives with potent class I histone deacetylase inhibitory activities.

PubMed

Chao, Shi-Wei; Chen, Liang-Chieh; Yu, Chia-Chun; Liu, Chang-Yi; Lin, Tony Eight; Guh, Jih-Hwa; Wang, Chen-Yu; Chen, Chun-Yung; Hsu, Kai-Cheng; Huang, Wei-Jan

2018-01-01

Histone deacetylase (HDAC) is a validated drug target for various diseases. This study combined indole recognition cap with SAHA, an FDA-approved HDAC inhibitor used to treat cutaneous T-cell lymphoma (CTCL). The structure activity relationship of the resulting compounds that inhibited HDAC was disclosed as well. Some compounds exhibited much stronger inhibitory activities than SAHA. We identified two meta-series compounds 6j and 6k with a two-carbon linker had IC 50 values of 3.9 and 4.5 nM for HDAC1, respectively. In contrast, the same oriented compounds with longer carbon chain linkers showed weaker inhibition. The result suggests that the linker chain length greatly contributed to enzyme inhibitory potency. In addition, comparison of enzyme-inhibiting activity between the compounds and SAHA showed that compounds 6j and 6k displayed higher inhibiting activity for class I (HDAC1, -2, -3 and -8). The molecular docking and structure analysis revealed structural differences with the inhibitor cap and metal-binding regions between the HDAC isozymes that affect interactions with the inhibitors and play a key role for selectivity. Further biological evaluation showed multiple cellular effects associated with compounds 6j- and 6k-induced HDAC inhibitory activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Investigating the role of chain and linker length on the catalytic activity of an H 2 production catalyst containing a β-hairpin peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reback, Matthew L.; Ginovska, Bojana; Buchko, Garry W.

Building on our recent report of an active H2 production catalyst [Ni(PPh2NProp-peptide)2]2+ (Prop=para-phenylpropionic acid, peptide (R10)=WIpPRWTGPR-NH2, p=D-proline, and P2N=1-aza-3,6-diphosphacycloheptane) that contains structured -hairpin peptides, here we investigate how H2 production is effected by: (1) the length of the hairpin (eight or ten residues) and (2) limiting the flexibility between the peptide and the core complex by altering the length of the linker: para-phenylpropionic acid (three carbons) or para-benzoic acid (one carbon). Reduction of the peptide chain length from ten to eight residues increases or maintains the catalytic current for H2 production for all complexes, suggesting a non-productive steric interaction atmore » longer peptide lengths. While the structure of the hairpin appears largely intact for the complexes, NMR data are consistent with differences in dynamic behavior which may contribute to the observed differences in catalytic activity. Molecular dynamics simulations demonstrate that complexes with a one-carbon linker have the desired effect of restricting the motion of the hairpin relative to the complex; however, the catalytic currents are significantly reduced compared to complexes containing a three-carbon linker as a result of the electron withdrawing nature of the -COOH group. These results demonstrate the complexity and interrelated nature of the outer coordination sphere on catalysis.« less

Design and synthesis of small molecule agonists of EphA2 receptor.

PubMed

Petty, Aaron; Idippily, Nethrie; Bobba, Viharika; Geldenhuys, Werner J; Zhong, Bo; Su, Bin; Wang, Bingcheng

2018-01-01

Ligand-independent activation of EphA2 receptor kinase promotes cancer metastasis and invasion. Activating EphA2 receptor tyrosine kinase with small molecule agonist is a novel strategy to treat EphA2 overexpressing cancer. In this study, we performed a lead optimization of a small molecule Doxazosin that was identified as an EphA2 receptor agonist. 33 new analogs were developed and evaluated; a structure-activity relationship was summarized based on the EphA2 activation of these derivatives. Two new derivative compounds 24 and 27 showed much improved activity compared to Doxazosin. Compound 24 possesses a bulky amide moiety, and compound 27 has a dimeric structure that is very different to the parental compound. Compound 27 with a twelve-carbon linker of the dimer activated the kinase and induced receptor internalization and cell death with the best potency. Another dimer with a six-carbon linker has significantly reduced potency compared to the dimer with a longer linker, suggesting that the length of the linker is critical for the activity of the dimeric agonist. To explore the receptor binding characteristics of the new molecules, we applied a docking study to examine how the small molecule binds to the EphA2 receptor. The results reveal that compounds 24 and 27 form more hydrogen bonds to EphA2 than Doxazosin, suggesting that they may have higher binding affinity to the receptor. Published by Elsevier Masson SAS.

Insights into the Impact of Linker Flexibility and Fragment Ionization on the Design of CK2 Allosteric Inhibitors: Comparative Molecular Dynamics Simulation Studies.

PubMed

Zhou, Yue; Zhang, Na; Qi, Xiaoqian; Tang, Shan; Sun, Guohui; Zhao, Lijiao; Zhong, Rugang; Peng, Yongzhen

2018-01-01

Protein kinase is a novel therapeutic target for human diseases. The off-target and side effects of ATP-competitive inhibitors preclude them from the clinically relevant drugs. The compounds targeting the druggable allosteric sites outside the highly conversed ATP binding pocket have been identified as promising alternatives to overcome current barriers of ATP-competitive inhibitors. By simultaneously interacting with the αD region (new allosteric site) and sub-ATP binding pocket, the attractive compound CAM4066 was named as allosteric inhibitor of CK2α. It has been demonstrated that the rigid linker and non-ionizable substituted fragment resulted in significant decreased inhibitory activities of compounds. The molecular dynamics simulations and energy analysis revealed that the appropriate coupling between the linker and pharmacophore fragments were essential for binding of CAM4066 with CK2α. The lower flexible linker of compound 21 lost the capability of coupling fragments A and B to αD region and positive area, respectively, whereas the methyl benzoate of fragment B induced the re-orientated Pre-CAM4066 with the inappropriate polar interactions. Most importantly, the match between the optimized linker and pharmacophore fragments is the challenging work of fragment-linking based drug design. These results provide rational clues to further structural modification and development of highly potent allosteric inhibitors of CK2.

Insights into the Impact of Linker Flexibility and Fragment Ionization on the Design of CK2 Allosteric Inhibitors: Comparative Molecular Dynamics Simulation Studies

PubMed Central

Zhou, Yue; Zhang, Na; Qi, Xiaoqian; Tang, Shan; Zhao, Lijiao; Zhong, Rugang; Peng, Yongzhen

2018-01-01

Protein kinase is a novel therapeutic target for human diseases. The off-target and side effects of ATP-competitive inhibitors preclude them from the clinically relevant drugs. The compounds targeting the druggable allosteric sites outside the highly conversed ATP binding pocket have been identified as promising alternatives to overcome current barriers of ATP-competitive inhibitors. By simultaneously interacting with the αD region (new allosteric site) and sub-ATP binding pocket, the attractive compound CAM4066 was named as allosteric inhibitor of CK2α. It has been demonstrated that the rigid linker and non-ionizable substituted fragment resulted in significant decreased inhibitory activities of compounds. The molecular dynamics simulations and energy analysis revealed that the appropriate coupling between the linker and pharmacophore fragments were essential for binding of CAM4066 with CK2α. The lower flexible linker of compound 21 lost the capability of coupling fragments A and B to αD region and positive area, respectively, whereas the methyl benzoate of fragment B induced the re-orientated Pre-CAM4066 with the inappropriate polar interactions. Most importantly, the match between the optimized linker and pharmacophore fragments is the challenging work of fragment-linking based drug design. These results provide rational clues to further structural modification and development of highly potent allosteric inhibitors of CK2. PMID:29301250

A Series of Robust Copper-Based Triazolyl Isophthalate MOFs: Impact of Linker Functionalization on Gas Sorption and Catalytic Activity †

PubMed Central

Junghans, Ulrike; Kobalz, Merten; Erhart, Oliver; Preißler, Hannes; Lincke, Jörg; Möllmer, Jens; Krautscheid, Harald; Gläser, Roger

2017-01-01

The synthesis and characterization of an isomorphous series of copper-containing microporous metal-organic frameworks (MOFs) based on triazolyl isophthalate linkers with the general formula ∞3[Cu4(μ3-OH)2(R1-R2-trz-ia)3(H2O)x] are presented. Through size adjustment of the alkyl substituents R1 and/or R2 at the linker, the impact of linker functionalization on structure-property relationships was studied. Due to the arrangement of the substituents towards the cavities, the porosity (pore fraction 28%–39%), as well as the pore size can be adjusted by the size of the substituents of the triazole ring. Thermal analysis and temperature-dependent PXRD studies reveal a thermal stability of the MOFs up to 230 °C due to increasing framework stability through fine-tuning of the linker substitution pattern. Adsorption of CO2 (298 K) shows a decreasing maximum loading with increasing steric demand of the substituents of the triazole ring. Furthermore, the selective oxidation of cyclohexene with tert-butyl hydroperoxide (TBHP) is studied over the MOFs at 323 K in liquid chloroform. The catalytic activity increases with the steric demand of the substituents. Additionally, these isomorphous MOFs exhibit considerable robustness under oxidizing conditions confirmed by CO2 adsorption studies, as well as by the catalytic selective oxidation experiments. PMID:28772698

Surface morphology control of cross-linked polymer particles via dispersion polymerization.

PubMed

Peng, Bo; Imhof, Arnout

2015-05-14

Cross-linked polymer colloids (poly(methyl methacrylate) and polystyrene) with diverse shapes were prepared in polar solvents (ethanol, methanol and water) via dispersion polymerization, in which a linear addition of the cross-linker was used during reaction. Apart from spherical particles we found dented spheres or particles covered with nodules, or a combination of both. A comprehensive investigation was carried out, mainly concentrating on the effect of the experimental conditions (e.g., the addition start time and total addition time, cross-linker density and the solvency of the solvents) on particle morphologies. Consequently, we suggest a number of effective ways for the synthesis of regular (spherical) colloidal particles through maintaining a relatively low concentration of the cross-linker during the entire reaction, or forcing the co-polymerization (of monomer and cross-linker) locus to the continuous medium, or using a high quality or quantity of the stabilizer. Moreover, the size of the particles was also precisely manipulated by varying the polarity of the solvents, the concentration of the cross-linker, and the amount and average molecular weight of the stabilizer. In addition, the formation of the heavily dented particles with a very rough surface prepared under a pure or oxygen-'contaminated' nitrogen environment was monitored over time. The results accumulated in this article are of use for a better understanding of the mechanism of the polymerization and control over the structure and property of polymer particles.

Slowing Translation between Protein Domains by Increasing Affinity between mRNAs and the Ribosomal Anti-Shine-Dalgarno Sequence Improves Solubility.

PubMed

Vasquez, Kevin A; Hatridge, Taylor A; Curtis, Nicholas C; Contreras, Lydia M

2016-02-19

Recent studies have demonstrated that effective protein production requires coordination of multiple cotranslational cellular processes, which are heavily affected by translation timing. Until recently, protein engineering has focused on codon optimization to maximize protein production rates, mostly considering the effect of tRNA abundance. However, as it relates to complex multidomain proteins, it has been hypothesized that strategic translational pauses between domains and between distinct individual structural motifs can prevent interactions between nascent chain fragments that generate kinetically trapped misfolded peptides and thereby enhance protein yields. In this study, we introduce synthetic transient pauses between structural domains in a heterologous model protein based on designed patterns of affinity between the mRNA and the anti-Shine-Dalgarno (aSD) sequence on the ribosome. We demonstrate that optimizing translation attenuation at domain boundaries can predictably affect solubility patterns in bacteria. Exploration of the affinity space showed that modifying less than 1% of the nucleotides (on a small 12 amino acid linker) can vary soluble protein yields up to ∼7-fold without altering the primary sequence of the protein. In the context of longer linkers, where a larger number of distinct structural motifs can fold outside the ribosome, optimal synonymous codon variations resulted in an additional 2.1-fold increase in solubility, relative to that of nonoptimized linkers of the same length. While rational construction of 54 linkers of various affinities showed a significant correlation between protein solubility and predicted affinity, only weaker correlations were observed between tRNA abundance and protein solubility. We also demonstrate that naturally occurring high-affinity clusters are present between structural domains of β-galactosidase, one of Escherichia coli's largest native proteins. Interdomain ribosomal affinity is an important factor that has not previously been explored in the context of protein engineering.

Adenovirus fibre shaft sequences fold into the native triple beta-spiral fold when N-terminally fused to the bacteriophage T4 fibritin foldon trimerisation motif.

PubMed

Papanikolopoulou, Katerina; Teixeira, Susana; Belrhali, Hassan; Forsyth, V Trevor; Mitraki, Anna; van Raaij, Mark J

2004-09-03

Adenovirus fibres are trimeric proteins that consist of a globular C-terminal domain, a central fibrous shaft and an N-terminal part that attaches to the viral capsid. In the presence of the globular C-terminal domain, which is necessary for correct trimerisation, the shaft segment adopts a triple beta-spiral conformation. We have replaced the head of the fibre by the trimerisation domain of the bacteriophage T4 fibritin, the foldon. Two different fusion constructs were made and crystallised, one with an eight amino acid residue linker and one with a linker of only two residues. X-ray crystallographic studies of both fusion proteins shows that residues 319-391 of the adenovirus type 2 fibre shaft fold into a triple beta-spiral fold indistinguishable from the native structure, although this is now resolved at a higher resolution of 1.9 A. The foldon residues 458-483 also adopt their natural structure. The intervening linkers are not well ordered in the crystal structures. This work shows that the shaft sequences retain their capacity to fold into their native beta-spiral fibrous fold when fused to a foreign C-terminal trimerisation motif. It provides a structural basis to artificially trimerise longer adenovirus shaft segments and segments from other trimeric beta-structured fibre proteins. Such artificial fibrous constructs, amenable to crystallisation and solution studies, can offer tractable model systems for the study of beta-fibrous structure. They can also prove useful for gene therapy and fibre engineering applications.

Deanne W. Sammond | NREL

Science.gov Websites

Interactions," Journal of Biomolecular Structure & Dynamics (2009) "Structure-Based Protocol for from left to right with several dots of multiple colors. "Cellulase Linkers Are Optimized Based on the Sequence and Structure of a Protein-Binding Peptide," Journal of the American Chemical

Structural Basis of Interdomain Communication in the Hsc70 Chaperone

PubMed Central

Jiang, Jianwen; Prasad, Kondury; Lafer, Eileen M.; Sousa, Rui

2015-01-01

Summary Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. Were-port here the 2.6 Å structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds. PMID:16307916

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A.

PubMed

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-12-09

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A*

PubMed Central

Kont, Riin; Kari, Jeppe; Borch, Kim; Westh, Peter; Väljamäe, Priit

2016-01-01

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase. PMID:27780868

Functional characterization of Kv11.1 (hERG) potassium channels split in the voltage-sensing domain.

PubMed

de la Peña, Pilar; Domínguez, Pedro; Barros, Francisco

2018-03-23

Voltage-dependent KCNH family potassium channel functionality can be reconstructed using non-covalently linked voltage-sensing domain (VSD) and pore modules (split channels). However, the necessity of a covalent continuity for channel function has not been evaluated at other points within the two functionally independent channel modules. We find here that by cutting Kv11.1 (hERG, KCNH2) channels at the different loops linking the transmembrane spans of the channel core, not only channels split at the S4-S5 linker level, but also those split at the intracellular S2-S3 and the extracellular S3-S4 loops, yield fully functional channel proteins. Our data indicate that albeit less markedly, channels split after residue 482 in the S2-S3 linker resemble the uncoupled gating phenotype of those split at the C-terminal end of the VSD S4 transmembrane segment. Channels split after residues 514 and 518 in the S3-S4 linker show gating characteristics similar to those of the continuous wild-type channel. However, breaking the covalent link at this level strongly accelerates the voltage-dependent accessibility of a membrane impermeable methanethiosulfonate reagent to an engineered cysteine at the N-terminal region of the S4 transmembrane helix. Thus, besides that of the S4-S5 linker, structural integrity of the intracellular S2-S3 linker seems to constitute an important factor for proper transduction of VSD rearrangements to opening and closing the cytoplasmic gate. Furthermore, our data suggest that the short and probably rigid characteristics of the extracellular S3-S4 linker are not an essential component of the Kv11.1 voltage sensing machinery.

SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

PubMed

Register, A C; Leonard, Stephen E; Maly, Dustin J

2014-11-11

Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

Cross-Linker Unbinding and Self-Similarity in Bundled Cytoskeletal Networks

NASA Astrophysics Data System (ADS)

Lieleg, O.; Bausch, A. R.

2007-10-01

The macromechanical properties of purely bundled in vitro actin networks are not only determined by the micromechanical properties of individual bundles but also by molecular unbinding events of the actin-binding protein (ABP) fascin. Under high mechanical load the network elasticity depends on the forced unbinding of individual ABPs in a rate dependent manner. Cross-linker unbinding in combination with the structural self-similarity of the network enables the introduction of a concentration-time superposition principle—broadening the mechanically accessible frequency range over 8 orders of magnitude.

Atomic structure of the vimentin central α-helical domain and its implications for intermediate filament assembly.

PubMed

Chernyatina, Anastasia A; Nicolet, Stefan; Aebi, Ueli; Herrmann, Harald; Strelkov, Sergei V

2012-08-21

Together with actin filaments and microtubules, intermediate filaments (IFs) are the basic cytoskeletal components of metazoan cells. Over 80 human diseases have been linked to mutations in various IF proteins to date. However, the filament structure is far from being resolved at the atomic level, which hampers rational understanding of IF pathologies. The elementary building block of all IF proteins is a dimer consisting of an α-helical coiled-coil (CC) "rod" domain flanked by the flexible head and tail domains. Here we present three crystal structures of overlapping human vimentin fragments that comprise the first half of its rod domain. Given the previously solved fragments, a nearly complete atomic structure of the vimentin rod has become available. It consists of three α-helical segments (coils 1A, 1B, and 2) interconnected by linkers (L1 and L12). Most of the CC structure has a left-handed twist with heptad repeats, but both coil 1B and coil 2 also exhibit untwisted, parallel stretches with hendecad repeats. In the crystal structure, linker L1 was found to be α-helical without being involved in the CC formation. The available data allow us to construct an atomic model of the antiparallel tetramer representing the second level of vimentin assembly. Although the presence of the nonhelical head domains is essential for proper tetramer stabilization, the precise alignment of the dimers forming the tetramer appears to depend on the complementarity of their surface charge distribution patterns, while the structural plasticity of linker L1 and coil 1A plays a role in the subsequent IF assembly process.

The central globular domain of the nucleocapsid protein of human immunodeficiency virus type 1 is critical for virion structure and infectivity.

PubMed

Ottmann, M; Gabus, C; Darlix, J L

1995-03-01

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is a 72-amino-acid peptide containing two CCHC-type zinc fingers linked by a short basic sequence, 29RAPRKKG35, which is conserved in HIV-1 and simian immunodeficiency virus. The complete three-dimensional structure of NCp7 has been determined by 1H-nuclear magnetic resonance spectroscopy (N. Morellet, H. de Rocquigny, Y. Mely, N. Jullian, H. Demene, M. Ottmann, D. Gerard, J. L. Darlix, M. C. Fournié-Zaluski, and B. P. Roques, J. Mol. Biol. 235:287-301, 1994) and revealed a central globular domain where the two zinc fingers are brought in close proximity by the RAPRKKG linker. To examine the role of this globular structure and more precisely of the RAPRKKG linker in virion structure and infectivity, we generated HIV-1 DNA mutants in the RAPRKK sequence of NCp7 and analyzed the mutant virions produced by transfected cells. Mutations that probably alter the structure of NCp7 structure led to the formation of very poorly infectious virus (A30P) or noninfectious virus (P31L and R32G). In addition, the P31L mutant did not contain detectable amounts of reverse transcriptase and had an immature core morphology, as determined by electron microscopy. On the other hand, mutations changing the basic nature of NCp7 had poor effect. R29S had a wild-type phenotype, and the replacement of 32RKK34 by SSS (S3 mutant) resulted in a decrease by no more than 100-fold of the virus titer. These results clearly show that the RAPRKKG linker contains residues that are critical for virion structure and infectivity.

The central globular domain of the nucleocapsid protein of human immunodeficiency virus type 1 is critical for virion structure and infectivity.

PubMed Central

Ottmann, M; Gabus, C; Darlix, J L

1995-01-01

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is a 72-amino-acid peptide containing two CCHC-type zinc fingers linked by a short basic sequence, 29RAPRKKG35, which is conserved in HIV-1 and simian immunodeficiency virus. The complete three-dimensional structure of NCp7 has been determined by 1H-nuclear magnetic resonance spectroscopy (N. Morellet, H. de Rocquigny, Y. Mely, N. Jullian, H. Demene, M. Ottmann, D. Gerard, J. L. Darlix, M. C. Fournié-Zaluski, and B. P. Roques, J. Mol. Biol. 235:287-301, 1994) and revealed a central globular domain where the two zinc fingers are brought in close proximity by the RAPRKKG linker. To examine the role of this globular structure and more precisely of the RAPRKKG linker in virion structure and infectivity, we generated HIV-1 DNA mutants in the RAPRKK sequence of NCp7 and analyzed the mutant virions produced by transfected cells. Mutations that probably alter the structure of NCp7 structure led to the formation of very poorly infectious virus (A30P) or noninfectious virus (P31L and R32G). In addition, the P31L mutant did not contain detectable amounts of reverse transcriptase and had an immature core morphology, as determined by electron microscopy. On the other hand, mutations changing the basic nature of NCp7 had poor effect. R29S had a wild-type phenotype, and the replacement of 32RKK34 by SSS (S3 mutant) resulted in a decrease by no more than 100-fold of the virus titer. These results clearly show that the RAPRKKG linker contains residues that are critical for virion structure and infectivity. PMID:7853517

Malleable machines in transcription regulation: the mediator complex.

PubMed

Tóth-Petróczy, Agnes; Oldfield, Christopher J; Simon, István; Takagi, Yuichiro; Dunker, A Keith; Uversky, Vladimir N; Fuxreiter, Monika

2008-12-01

The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the disordered regions and their embedded interaction sites are quite similar in the two organisms. All of these data suggest an integral role for intrinsic disorder in Mediator's function.

Defect Creation by Linker Fragmentation in Metal-Organic Frameworks and Its Effects on Gas Uptake Properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barin, G; Krungleviciute, V; Gutov, O

2014-07-07

We successfully demonstrate an approach based on linker fragmentation to create defects and tune the pore volumes and surface areas of two metal-organic frameworks, NU-125 and HKUST-1, both of which feature copper paddlewheel nodes. Depending on the linker fragment composition, the defect can be either a vacant site or a functional group that the original linker does not have. In the first case, we show that both surface area and pore volume increase, while in the second case they decrease. The effect of defects on the high-pressure gas uptake is also studied over a large temperature and pressure range formore » different gases. We found that despite an increase in pore volume and surface area in structures with vacant sites, the absolute adsorption for methane decreases for HKUST-1 and slightly increases for NU-125. However, the working capacity (deliverable amount between 65 and 5 bar) in both cases remains similar to parent frameworks due to lower uptakes at low pressures. In the case of NU-125, the effect of defects became more pronounced at lower temperatures, reflecting the greater surface areas and pore volumes of the altered forms.« less

Fivefold increase of hydrogen uptake in MOF74 through linker decorations

NASA Astrophysics Data System (ADS)

Arter, C. A.; Zuluaga, S.; Harrison, D.; Welchman, E.; Thonhauser, T.

2016-10-01

We present ab initio results for linker decorations in Mg-MOF74, i.e., attaching various metals M =Li, Na, K, Sc, Cr, Mn, Fe, Ni, Cu, Zn, Rb, Pd, Ag, and Pt near the ring of the linker, creating new strong adsorption sites and thus maximizing small-molecule uptake. We find that in most cases these decorations influence the overall form and structure of Mg-MOF74 only marginally. After the initial screening, we chose metals that bind favorably to the linker and further investigated adsorption of H2,CO2, and H2O for M =Li , Na, K, and Sc. For the case of H2 we show that up to 24 additional guest molecules can be adsorbed in the metal-organic framework (MOF) unit cell, with binding energies comparable to the original open-metal sites at the six corners of the channel. This leads to a fivefold increase of the molecule uptake in Mg-MOF74, with tremendous impact on many applications in general and hydrogen storage in particular, where the gravimetric hydrogen density increases from 1.63 to 7.28 mass % and the volumetric density increases from 15.10 to 75.50 g H2L-1 .

Fast antibody fragment motion: flexible linkers act as entropic spring

PubMed Central

Stingaciu, Laura R.; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

2016-01-01

A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. The Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function. PMID:27020739

Fast antibody fragment motion: flexible linkers act as entropic spring

DOE PAGES

Stingaciu, Laura R.; Ivanova, Oxana; Ohl, Michael; ...

2016-03-29

A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unboundmore » state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. In conclusion, the Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function.« less

Fast antibody fragment motion: flexible linkers act as entropic spring.

PubMed

Stingaciu, Laura R; Ivanova, Oxana; Ohl, Michael; Biehl, Ralf; Richter, Dieter

2016-03-29

A flexible linker region between three fragments allows antibodies to adjust their binding sites to an antigen or receptor. Using Neutron Spin Echo Spectroscopy we observed fragment motion on a timescale of 7 ns with motional amplitudes of about 1 nm relative to each other. The mechanistic complexity of the linker region can be described by a spring model with Brownian motion of the fragments in a harmonic potential. Displacements, timescale, friction and force constant of the underlying dynamics are accessed. The force constant exhibits a similar strength to an entropic spring, with friction of the fragment matching the unbound state. The observed fast motions are fluctuations in pre-existing equilibrium configurations. The Brownian motion of domains in a harmonic potential is the appropriate model to examine functional hinge motions dependent on the structural topology and highlights the role of internal forces and friction to function.

Automated Assignment of MS/MS Cleavable Cross-Links in Protein 3D-Structure Analysis

NASA Astrophysics Data System (ADS)

Götze, Michael; Pettelkau, Jens; Fritzsche, Romy; Ihling, Christian H.; Schäfer, Mathias; Sinz, Andrea

2015-01-01

CID-MS/MS cleavable cross-linkers hold an enormous potential for an automated analysis of cross-linked products, which is essential for conducting structural proteomics studies. The created characteristic fragment ion patterns can easily be used for an automated assignment and discrimination of cross-linked products. To date, there are only a few software solutions available that make use of these properties, but none allows for an automated analysis of cleavable cross-linked products. The MeroX software fills this gap and presents a powerful tool for protein 3D-structure analysis in combination with MS/MS cleavable cross-linkers. We show that MeroX allows an automatic screening of characteristic fragment ions, considering static and variable peptide modifications, and effectively scores different types of cross-links. No manual input is required for a correct assignment of cross-links and false discovery rates are calculated. The self-explanatory graphical user interface of MeroX provides easy access for an automated cross-link search platform that is compatible with commonly used data file formats, enabling analysis of data originating from different instruments. The combination of an MS/MS cleavable cross-linker with a dedicated software tool for data analysis provides an automated workflow for 3D-structure analysis of proteins. MeroX is available at www.StavroX.com .

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

PubMed Central

Taraska, Justin W.; Puljung, Michael C.; Zagotta, William N.

2009-01-01

The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger fluorophores and longer linkers, this donor–acceptor pair accurately reports short-range distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements. PMID:19805285

NMR solution structure of the N-terminal domain of hERG and its interaction with the S4-S5 linker

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Qingxin; Gayen, Shovanlal; Chen, Angela Shuyi

Research highlights: {yields} The N-terminal domain (NTD, eag domain) containing 135 residues of hERG was expressed and purified from E. coli cells. {yields} Solution structure of NTD was determined with NMR spectroscopy. {yields} The alpha-helical region (residues 13-23) was demonstrated to possess the characteristics of an amphipathic helix. {yields} NMR titration confirmed the interaction between NTD and the peptide from the S4-S5 linker. -- Abstract: The human Ether-a-go-go Related Gene (hERG) potassium channel mediates the rapid delayed rectifier current (IKr) in the cardiac action potential. Mutations in the 135 amino acid residue N-terminal domain (NTD) cause channel dysfunction or mis-translocation.more » To study the structure of NTD, it was overexpressed and purified from Escherichia coli cells using affinity purification and gel filtration chromatography. The purified protein behaved as a monomer under purification conditions. Far- and near-UV, circular dichroism (CD) and solution nuclear magnetic resonance (NMR) studies showed that the purified protein was well-folded. The solution structure of NTD was obtained and the N-terminal residues 13-23 forming an amphipathic helix which may be important for the protein-protein or protein-membrane interactions. NMR titration experiment also demonstrated that residues from 88 to 94 in NTD are important for the molecular interaction with the peptide derived from the S4-S5 linker.« less

Low resolution crystal structure of Arenicola erythrocruorin: influence of coiled coils on the architecture of a megadalton respiratory protein.

PubMed

Royer, William E; Omartian, Michael N; Knapp, James E

2007-01-05

Annelid erythrocruorins are extracellular respiratory complexes assembled from 180 subunits into hexagonal bilayers. Cryo-electron microscopic experiments have identified two different architectural classes. In one, designated type I, the vertices of the two hexagonal layers are partially staggered, with one hexagonal layer rotated by about 16 degrees relative to the other layer, whereas in the other class, termed type II, the vertices are essentially eclipsed. We report here the first crystal structure of a type II erythrocruorin, that from Arenicola marina, at 6.2 A resolution. The structure reveals the presence of long continuous triple-stranded coiled-coil "spokes" projecting towards the molecular center from each one-twelfth unit; interdigitation of these spokes provides the only contacts between the two hexagonal layers of the complex. This arrangement contrasts with that of a type I erythrocruorin from Lumbricus terrestris in which the spokes are broken into two triple-stranded coiled coils with a disjointed connection. The disjointed connection allows formation of a more compact structure in the type I architecture, with the two hexagonal layers closer together and additional extensive contacts between the layers. Comparison of sequences of the coiled-coil regions of various linker subunits shows that the linker subunits from type II erythrocruorins possess continuous heptad repeats, whereas a sequence gap places these repeats out of register in the type I linker subunits, consistent with a disjointed coiled-coil arrangement.

Structure of RDE-4 dsRBDs and mutational studies provide insights into dsRNA recognition in the Caenorhabditis elegans RNAi pathway.

PubMed

Chiliveri, Sai Chaitanya; Deshmukh, Mandar V

2014-02-15

The association of RDE-4 (RNAi defective 4), a protein containing two dsRBDs (dsRNA-binding domains), with long dsRNA and Dcr-1 (Dicer1 homologue) initiates the siRNA pathway in Caenorhabditis elegans. Unlike its homologues in higher eukaryotes, RDE-4 dsRBDs possess weak (micromolar) affinity for short dsRNA. With increasing length of dsRNA, RDE-4 exhibits enhanced affinity due to co-operativity. The linker and dsRBD2 are indispensable for RDE-4's simultaneous interaction with dsRNA and Dcr-1. In the present study, we have determined the solution structures of RDE-4 constructs that contain both dsRBDs and the linker region. In addition to the canonical dsRBD fold, both dsRBDs of RDE-4 show modified structural features such as truncation in the β1-β2 loop that rationalize RDE-4's relatively weak dsRNA affinity. Structure and binding studies demonstrate that dsRBD2 plays a decisive role in the RDE-4-dsRNA interaction; however, in contrast with previous findings, we found ephemeral interaction of RDE-4 dsRBD1 with dsRNA. More importantly, mutations in two tandem lysine residues (Lys217 and Lys218) in dsRBD2 impair RDE-4's dsRNA-binding ability and could obliterate RNAi initiation in C. elegans. Additionally, we postulate a structural basis for the minimal requirement of linker and dsRBD2 for RDE-4's association with dsRNA and Dcr-1.

Germline-specific H1 variants: the "sexy" linker histones.

PubMed

Pérez-Montero, Salvador; Carbonell, Albert; Azorín, Fernando

2016-03-01

The eukaryotic genome is packed into chromatin, a nucleoprotein complex mainly formed by the interaction of DNA with the abundant basic histone proteins. The fundamental structural and functional subunit of chromatin is the nucleosome core particle, which is composed by 146 bp of DNA wrapped around an octameric protein complex formed by two copies of each core histone H2A, H2B, H3, and H4. In addition, although not an intrinsic component of the nucleosome core particle, linker histone H1 directly interacts with it in a monomeric form. Histone H1 binds nucleosomes near the exit/entry sites of linker DNA, determines nucleosome repeat length and stabilizes higher-order organization of nucleosomes into the ∼30 nm chromatin fiber. In comparison to core histones, histone H1 is less well conserved through evolution. Furthermore, histone H1 composition in metazoans is generally complex with most species containing multiple variants that play redundant as well as specific functions. In this regard, a characteristic feature is the presence of specific H1 variants that replace somatic H1s in the germline and during early embryogenesis. In this review, we summarize our current knowledge about their structural and functional properties.

The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies

NASA Astrophysics Data System (ADS)

Iacobucci, Claudio; Piotrowski, Christine; Rehkamp, Anne; Ihling, Christian H.; Sinz, Andrea

2018-04-01

Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal "click reaction" and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2. [Figure not available: see fulltext.

STED nanoscopy of the centrosome linker reveals a CEP68-organized, periodic rootletin network anchored to a C-Nap1 ring at centrioles.

PubMed

Vlijm, Rifka; Li, Xue; Panic, Marko; Rüthnick, Diana; Hata, Shoji; Herrmannsdörfer, Frank; Kuner, Thomas; Heilemann, Mike; Engelhardt, Johann; Hell, Stefan W; Schiebel, Elmar

2018-03-06

The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is ∼35 to 40 nm, leading to an estimated minimal rootletin length of ∼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator. Copyright © 2018 the Author(s). Published by PNAS.

Potential Pitfalls and Solutions for Use of Fluorescent Fusion Proteins to Study the Lysosome

PubMed Central

Huang, Ling; Pike, Douglas; Sleat, David E.; Nanda, Vikas; Lobel, Peter

2014-01-01

Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2), a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr) fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6-phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1), while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications. PMID:24586430

Kinetics and Thermodynamics of Watson-Crick Base Pairing Driven DNA Origami Dimerization.

PubMed

Zenk, John; Tuntivate, Chanon; Schulman, Rebecca

2016-03-16

We investigate the kinetics and thermodynamics of DNA origami dimerization using flat rectangle origami components and different architectures of Watson-Crick complementary single-stranded DNA ("sticky end") linking strategies. We systematically vary the number of linkers, the length of the sticky ends on the linker, and linker architecture and measure the corresponding yields as well as forward and reverse reaction rate constants through fluorescence quenching assays. Yields were further verified using atomic force microscopy. We calculate values of H° and ΔS° for various interface designs and find nonlinear van't Hoff behavior, best described by two linear equations, suggesting distinct regimes of dimerization between those with and those without well-formed interfaces. We find that self-assembly reactions can be tuned by manipulating the interface architecture without suffering a loss in yield, even when yield is high, ∼75-80%. We show that the second-order forward reaction rate constant (k(on)) depends on both linker architecture and number of linkers used, with typical values on the order of 10(5)-10(6) (M·s)(-1), values that are similar to those of bimolecular association of small, complementary DNA strands. The k(on) values are generally non-Arrhenius, tending to increase with decreasing temperature. Finally, we use kinetic and thermodynamic information about the optimal linking architecture to extend the system to an infinite, two-component repeating lattice system and show that we can form micron-sized lattices, with well-formed structures up to 8 μm(2).

Novel Fusion Protein Approach for Efficient High-Throughput Screening of Small Molecule–Mediating Protein-Protein Interactions in Cells and Living Animals

PubMed Central

Paulmurugan, Ramasamy; Gambhir, Sanjiv S.

2014-01-01

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule–mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction–mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGS-FACGSLSCGSF. A 9 ± 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation. PMID:16103094

Novel fusion protein approach for efficient high-throughput screening of small molecule-mediating protein-protein interactions in cells and living animals.

PubMed

Paulmurugan, Ramasamy; Gambhir, Sanjiv S

2005-08-15

Networks of protein interactions execute many different intracellular pathways. Small molecules either synthesized within the cell or obtained from the external environment mediate many of these protein-protein interactions. The study of these small molecule-mediated protein-protein interactions is important in understanding abnormal signal transduction pathways in a variety of disorders, as well as in optimizing the process of drug development and validation. In this study, we evaluated the rapamycin-mediated interaction of the human proteins FK506-binding protein (FKBP12) rapamycin-binding domain (FRB) and FKBP12 by constructing a fusion of these proteins with a split-Renilla luciferase or a split enhanced green fluorescent protein (split-EGFP) such that complementation of the reporter fragments occurs in the presence of rapamycin. Different linker peptides in the fusion protein were evaluated for the efficient maintenance of complemented reporter activity. This system was studied in both cell culture and xenografts in living animals. We found that peptide linkers with two or four EAAAR repeat showed higher protein-protein interaction-mediated signal with lower background signal compared with having no linker or linkers with amino acid sequences GGGGSGGGGS, ACGSLSCGSF, and ACGSLSCGSFACGSLSCGSF. A 9 +/- 2-fold increase in signal intensity both in cell culture and in living mice was seen compared with a system that expresses both reporter fragments and the interacting proteins separately. In this fusion system, rapamycin induced heterodimerization of the FRB and FKBP12 moieties occurred rapidly even at very lower concentrations (0.00001 nmol/L) of rapamycin. For a similar fusion system employing split-EGFP, flow cytometry analysis showed significant level of rapamycin-induced complementation.

A mechanism for acetylcholine receptor gating based on structure, coupling, phi, and flip.

PubMed

Gupta, Shaweta; Chakraborty, Srirupa; Vij, Ridhima; Auerbach, Anthony

2017-01-01

Nicotinic acetylcholine receptors are allosteric proteins that generate membrane currents by isomerizing ("gating") between resting and active conformations under the influence of neurotransmitters. Here, to explore the mechanisms that link the transmitter-binding sites (TBSs) with the distant gate, we use mutant cycle analyses to measure coupling between residue pairs, phi value analyses to sequence domain rearrangements, and current simulations to reproduce a microsecond shut component ("flip") apparent in single-channel recordings. Significant interactions between amino acids separated by >15 Å are rare; an exception is between the αM2-M3 linkers and the TBSs that are ∼30 Å apart. Linker residues also make significant, local interactions within and between subunits. Phi value analyses indicate that without agonists, the linker is the first region in the protein to reach the gating transition state. Together, the phi pattern and flip component suggest that a complete, resting↔active allosteric transition involves passage through four brief intermediate states, with brief shut events arising from sojourns in all or a subset. We derive energy landscapes for gating with and without agonists, and propose a structure-based model in which resting→active starts with spontaneous rearrangements of the M2-M3 linkers and TBSs. These conformational changes stabilize a twisted extracellular domain to promote transmembrane helix tilting, gate dilation, and the formation of a "bubble" that collapses to initiate ion conduction. The energy landscapes suggest that twisting is the most energetically unfavorable step in the resting→active conformational change and that the rate-limiting step in the reverse process is bubble formation. © 2017 Gupta et al.

Dissociation Behavior of a TEMPO-Active Ester Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS) in Negative ESI-MS.

PubMed

Hage, Christoph; Ihling, Christian H; Götze, Michael; Schäfer, Mathias; Sinz, Andrea

2017-01-01

We have synthesized a homobifunctional amine-reactive cross-linking reagent, containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) and a benzyl group (Bz), termed TEMPO-Bz-linker, to derive three-dimensional structural information of proteins. The aim for designing this novel cross-linker was to facilitate the mass spectrometric analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). In an initial study, we had investigated the fragmentation behavior of TEMPO-Bz-derivatized peptides upon collision activation in (+)-electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) experiments. In addition to the homolytic NO-C bond cleavage FRIPS pathway delivering the desired odd-electron product ions, an alternative heterolytic NO-C bond cleavage, resulting in even-electron product ions mechanism was found to be relevant. The latter fragmentation route clearly depends on the protonation of the TEMPO-Bz-moiety itself, which motivated us to conduct (-)-ESI-MS, CID-MS/MS, and MS 3 experiments of TEMPO-Bz-cross-linked peptides to further clarify the fragmentation behavior of TEMPO-Bz-peptide molecular ions. We show that the TEMPO-Bz-linker is highly beneficial for conducting FRIPS in negative ionization mode as the desired homolytic cleavage of the NO-C bond is the major fragmentation pathway. Based on characteristic fragments, the isomeric amino acids leucine and isoleucine could be discriminated. Interestingly, we observed pronounced amino acid side chain losses in cross-linked peptides if the cross-linked peptides contain a high number of acidic amino acids. Graphical Abstract ᅟ.

Multiple-component covalent organic frameworks

PubMed Central

Huang, Ning; Zhai, Lipeng; Coupry, Damien E.; Addicoat, Matthew A.; Okushita, Keiko; Nishimura, Katsuyuki; Heine, Thomas; Jiang, Donglin

2016-01-01

Covalent organic frameworks are a class of crystalline porous polymers that integrate molecular building blocks into periodic structures and are usually synthesized using two-component [1+1] condensation systems comprised of one knot and one linker. Here we report a general strategy based on multiple-component [1+2] and [1+3] condensation systems that enable the use of one knot and two or three linker units for the synthesis of hexagonal and tetragonal multiple-component covalent organic frameworks. Unlike two-component systems, multiple-component covalent organic frameworks feature asymmetric tiling of organic units into anisotropic skeletons and unusually shaped pores. This strategy not only expands the structural complexity of skeletons and pores but also greatly enhances their structural diversity. This synthetic platform is also widely applicable to multiple-component electron donor–acceptor systems, which lead to electronic properties that are not simply linear summations of those of the conventional [1+1] counterparts. PMID:27460607

Multiple-component covalent organic frameworks

NASA Astrophysics Data System (ADS)

Huang, Ning; Zhai, Lipeng; Coupry, Damien E.; Addicoat, Matthew A.; Okushita, Keiko; Nishimura, Katsuyuki; Heine, Thomas; Jiang, Donglin

2016-07-01

Covalent organic frameworks are a class of crystalline porous polymers that integrate molecular building blocks into periodic structures and are usually synthesized using two-component [1+1] condensation systems comprised of one knot and one linker. Here we report a general strategy based on multiple-component [1+2] and [1+3] condensation systems that enable the use of one knot and two or three linker units for the synthesis of hexagonal and tetragonal multiple-component covalent organic frameworks. Unlike two-component systems, multiple-component covalent organic frameworks feature asymmetric tiling of organic units into anisotropic skeletons and unusually shaped pores. This strategy not only expands the structural complexity of skeletons and pores but also greatly enhances their structural diversity. This synthetic platform is also widely applicable to multiple-component electron donor-acceptor systems, which lead to electronic properties that are not simply linear summations of those of the conventional [1+1] counterparts.

Linker DNA accessibility in chromatin fibers of different conformations: a reevaluation.

PubMed Central

Zlatanova, J; Leuba, S H; Yang, G; Bustamante, C; van Holde, K

1994-01-01

New studies on chromatin fiber morphology, using the technique of scanning force microscopy (SFM), have caused us to reexamine recent analysis of nuclease digestion of chromatin. Chicken erythrocyte chromatin fibers, glutaraldehyde-fixed at 0, 10, and 80 mM NaCl, were imaged with the help of SFM. The chromatin fibers possessed a loose three-dimensional 30-nm structure even in the absence of added salt. This structure slightly condensed upon addition of 10 mM NaCl, and highly compacted, irregularly segmented fibers were observed at 80 mM NaCl. This sheds new light upon our previously reported analysis of the kinetics of digestion by soluble and membrane-immobilized micrococcal nuclease [Leuba, S. H., Zlatanova, J. & van Holde, K. (1994) J. Mol. Biol. 235, 871-880]. While the low-ionic-strength fibers were readily digested, the highly compacted structure formed at 80 mM NaCl was refractory to nuclease attack, implying that the linkers were fully accessible in the low-ionic-strength conformation but not in the condensed fibers. We now find that cleavage of the linker DNA by a small molecule, methidiumpropyl-EDTA-Fe(II), proceeds for all types of conformations at similar rates. Thus, steric hindrance is responsible for the lack of accessibility to micrococcal nuclease in the condensed fiber. Taken in total the data suggest that reexamination of existing models of chromatin conformation is warranted. Images PMID:8202481

Integrity of N- and C-termini is important for E. coli Hsp31 chaperone activity

PubMed Central

Sastry, M S R; Zhou, Weibin; Baneyx, François

2009-01-01

Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging. PMID:19517531

High-resolution solid-state 13C NMR spectroscopy of the paramagnetic metal-organic frameworks, STAM-1 and HKUST-1.

PubMed

Dawson, Daniel M; Jamieson, Lauren E; Mohideen, M Infas H; McKinlay, Alistair C; Smellie, Iain A; Cadou, Romain; Keddie, Neil S; Morris, Russell E; Ashbrook, Sharon E

2013-01-21

Solid-state (13)C magic-angle spinning (MAS) NMR spectroscopy is used to investigate the structure of the Cu(II)-based metal-organic frameworks (MOFs), HKUST-1 and STAM-1, and the structural changes occurring within these MOFs upon activation (dehydration). NMR spectroscopy is an attractive technique for the investigation of these materials, owing to its high sensitivity to local structure, without any requirement for longer-range order. However, interactions between nuclei and unpaired electrons in paramagnetic systems (e.g., Cu(II)-based MOFs) pose a considerable challenge, not only for spectral acquisition, but also in the assignment and interpretation of the spectral resonances. Here, we exploit the rapid T(1) relaxation of these materials to obtain (13)C NMR spectra using a spin-echo pulse sequence at natural abundance levels, and employ frequency-stepped acquisition to ensure uniform excitation of resonances over a wide frequency range. We then utilise selective (13)C isotopic labelling of the organic linker molecules to enable an unambiguous assignment of NMR spectra of both MOFs for the first time. We show that the monomethylated linker can be recovered from STAM-1 intact, demonstrating not only the interesting use of this MOF as a protecting group, but also the ability (for both STAM-1 and HKUST-1) to recover isotopically-enriched linkers, thereby reducing significantly the overall cost of the approach.

Crystallization of bi-functional ligand protein complexes.

PubMed

Antoni, Claudia; Vera, Laura; Devel, Laurent; Catalani, Maria Pia; Czarny, Bertrand; Cassar-Lajeunesse, Evelyn; Nuti, Elisa; Rossello, Armando; Dive, Vincent; Stura, Enrico Adriano

2013-06-01

Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects. Copyright © 2013 Elsevier Inc. All rights reserved.

Crystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design.

PubMed

Corradi, Hazel R; Schwager, Sylva L U; Nchinda, Aloysius T; Sturrock, Edward D; Acharya, K Ravi

2006-03-31

Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pallan, Pradeep S.; Marshall, William S.; Harp, Joel

To understand the role of structural elements of RNA pseudoknots in controlling the extent of -1-type ribosomal frameshifting, we determined the crystal structure of a high-efficiency frameshifting mutant of the pseudoknot from potato leaf roll virus (PLRV). Correlations of the structure with available in vitro frameshifting data for PLRV pseudoknot mutants implicate sequence and length of a stem-loop linker as modulators of frameshifting efficiency. Although the sequences and overall structures of the RNA pseudoknots from PLRV and beet western yellow virus (BWYV) are similar, nucleotide deletions in the linker and adjacent minor groove loop abolish frameshifting only with the latter.more » Conversely, mutant PLRV pseudoknots with up to four nucleotides deleted in this region exhibit nearly wild-type frameshifting efficiencies. The crystal structure helps rationalize the different tolerances for deletions in the PLRV and BWYV RNAs, and we have used it to build a three-dimensional model of the PRLV pseudoknot with a four-nucleotide deletion. The resulting structure defines a minimal RNA pseudoknot motif composed of 22 nucleotides capable of stimulating -1-type ribosomal frameshifts.« less

Structural features of LC8-induced self-association of swallow.

PubMed

Kidane, Ariam I; Song, Yujuan; Nyarko, Afua; Hall, Justin; Hare, Michael; Löhr, Frank; Barbar, Elisar

2013-09-03

Cell functions depend on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than that of dynein, and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the energetics and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, intermonomer nuclear Overhauser effects assigned to residues expected at interface positions, and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled coil. Secondary chemical shifts and (15)N backbone relaxation identify the boundaries and distinguishing structural features of the coiled coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled coil is critical for bicoid mRNA localization because it favors structural organization of Swallow, which except for the central LC8-promoted coiled coil is primarily disordered.

Structural Features of LC8-Induced Self Association of Swallow†

PubMed Central

Kidane, Ariam I.; Song, Yujuan; Nyarko, Afua; Hall, Justin; Hare, Michael; Löhr, Frank; Barbar, Elisar

2013-01-01

Cell function depends on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than dynein and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the mechanistic and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, inter-monomer NOEs assigned to residues expected at interface positions and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled-coil. Secondary chemical shifts and 15N backbone relaxation identify the boundaries and distinguishing structural features of the coiled-coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled-coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled-coil is critical for bicoid mRNA localization because it could induce structural organization of Swallow, which except for the central LC8-promoted coiled-coil is primarily disordered. PMID:23914803

Structural insights into the histone H1-nucleosome complex

PubMed Central

Zhou, Bing-Rui; Feng, Hanqiao; Kato, Hidenori; Dai, Liang; Yang, Yuedong; Zhou, Yaoqi; Bai, Yawen

2013-01-01

Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1–nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo. PMID:24218562

F-actin cross-linking enhances the stability of force generation in disordered actomyosin networks

NASA Astrophysics Data System (ADS)

Jung, Wonyeong; Murrell, Michael P.; Kim, Taeyoon

2015-12-01

Myosin molecular motors and actin cross-linking proteins (ACPs) are known to mediate the generation and transmission of mechanical forces within the cortical F-actin cytoskeleton that drive major cellular processes such as cell division and migration. However, how motors and ACPs interact collectively over diverse timescales to modulate the time-dependent mechanical properties of the cytoskeleton remains unclear. In this study, we present a three-dimensional agent-based computational model of the cortical actomyosin network to quantitatively determine the effects of motor activity and the density and kinetics of ACPs on the accumulation and maintenance of mechanical tension within a disordered actomyosin network. We found that motors accumulate large stress quickly by behaving as temporary cross-linkers although this stress is relaxed over time unless there are sufficient passive ACPs to stabilize the network. Stabilization by ACPs helps motors to generate forces up to their maximum potential, leading to significant enhancement of the efficiency and stability of stress generation. Thus, we demonstrated that the force-dependent kinetics of ACP dissociation plays a critical role for the accumulation and sustainment of stress and the structural remodeling of networks.

Alkyl piperidine and piperazine hydroxamic acids as HDAC inhibitors.

PubMed

Rossi, Cristina; Porcelloni, Marina; D'Andrea, Piero; Fincham, Christopher I; Ettorre, Alessandro; Mauro, Sandro; Squarcia, Antonella; Bigioni, Mario; Parlani, Massimo; Nardelli, Federica; Binaschi, Monica; Maggi, Carlo A; Fattori, Daniela

2011-04-15

We report here the strategy used in our research group to find a new class of histone deacetylase (HDAC) inhibitors. A series of N-substituted 4-alkylpiperazine and 4-alkylpiperidine hydroxamic acids, corresponding to the basic structure of HDAC inhibitors (zinc binding moiety-linker-capping group) has been designed, prepared, and tested for HDAC inhibition. Linker length and aromatic capping group connection were systematically varied to find the optimal geometric parameters. A new series of submicromolar inhibitors was thus identified, which showed antiproliferative activity on HCT-116 colon carcinoma cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Smart nanogels at the air/water interface: structural studies by neutron reflectivity

NASA Astrophysics Data System (ADS)

Zielińska, Katarzyna; Sun, Huihui; Campbell, Richard A.; Zarbakhsh, Ali; Resmini, Marina

2016-02-01

The development of effective transdermal drug delivery systems based on nanosized polymers requires a better understanding of the behaviour of such nanomaterials at interfaces. N-Isopropylacrylamide-based nanogels synthesized with different percentages of N,N'-methylenebisacrylamide as cross-linker, ranging from 10 to 30%, were characterized at physiological temperature at the air/water interface, using neutron reflectivity (NR), with isotopic contrast variation, and surface tension measurements; this allowed us to resolve the adsorbed amount and the volume fraction of nanogels at the interface. A large conformational change for the nanogels results in strong deformations at the interface. As the percentage of cross-linker incorporated in the nanogels becomes higher, more rigid matrices are obtained, although less deformed, and the amount of adsorbed nanogels is increased. The data provide the first experimental evidence of structural changes of nanogels as a function of the degree of cross-linking at the air/water interface.The development of effective transdermal drug delivery systems based on nanosized polymers requires a better understanding of the behaviour of such nanomaterials at interfaces. N-Isopropylacrylamide-based nanogels synthesized with different percentages of N,N'-methylenebisacrylamide as cross-linker, ranging from 10 to 30%, were characterized at physiological temperature at the air/water interface, using neutron reflectivity (NR), with isotopic contrast variation, and surface tension measurements; this allowed us to resolve the adsorbed amount and the volume fraction of nanogels at the interface. A large conformational change for the nanogels results in strong deformations at the interface. As the percentage of cross-linker incorporated in the nanogels becomes higher, more rigid matrices are obtained, although less deformed, and the amount of adsorbed nanogels is increased. The data provide the first experimental evidence of structural changes of nanogels as a function of the degree of cross-linking at the air/water interface. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07538f

Effect of the linkers between the zinc fingers in zinc finger protein 809 on gene silencing and nuclear localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ichida, Yu, E-mail: ichida-y@ncchd.go.jp; Utsunomiya, Yuko; Onodera, Masafumi

2016-03-18

Zinc finger protein 809 (ZFP809) belongs to the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV). ZFP809 binds to the primer-binding site (PBS)located downstream of the MoMLV-long terminal repeat (LTR) and induces epigenetic modifications at integration sites, such as repressive histone modifications and de novo DNA methylation. KRAB-ZFPs contain consensus TGEKP linkers between C2H2 zinc fingers. The phosphorylation of threonine residues within linkers leads to the inactivation of zinc finger binding to target sequences. ZFP809 also contains consensus linkers between zinc fingers. However, the function of ZFP809 linkers remainsmore » unknown. In the present study, we constructed ZFP809 proteins containing mutated linkers and examined their ability to silence transgene expression driven by MLV, binding ability to MLV PBS, and cellular localization. The results of the present study revealed that the linkers affected the ability of ZFP809 to silence transgene expression. Furthermore, this effect could be partly attributed to changes in the localization of ZFP809 proteins containing mutated linkers. Further characterization of ZFP809 linkers is required for understanding the functions and features of KRAB-ZFP-containing linkers. - Highlights: • ZFP809 has three consensus linkers between the zinc fingers. • Linkers are required for ZFP809 to silence transgene expression driven by MLV-LTR. • Linkers affect the precise nuclear localization of ZFP809.« less

H3 Histone Tail Conformation within the Nucleosome and the Impact of K14 Acetylation Studied Using Enhanced Sampling Simulation

PubMed Central

Ikebe, Jinzen; Sakuraba, Shun; Kono, Hidetoshi

2016-01-01

Acetylation of lysine residues in histone tails is associated with gene transcription. Because histone tails are structurally flexible and intrinsically disordered, it is difficult to experimentally determine the tail conformations and the impact of acetylation. In this work, we performed simulations to sample H3 tail conformations with and without acetylation. The results show that irrespective of the presence or absence of the acetylation, the H3 tail remains in contact with the DNA and assumes an α-helix structure in some regions. Acetylation slightly weakened the interaction between the tail and DNA and enhanced α-helix formation, resulting in a more compact tail conformation. We inferred that this compaction induces unwrapping and exposure of the linker DNA, enabling DNA-binding proteins (e.g., transcription factors) to bind to their target sequences. In addition, our simulation also showed that acetylated lysine was more often exposed to the solvent, which is consistent with the fact that acetylation functions as a post-translational modification recognition site marker. PMID:26967163

Structural Basis of Vta1 Function in the Multivesicular Body Sorting Pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Junyu; Xia, Hengchuan; Zhou, Jiahai

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity, but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domainmore » stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif-containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly.« less

Structural basis of Vta1 function in the multi-vesicular body sorting pathway

PubMed Central

Xiao, Junyu; Xia, Hengchuan; Zhou, Jiahai; Azmi, Ishara; Davies, Brian A.; Katzmann, David J.; Xu, Zhaohui

2009-01-01

Summary The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domain stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly. PMID:18194651

Construction, Structural Diversity and Properties of Five Coordination Polymers Based on 5-Nitroisophthalate and Bis(imidazole) Linkers

NASA Astrophysics Data System (ADS)

Arıcı, Mürsel

2018-06-01

Five coordination polymers, namely, [Cd(μ3-5-nip)(μ-obix)]n (1), [Co(μ3-5-nip)(μ-obix)]n (2), [Zn(μ-5-nip)(μ-obix)]n (3 and 4) and [Cd(μ-5-nip)(μ-bisobix)]n (5) (5-nip: 5-nitroisophthalate, obix: 1,2-bis(imidazol-1ylmethyl)benzene, bisobix: 1,2-bis(2-isopropylimidazol-1ylmethyl)benzene) were hydrothermally synthesized and characterized by IR spectroscopy, elemental analysis, single crystal and powder X-ray diffraction and thermal analysis (TG/DTA). X-ray results showed that the complexes displayed structural diversity depending on metal ions and conformations of bis(imidazole) linkers. Complexes 1 and 2 were 1D structures and obix ligand displayed cis-conformation. Complexes 3 and 4 exhibited 2D and 3D structures with same components depending on obix conformation. In complex 5, 3D+3D→3D interpenetrated structure was obtained with dia topology when bisobix having sterically hindered groups on imidazole rings was used. Moreover, thermal, photoluminescence and optical properties of the complexes were also investigated.

Quantitation of fixative-induced morphologic and antigenic variation in mouse and human breast cancers

PubMed Central

Cardiff, Robert D; Hubbard, Neil E; Engelberg, Jesse A; Munn, Robert J; Miller, Claramae H; Walls, Judith E; Chen, Jane Q; Velásquez-García, Héctor A; Galvez, Jose J; Bell, Katie J; Beckett, Laurel A; Li, Yue-Ju; Borowsky, Alexander D

2013-01-01

Quantitative Image Analysis (QIA) of digitized whole slide images for morphometric parameters and immunohistochemistry of breast cancer antigens was used to evaluate the technical reproducibility, biological variability, and intratumoral heterogeneity in three transplantable mouse mammary tumor models of human breast cancer. The relative preservation of structure and immunogenicity of the three mouse models and three human breast cancers was also compared when fixed with representatives of four distinct classes of fixatives. The three mouse mammary tumor cell models were an ER + /PR + model (SSM2), a Her2 + model (NDL), and a triple negative model (MET1). The four breast cancer antigens were ER, PR, Her2, and Ki67. The fixatives included examples of (1) strong cross-linkers, (2) weak cross-linkers, (3) coagulants, and (4) combination fixatives. Each parameter was quantitatively analyzed using modified Aperio Technologies ImageScope algorithms. Careful pre-analytical adjustments to the algorithms were required to provide accurate results. The QIA permitted rigorous statistical analysis of results and grading by rank order. The analyses suggested excellent technical reproducibility and confirmed biological heterogeneity within each tumor. The strong cross-linker fixatives, such as formalin, consistently ranked higher than weak cross-linker, coagulant and combination fixatives in both the morphometric and immunohistochemical parameters. PMID:23399853

5-fold increase of hydrogen uptake in MOF74 through linker decorations

NASA Astrophysics Data System (ADS)

Thonhauser, T.; Zuluaga, S.; Harrison, D.; Welchman, E.; Arter, C.

We present ab initio results for linker decorations in Mg-MOF74-i.e. attaching various metals  = Li, Na, K, Sc, Cr, Mn, Fe, Ni, Cu, Zn, Rb, Pd, Ag, and Pt near the ring of the linker-creating new strong adsorption sites and thus maximizing small molecule uptake. We find that in most cases these decorations influence the overall form and structure of Mg-MOF74 only marginally. After the initial screening we chose metals that bind favorably to the linker and further investigate adsorption of H2, CO2, and H2O for  = Li, Na, K, and Sc. For the case of H2 we show that up to 24 additional guest molecules can be adsorbed in the MOF unit cell, with binding energies comparable to the original open-metal sites at the six corners of the channel. This leads to a 5-fold increase of the molecule uptake in Mg-MOF74, with tremendous impact on many applications in general and hydrogen storage in particular-where the gravimetric hydrogen density increases from 1 . 63 mass% to 7 . 28 mass% and the volumetric density from 15.10 g H2 L-1 to 75.50 g H2 L-1. This work was supported by NSF Grant No. DMR-1145968.

Lignin-Based Materials Through Thiol-Maleimide "Click" Polymerization.

PubMed

Buono, Pietro; Duval, Antoine; Averous, Luc; Habibi, Youssef

2017-03-09

In the present report an environmentally friendly approach to transforming renewable feedstocks into value-added materials is proposed. This transformation pathway was conducted under green conditions, without the use of solvents or catalyst. First, controlled modification of lignin, a major biopolymer present in wood and plants, was achieved by esterification with 11-maleimidoundecylenic acid (11-MUA), a derivative from castor oil that contains maleimide groups, following its transformation into 11-maleimidoundecanoyl chloride (11-MUC). Different degrees of substitution were achieved by using various amounts of the 11-MUC, leading to an efficient conversion of lignin hydroxy groups, as demonstrated by 1 H and 31 P NMR analyses. These fully biobased maleimide-lignin derivatives were subjected to an extremely fast (ca. 1 min) thiol-ene "click" polymerization with thiol-containing linkers. Aliphatic and aromatic thiol linkers bearing two to four thiol groups were used to tune the reactivity and crosslink density. The properties of the resulting materials were evaluated by swelling tests and thermal and mechanical analyses, which showed that varying the degree of functionality of the linker and the linker structure allowed accurate tailoring of the thermal and mechanical properties of the final materials, thus providing interesting perspectives for lignin in functional aromatic polymers. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cd(II)-coordination polymers based on tetracarboxylic acid and diverse bis(imidazole) ligands: Synthesis, structural diversity and photoluminescence properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arıcı, Mürsel, E-mail: marici@ogu.edu.tr; Yeşilel, Okan Zafer; Taş, Murat

Three new Cd(II)-coordination polymers, namely, ([Cd{sub 2}(μ{sub 6}-ao{sub 2}btc)(μ-1,5-bipe){sub 2}]·2H{sub 2}O){sub n} (1), ([Cd{sub 2}(μ{sub 6}-ao{sub 2}btc)(μ-1,4-bix){sub 2}]{sub n}·2DMF) (2) and ([Cd{sub 2}(μ{sub 8}-abtc)(μ-1,4-betix)]·DMF·H{sub 2}O){sub n} (3) (ao{sub 2}btc=di-oxygenated form of 3,3′,5,5′-azobenzenetetracarboxylate, 1,5-bipe: 1,5-bis(imidazol-1yl)pentane, 1,4-bix=1,4-bis(imidazol-1ylmethyl)benzene, 1,4-betix=1,4-bis(2-ethylimidazol-1ylmethyl)benzene) were synthesized with 3,3′,5,5′-azobenzenetetracarboxylic acid and flexible, semi-flexible and semi-flexible substituted bis(imidazole) linkers. They were characterized by IR spectroscopy, elemental analysis, single-crystal X-ray diffraction, powder X-ray diffractions (PXRD) and thermal analyses (TG/DTA). Complexes 1–3 exhibited structural diversities depending on flexible, semi-flexible and semi-flexible substituted bis(imidazole) ligands. Complex 1 was 2D structure with 3,6L18 topology. Complex 2 had a 3D pillar-layered framework with the raremore » sqc27 topology. When semi-flexible substituted bis(imidazole) linker was used, 3D framework of complex 3 was obtained with the paddlewheel Cd{sub 2}(CO{sub 2}){sub 4}-type binuclear SBU. Moreover, thermal and photoluminescence properties of the complexes were determined in detailed. - Graphical abstract: In this study, three novel Cd(II)-coordination polymers were synthesized with 3,3′,5,5′-azobenzenetetracarboxylic acid and flexible, semi-flexible and semi-flexible substituted bis(imidazole) linkers. They were characterized by IR spectroscopy, elemental analysis, single-crystal X-ray diffraction, powder X-ray diffractions (PXRD) and thermal analyses (TG/DTA). Complexes 1–3 exhibited structural diversities depending on flexible, semi-flexible and semi-flexible substituted bis(imidazole) ligands. Complex 1 was 2D structure with 3,6L18 topology. Complex 2 had a 3D pillar-layered framework with the rare sqc27 topology. When semi-flexible substituted bis(imidazole) linker was used, 3D framework of complex 3 was obtained with the paddlewheel Cd{sub 2}(CO{sub 2}){sub 4}-type binuclear SBU. - Highlights: • Three new Cd(II)-coordination polymers with azobenzenetetracarboxylic acid and diverse bis(imidazole) linkers. • Complex 1 is 2D structure with 3,6L18 topology. • 3D pillar-layered framework of 2 with the rare sqc27 topology. • 3D framework of 3 with the paddlewheel Cd{sub 2}(CO{sub 2}){sub 4}-type SBU.« less

A mechanism for acetylcholine receptor gating based on structure, coupling, phi, and flip

PubMed Central

Gupta, Shaweta; Chakraborty, Srirupa; Vij, Ridhima

2017-01-01

Nicotinic acetylcholine receptors are allosteric proteins that generate membrane currents by isomerizing (“gating”) between resting and active conformations under the influence of neurotransmitters. Here, to explore the mechanisms that link the transmitter-binding sites (TBSs) with the distant gate, we use mutant cycle analyses to measure coupling between residue pairs, phi value analyses to sequence domain rearrangements, and current simulations to reproduce a microsecond shut component (“flip”) apparent in single-channel recordings. Significant interactions between amino acids separated by >15 Å are rare; an exception is between the αM2–M3 linkers and the TBSs that are ∼30 Å apart. Linker residues also make significant, local interactions within and between subunits. Phi value analyses indicate that without agonists, the linker is the first region in the protein to reach the gating transition state. Together, the phi pattern and flip component suggest that a complete, resting↔active allosteric transition involves passage through four brief intermediate states, with brief shut events arising from sojourns in all or a subset. We derive energy landscapes for gating with and without agonists, and propose a structure-based model in which resting→active starts with spontaneous rearrangements of the M2–M3 linkers and TBSs. These conformational changes stabilize a twisted extracellular domain to promote transmembrane helix tilting, gate dilation, and the formation of a “bubble” that collapses to initiate ion conduction. The energy landscapes suggest that twisting is the most energetically unfavorable step in the resting→active conformational change and that the rate-limiting step in the reverse process is bubble formation. PMID:27932572

Dynamics differentiate between active and inactive inteins

PubMed Central

Cronin, Melissa; Coolbaugh, Michael J; Nellis, David; Zhu, Jianwei; Wood, David W.; Nussinov, Ruth; Ma, Buyong

2014-01-01

The balance between stability and dynamics for active enzymes can be somewhat quantified by studies of intein splicing and cleaving reactions. Inteins catalyze the ligation of flanking host exteins while excising themselves. The potential for applications led to engineering of a mini-intein splicing domain, where the homing endonuclease domain of the Mycobacterium tuberculosis RecA (Mtu recA) intein was removed. The remaining domains were linked by several short peptides, but splicing activity in all was substantially lower than the full-length intein. Native splicing activity was restored in some cases by a V67L mutation. Using computations and experiments, we examine the impact of this mutation on the stability and conformational dynamics of the mini-intein splicing domain. Molecular dynamics simulations were used to delineate the factors that determine the active state, including the V67L mini-intein mutant, and peptide linker. We found that (1) the V67L mutation lowers the global fluctuations in all modeled mini-inteins, stabilizing the mini-intein constructs; (2) the connecting linker length affects intein dynamics; and (3) the flexibilities of the linker and intein core are higher in the active structure. We have observed that the interaction of the linker region and a turn region around residues 35-41 provides the pathway for the allostery interaction. Our experiments reveal that intein catalysis is characterized by non-linear Arrhenius plot, confirming the significant contribution of protein conformational dynamics to intein function. We conclude that while the V67L mutation stabilizes the global structure, cooperative dynamics of all intein regions appear more important for intein function than high stability. Our studies suggest that effectively quenching the conformational dynamics of an intein through engineered allosteric interactions could deactivate intein splicing or cleaving. PMID:25087201

Self-assembling complexes between binary mixtures of lipids with different linkers and nucleic acids promote universal mRNA, DNA and siRNA delivery.

PubMed

Colombani, Thibault; Peuziat, Pauline; Dallet, Laurence; Haudebourg, Thomas; Mével, Mathieu; Berchel, Mathieu; Lambert, Olivier; Habrant, Damien; Pitard, Bruno

2017-03-10

Protein expression and RNA interference require efficient delivery of DNA or mRNA and small double stranded RNA into cells, respectively. Although cationic lipids are the most commonly used synthetic delivery vectors, a clear need still exists for a better delivery of various types of nucleic acids molecules to improve their biological activity. To optimize the transfection efficiency, a molecular approach consisting in modifying the chemical structure of a given cationic lipid is usually performed, but an alternative strategy could rely on modulating the supramolecular assembly of lipidic lamellar phases sandwiching the nucleic acids molecules. To validate this new concept, we synthesized on one hand two paromomycin-based cationic lipids, with either an amide or a phosphoramide linker, and on the other hand two imidazole-based neutral lipids, having as well either an amide or a phosphoramide function as linker. Combinations of cationic and helper lipids containing the same amide or phosphoramide linkers led to the formation of homogeneous lamellar phases, while hybrid lamellar phases were obtained when the linkers on the cationic and helper lipids were different. Cryo-transmission electron microscopy and fluorescence experiments showed that liposomes/nucleic acids complexes resulting from the association of nucleic acids with hybrid lamellar phases led to complexes that were more stable in the extracellular compartment compared to those obtained with homogeneous systems. In addition, we observed that the most active supramolecular assemblies for the delivery of DNA, mRNA and siRNA were obtained when the cationic and helper lipids possess linkers of different natures. The results clearly show that this supramolecular strategy modulating the property of the lipidic lamellar phase constitutes a new approach for increasing the delivery of various types of nucleic acid molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

Design and development of novel linker for PbS quantum dots/TiO₂ mesoscopic solar cell.

PubMed

Etgar, Lioz; Park, Jinhyung; Barolo, Claudia; Nazeeruddin, Md K; Viscardi, Guido; Graetzel, Michael

2011-09-01

A novel bifunctional linker molecule, bis(4-mercaptophenyl)phosphinic acid, is designed to be used in a QDs solar cells. The linker anchors to TiO(2) mesoporous film through the phosphinic acid functional group and to the PbS QDs through the two thiol groups. The way of attachment of this new linker molecule in a photovoltaic PbS QDs/TiO(2) mesoporous device was studied by FTIR measurements. The photovoltaic performance of this new linker in a heterojunction PbS QDs solar cell show high V(oc) relative to QDs based solar cells, which will allow to receive high power conversion efficiency using this novel designed linker. This novel bifunctional linker molecule should pave the way for enhancing binding strength, and efficiency of QDs solar cells compared to the state-of-the-art linkers.

From force-fields to photons: MD simulations of dye-labeled nucleic acids and Monte Carlo modeling of FRET

NASA Astrophysics Data System (ADS)

Goldner, Lori

2012-02-01

Fluorescence resonance energy transfer (FRET) is a powerful technique for understanding the structural fluctuations and transformations of RNA, DNA and proteins. Molecular dynamics (MD) simulations provide a window into the nature of these fluctuations on a different, faster, time scale. We use Monte Carlo methods to model and compare FRET data from dye-labeled RNA with what might be predicted from the MD simulation. With a few notable exceptions, the contribution of fluorophore and linker dynamics to these FRET measurements has not been investigated. We include the dynamics of the ground state dyes and linkers in our study of a 16mer double-stranded RNA. Water is included explicitly in the simulation. Cyanine dyes are attached at either the 3' or 5' ends with a 3 carbon linker, and differences in labeling schemes are discussed.[4pt] Work done in collaboration with Peker Milas, Benjamin D. Gamari, and Louis Parrot.

Tetrahymena Poc1 ensures proper intertriplet microtubule linkages to maintain basal body integrity

PubMed Central

Meehl, Janet B.; Bayless, Brian A.; Giddings, Thomas H.; Pearson, Chad G.; Winey, Mark

2016-01-01

Basal bodies comprise nine symmetric triplet microtubules that anchor forces produced by the asymmetric beat pattern of motile cilia. The ciliopathy protein Poc1 stabilizes basal bodies through an unknown mechanism. In poc1∆ cells, electron tomography reveals subtle defects in the organization of intertriplet linkers (A-C linkers) that connect adjacent triplet microtubules. Complete triplet microtubules are lost preferentially near the posterior face of the basal body. Basal bodies that are missing triplets likely remain competent to assemble new basal bodies with nine triplet microtubules, suggesting that the mother basal body microtubule structure does not template the daughter. Our data indicate that Poc1 stabilizes basal body triplet microtubules through linkers between neighboring triplets. Without this stabilization, specific triplet microtubules within the basal body are more susceptible to loss, probably due to force distribution within the basal body during ciliary beating. This work provides insights into how the ciliopathy protein Poc1 maintains basal body integrity. PMID:27251062

Liquid droplets of cross-linked actin filaments

NASA Astrophysics Data System (ADS)

Weirich, Kimberly; Banerjee, Shiladitya; Dasbiswas, Kinjal; Vaikuntanathan, Suriyanarayan; Gardel, Margaret

Soft materials constructed from biomolecules self-assemble into a myriad of structures that work in concert to support cell physiology. One critical soft material is the actin cytoskeleton, a viscoelastic gel composed of cross-linked actin filaments. Although actin networks are primarily known for their elastic properties, which are crucial to regulating cell mechanics, the viscous behavior has been theorized to enable shape changes and flows. We experimentally demonstrate a fluid phase of cross-linked actin, where cross-linker condenses dilute short actin filaments into spindle-shaped droplets, or tactoids. Tactoids have shape dynamics consistent with a continuum model of liquid crystal droplets. The cross-linker, which acts as a long range attractive interaction, analogous to molecular cohesion, controls the tactoid shape and dynamics, which reports on the liquid's interfacial tension and viscosity. We investigate how the cross-linker properties and filament length influence the liquid properties. These results demonstrate a novel mechanism to control organization of the actin cytoskeleton and provide insight into design principles for complex, macromolecular liquid phases.

Protecting group and switchable pore-discriminating adsorption properties of a hydrophilic-hydrophobic metal-organic framework.

PubMed

Mohideen, M Infas H; Xiao, Bo; Wheatley, Paul S; McKinlay, Alistair C; Li, Yang; Slawin, Alexandra M Z; Aldous, David W; Cessford, Naomi F; Düren, Tina; Zhao, Xuebo; Gill, Rachel; Thomas, K Mark; Griffin, John M; Ashbrook, Sharon E; Morris, Russell E

2011-04-01

Formed by linking metals or metal clusters through organic linkers, metal-organic frameworks are a class of solids with structural and chemical properties that mark them out as candidates for many emerging gas storage, separation, catalysis and biomedical applications. Important features of these materials include their high porosity and their flexibility in response to chemical or physical stimuli. Here, a copper-based metal-organic framework has been prepared in which the starting linker (benzene-1,3,5-tricarboxylic acid) undergoes selective monoesterification during synthesis to produce a solid with two different channel systems, lined by hydrophilic and hydrophobic surfaces, respectively. The material reacts differently to gases or vapours of dissimilar chemistry, some stimulating subtle framework flexibility or showing kinetic adsorption effects. Adsorption can be switched between the two channels by judicious choice of the conditions. The monoesterified linker is recoverable in quantitative yield, demonstrating possible uses of metal-organic frameworks in molecular synthetic chemistry as 'protecting groups' to accomplish selective transformations that are difficult using standard chemistry techniques.

Synthesis, kinetic studies and molecular modeling of novel tacrine dimers as cholinesterase inhibitors.

PubMed

de Aquino, Roney Anderson Nascimento; Modolo, Luzia Valentina; Alves, Rosemeire Brondi; de Fátima, Ângelo

2013-12-28

This study presents the synthesis of 15 new tacrine dimers as well as the Ki and IC50 results, studies of the kinetic mechanism, and molecular docking analysis of the dimers in relation to the cholinesterases hAChE, hBChE, EeAChE and eqBChE. In addition to spectroscopic characterization, X-ray structure determination was performed for two of the new compounds. These new dimers were found to be mixed nanomolar inhibitors of the evaluated targets with a broad and significant selectivity profile, and these properties are dependent on both the type of the linker and the volume of the hydroacridine alicyclic ring. The results indicate that the aromatic linkers play a significant role in generating specific interactions with the half-gorge region of the catalytic center. Thus, these types of linkers can positively modulate the electronic properties of the tacrine dimers studied with an improvement of their cholinesterase inhibition activity.

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain

PubMed Central

Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J.; Polo, Simona

2016-01-01

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VIshort and myosin VIlong, which differ in the C-terminal region. Their physiological and pathological role remains unknown. Here we identified an isoform-specific regulatory helix, named α2-linker that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a novel clathrin-binding domain that is unique to myosin VIlong and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, where alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VIshort for tumor cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VIshort. Thus the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VIlong) or migratory (myosin VIshort) functional roles. PMID:26950368

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

PubMed

Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J; Polo, Simona

2016-04-01

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.

Acid-Labile Poly(glycidyl methacrylate)-Based Star Gene Vectors.

PubMed

Yang, Yan-Yu; Hu, Hao; Wang, Xing; Yang, Fei; Shen, Hong; Xu, Fu-Jian; Wu, De-Cheng

2015-06-10

It was recently reported that ethanolamine-functionalized poly(glycidyl methacrylate) (PGEA) possesses great potential applications in gene therapy due to its good biocompatibility and high transfection efficiency. Importing responsivity into PGEA vectors would further improve their performances. Herein, a series of responsive star-shaped vectors, acetaled β-cyclodextrin-PGEAs (A-CD-PGEAs) consisting of a β-CD core and five PGEA arms linked by acid-labile acetal groups, were proposed and characterized as therapeutic pDNA vectors. The A-CD-PGEAs owned abundant hydroxyl groups to shield extra positive charges of A-CD-PGEAs/pDNA complexes, and the star structure could decrease charge density. The incorporation of acetal linkers endowed A-CD-PGEAs with pH responsivity and degradation. In weakly acidic endosome, the broken acetal linkers resulted in decomposition of A-CD-PGEAs and morphological transformation of A-CD-PGEAs/pDNA complexes, lowering cytotoxicity and accelerating release of pDNA. In comparison with control CD-PGEAs without acetal linkers, A-CD-PGEAs exhibited significantly better transfection performances.

Assembly of porous hierarchical copolymers/resin proppants: New approaches to smart proppant immobilization via molecular anchors.

PubMed

Alexander, Shirin; Dunnill, Charles W; Barron, Andrew R

2016-03-15

The assembly of temperature/pH sensitive complex microparticle structures through chemisorption and physisorption provides a responsive system that offers application as routes to immobilization of proppants in-situ. Thermogravimetric analysis (TGA) and scanning electron microscopy (SEM) along with energy dispersive X-ray analysis (EDX) have been used to characterize a series of bi-functionalized monolayers and/or multilayers grown on alumina microparticles and investigate the reactive nature of both temperature sensitive cross-linker (epoxy resin) with the layers and pH-responsive bridging layer (polyetheramine). The bifunctional acids, behaving as molecular anchors, allow for a controlled reaction with a cross-linker (resin or polymer) with the formation of networks, which is either irreversible or reversible based on the nature of the cross-linker. The networks results in formation of porous hierarchical particles that offer a potential route to the creation of immobile proppant pack. Copyright © 2015 Elsevier Inc. All rights reserved.

Label-free and pH-sensitive colorimetric materials for the sensing of urea

NASA Astrophysics Data System (ADS)

Li, Lu; Long, Yue; Gao, Jin-Ming; Song, Kai; Yang, Guoqiang

2016-02-01

This communication demonstrates a facile method for naked-eye detection of urea based on the structure color change of pH-sensitive photonic crystals. The insertion of urease provides excellent selectivity over other molecules. The detection of urea in different concentration ranges could be realized by changing the molar ratio between the functional monomer and cross-linker.This communication demonstrates a facile method for naked-eye detection of urea based on the structure color change of pH-sensitive photonic crystals. The insertion of urease provides excellent selectivity over other molecules. The detection of urea in different concentration ranges could be realized by changing the molar ratio between the functional monomer and cross-linker. Electronic supplementary information (ESI) available: Materials and chemicals, characterization, experimental details, and SEM images. See DOI: 10.1039/c5nr07690k

Investigating the geometrical preferences of a flexible benzimidazolone-based linker in the synthesis of coordination polymers

PubMed Central

Jones, Corey L.; Marsden, Elizabeth A.; Nevin, Adam C.; Kariuki, Benson M.; Bhadbhade, Mohan M.; Martin, Adam D.

2017-01-01

A series of new group 2 coordination polymers, MgL ={MgL(H2O)(DMF)0.75}∞, CaL = {CaL(DMF)2}∞, SrL = {SrL(H2O)0.5}∞ and BaL = {BaL(H2O)0.5}∞, were synthesized using a flexible benzimidazolone diacetic acid linker (H2L) in which the two carboxylic acid binding sites are connected to a planar core via {–CH2–} spacers that can freely rotate in solution. In a ‘curiosity-led' diversion from group 2 metals, the first row transition metal salts Mn2+, Cu2+ and Zn2+ were also reacted with L to yield crystals of MnL = {MnL(DMF)(H2O)3.33}∞, Cu3L2 = {Cu3L2(DMF)2(CHO2)2}∞ and ZnL = {ZnL(DMF)}∞. Crystal structures were obtained for all seven materials. All structures form as two-dimensional sheets and contain six-coordinate centres, with the exception of ZnL, which displays tetrahedrally coordinated metal centres, and Cu3L2, which contains square planar coordinated metal centres and Cu paddle-wheels. In each structure, the linker adopts one of two distinct conformations, with the carboxylate groups either cis or trans with respect to the planar core. All materials were also characterized by powder X-ray diffraction and thermogravimetric analysis. PMID:29308246

The Smad3 linker region contains a transcriptional activation domain

PubMed Central

2004-01-01

Transforming growth factor-β (TGF-β)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-β/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-β transcriptional activation responses, although it can be phosphorylated by the TGF-β receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-β. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control. PMID:15588252

The Smad3 linker region contains a transcriptional activation domain.

PubMed

Wang, Guannan; Long, Jianyin; Matsuura, Isao; He, Dongming; Liu, Fang

2005-02-15

Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.

Developing a Multiplexed Quantitative Cross-Linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes.

PubMed

Yu, Clinton; Huszagh, Alexander; Viner, Rosa; Novitsky, Eric J; Rychnovsky, Scott D; Huang, Lan

2016-10-18

Cross-linking mass spectrometry (XL-MS) represents a recently popularized hybrid methodology for defining protein-protein interactions (PPIs) and analyzing structures of large protein assemblies. In particular, XL-MS strategies have been demonstrated to be effective in elucidating molecular details of PPIs at the peptide resolution, providing a complementary set of structural data that can be utilized to refine existing complex structures or direct de novo modeling of unknown protein structures. To study structural and interaction dynamics of protein complexes, quantitative cross-linking mass spectrometry (QXL-MS) strategies based on isotope-labeled cross-linkers have been developed. Although successful, these approaches are mostly limited to pairwise comparisons. In order to establish a robust workflow enabling comparative analysis of multiple cross-linked samples simultaneously, we have developed a multiplexed QXL-MS strategy, namely, QMIX (Quantitation of Multiplexed, Isobaric-labeled cross (X)-linked peptides) by integrating MS-cleavable cross-linkers with isobaric labeling reagents. This study has established a new analytical platform for quantitative analysis of cross-linked peptides, which can be directly applied for multiplexed comparisons of the conformational dynamics of protein complexes and PPIs at the proteome scale in future studies.

Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication

NASA Astrophysics Data System (ADS)

Rustiguel, Joane K.; Soares, Ricardo O. S.; Meisburger, Steve P.; Davis, Katherine M.; Malzbender, Kristina L.; Ando, Nozomi; Dias-Baruffi, Marcelo; Nonato, Maria Cristina

2016-09-01

Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin-dependent kinase II holoenzyme

PubMed Central

Chao, Luke H.; Stratton, Margaret M.; Lee, Il-Hyung; Rosenberg, Oren S.; Levitz, Joshua; Mandell, Daniel J.; Kortemme, Tanja; Groves, Jay T.; Schulman, Howard; Kuriyan, John

2011-01-01

Summary Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains. PMID:21884935

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design.

PubMed

Dorywalska, Magdalena; Dushin, Russell; Moine, Ludivine; Farias, Santiago E; Zhou, Dahui; Navaratnam, Thayalan; Lui, Victor; Hasa-Moreno, Adela; Casas, Meritxell Galindo; Tran, Thomas-Toan; Delaria, Kathy; Liu, Shu-Hui; Foletti, Davide; O'Donnell, Christopher J; Pons, Jaume; Shelton, David L; Rajpal, Arvind; Strop, Pavel

2016-05-01

The degree of stability of antibody-drug linkers in systemic circulation, and the rate of their intracellular processing within target cancer cells are among the key factors determining the efficacy of antibody-drug conjugates (ADC) in vivo Previous studies demonstrated the susceptibility of cleavable linkers, as well as auristatin-based payloads, to enzymatic cleavage in rodent plasma. Here, we identify Carboxylesterase 1C as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-p-aminocarbamate (VC-PABC)-based linkers in mouse plasma. We further show that the activity of Carboxylesterase 1C towards VC-PABC-based linkers, and consequently the stability of ADCs in mouse plasma, can be effectively modulated by small chemical modifications to the linker. While the introduced modifications can protect the VC-PABC-based linkers from extracellular cleavage, they do not significantly alter the intracellular linker processing by the lysosomal protease Cathepsin B. The distinct substrate preference of the serum Carboxylesterase 1C offers the opportunity to modulate the extracellular stability of cleavable ADCs without diminishing the intracellular payload release required for ADC efficacy. Mol Cancer Ther; 15(5); 958-70. ©2016 AACR. ©2016 American Association for Cancer Research.

Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

PubMed Central

Wiedemann, Christoph; Szambowska, Anna; Häfner, Sabine; Ohlenschläger, Oliver; Gührs, Karl-Heinz; Görlach, Matthias

2015-01-01

The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain. PMID:25712103

Metal Ion Dependence, Thermodynamics, and Kinetics for Intramolecular Docking of a GAAA Tetraloop and Receptor Connected by a Flexible Linker†

PubMed Central

Downey, Christopher D.; Fiore, Julie L.; Stoddard, Colby D.; Hodak, Jose H.; Nesbitt, David J.; Pardi, Arthur

2008-01-01

The GAAA tetraloop-receptor is a commonly occurring tertiary interaction motif in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of an RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A7 linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking ([Co(NH3)63+] ≪ [Ca2+], [Mg2+], [Mn2+] ≪ [Na+], [K+]). Analysis of metal ion cooperativity yielded Hill coefficients of ≈ 2 for Na+- or K+-dependent docking versus ≈ 1 for the divalent ions and Co(NH3)63+. Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U7 and A14 single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed. PMID:16533049

Important role of phosphoramido linkage in imidazole-based dioleyl helper lipids for liposome stability and primary cell transfection.

PubMed

Mével, Mathieu; Haudebourg, Thomas; Colombani, Thibault; Peuziat, Pauline; Dallet, Laurence; Chatin, Benoît; Lambert, Olivier; Berchel, Mathieu; Montier, Tristan; Jaffrès, Paul-Alain; Lehn, Pierre; Pitard, Bruno

2016-01-01

To optimize synthetic gene delivery systems, there is a need to develop more efficient lipid formulations. Most cationic lipid formulations contain 'helper' neutral lipids because of their ability to increase DNA delivery, in particular by improving endosomal escape of DNA molecules via the pH-buffering effect of protonatable groups and/or fusion with the lipid bilayer of endosomes. We evaluated the influence of the linker structure between the two oleyl chains in the helper lipid on transfection efficiency in cell lines, as well as in primary cells (hepatocytes/cardiomyocytes). We reported the synthesis of two new pH-buffering imidazole helper lipids characterized by a polar headgroup containing one (compound 6) or two (compound 5) imidazole groups and two oleyl chains linked by an amide group. We studied their association with the aminoglycoside lipidic derivative dioleylsuccinylparomomycin (DOSP), which contains two oleyl chains linked to the aminoglycoside polar headgroup via an amide function. We compared the morphology and transfection properties of such binary liposomes of DOSP/5 and DOSP/6 with those of liposomes combining DOSP with another imidazole-based dioleyl helper lipid (MM27) in which a phosphoramido group acts as a linker between the two oleyl chains and imidazole function. The phosphoramido linker in the helper lipid induces a major difference in terms of morphology and resistance to decomplexation at physical pH for DOSP/helper lipid complexes. This hybrid dioleyl linker composition of DOSP/MM27 led to higher transfection efficiency in cell lines and in primary cells compared to complexes with homogeneous dioleyl linker. Copyright © 2015 John Wiley & Sons, Ltd.

Desmosine-Inspired Cross-Linkers for Hyaluronan Hydrogels

NASA Astrophysics Data System (ADS)

Hagel, Valentin; Mateescu, Markus; Southan, Alexander; Wegner, Seraphine V.; Nuss, Isabell; Haraszti, Tamás; Kleinhans, Claudia; Schuh, Christian; Spatz, Joachim P.; Kluger, Petra J.; Bach, Monika; Tussetschläger, Stefan; Tovar, Günter E. M.; Laschat, Sabine; Boehm, Heike

2013-06-01

We designed bioinspired cross-linkers based on desmosine, the cross-linker in natural elastin, to prepare hydrogels with thiolated hyaluronic acid. These short, rigid cross-linkers are based on pyridinium salts (as in desmosine) and can connect two polymer backbones. Generally, the obtained semi-synthetic hydrogels are form-stable, can withstand repeated stress, have a large linear-elastic range, and show strain stiffening behavior typical for biopolymer networks. In addition, it is possible to introduce a positive charge to the core of the cross-linker without affecting the gelation efficiency, or consequently the network connectivity. However, the mechanical properties strongly depend on the charge of the cross-linker. The properties of the presented hydrogels can thus be tuned in a range important for engineering of soft tissues by controlling the cross-linking density and the charge of the cross-linker.

Structure-guided design of novel Trypanosoma brucei Methionyl-tRNA synthetase inhibitors.

PubMed

Huang, Wenlin; Zhang, Zhongsheng; Barros-Álvarez, Ximena; Koh, Cho Yeow; Ranade, Ranae M; Gillespie, J Robert; Creason, Sharon A; Shibata, Sayaka; Verlinde, Christophe L M J; Hol, Wim G J; Buckner, Frederick S; Fan, Erkang

2016-11-29

A screening hit 1 against Trypanosoma brucei methionyl-tRNA synthetase was optimized using a structure-guided approach. The optimization led to the identification of two novel series of potent inhibitors, the cyclic linker and linear linker series. Compounds of both series were potent in a T. brucei growth inhibition assay while showing low toxicity to mammalian cells. The best compound of each series, 16 and 31, exhibited EC 50 s of 39 and 22 nM, respectively. Compounds 16 and 31 also exhibited promising PK properties after oral dosing in mice. Moreover, compound 31 had moderately good brain permeability, with a brain/plasma ratio of 0.27 at 60 min after IP injection. This study provides new lead compounds for arriving at new treatments of human African trypanosomiasis (HAT). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Molecular simulations bring new insights into flavonoid/quercetinase interaction modes.

PubMed

Fiorucci, Sébastien; Golebiowski, Jérôme; Cabrol-Bass, Daniel; Antonczak, Serge

2007-06-01

Molecular dynamics simulations, using the AMBER force field, were performed to study Quercetin 2,3-Dioxygenase enzyme (Quercetinase or 2,3QD). We have analyzed the structural modifications of the active site and of the linker region between the native enzyme and the enzyme-substrate complex. New structural informations, such as an allosteric effect in the presence of the substrate, as well as description of the enzyme-substrate interactions and values of binding free energies were brought out. All these results confirm the idea that the linker encloses the substrate in the active site and also enlighten the recognition role of the substrate B-ring by the enzyme. Moreover, a specific interaction scheme has been proposed to explain the relative degradation rate of various flavonoid compounds under the oxygenolysis reaction catalyzed by the Quercetin 2,3-Dioxygenase enzyme. 2007 Wiley-Liss, Inc.

Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions.

PubMed

Delaforge, Elise; Milles, Sigrid; Huang, Jie-Rong; Bouvier, Denis; Jensen, Malene Ringkjøbing; Sattler, Michael; Hart, Darren J; Blackledge, Martin

2016-01-01

Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales.

Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions

PubMed Central

Delaforge, Elise; Milles, Sigrid; Huang, Jie-rong; Bouvier, Denis; Jensen, Malene Ringkjøbing; Sattler, Michael; Hart, Darren J.; Blackledge, Martin

2016-01-01

Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales. PMID:27679800

Rad5, HLTF, and SHPRH: A Fresh View of an Old Story.

PubMed

Elserafy, Menattallah; Abugable, Arwa A; Atteya, Reham; El-Khamisy, Sherif F

2018-05-25

Not only have helicase-like transcription factor (HLTF) and SNF2 histone-linker PHD-finger RING-finger helicase (SHPRH) proved to be important players in post-replication repair like their yeast counterpart, Rad5, but they are also involved in multiple biological functions and are associated with several human disorders. We provide here an updated view of their functions, associated diseases, and potential therapeutic approaches. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Pore-Environment Engineering with Multiple Metal Sites in Rare-Earth Porphyrinic Metal-Organic Frameworks.

PubMed

Zhang, Liangliang; Yuan, Shuai; Feng, Liang; Guo, Bingbing; Qin, Jun-Sheng; Xu, Ben; Lollar, Christina; Sun, Daofeng; Zhou, Hong-Cai

2018-04-23

Multi-component metal-organic frameworks (MOFs) with precisely controlled pore environments are highly desired owing to their potential applications in gas adsorption, separation, cooperative catalysis, and biomimetics. A series of multi-component MOFs, namely PCN-900(RE), were constructed from a combination of tetratopic porphyrinic linkers, linear linkers, and rare-earth hexanuclear clusters (RE 6 ) under the guidance of thermodynamics. These MOFs exhibit high surface areas (up to 2523 cm 2  g -1 ) and unlimited tunability by modification of metal nodes and/or linker components. Post-synthetic exchange of linear linkers and metalation of two organic linkers were realized, allowing the incorporation of a wide range of functional moieties. Two different metal sites were sequentially placed on the linear linker and the tetratopic porphyrinic linker, respectively, giving rise to an ideal platform for heterogeneous catalysis. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural insights into the recognition of the internal A-rich linker from OxyS sRNA by Escherichia coli Hfq

PubMed Central

Wang, Lijun; Wang, Weiwei; Li, Fudong; Zhang, Jiahai; Wu, Jihui; Gong, Qingguo; Shi, Yunyu

2015-01-01

Small RNA OxyS is induced during oxidative stress in Escherichia coli and it is an Hfq-dependent negative regulator of mRNA translation. OxyS represses the translation of fhlA and rpoS mRNA, which encode the transcriptional activator and σs subunit of RNA polymerase, respectively. However, little is known regarding how Hfq, an RNA chaperone, interacts with OxyS at the atomic level. Here, using fluorescence polarization and tryptophan fluorescence quenching assays, we verified that the A-rich linker region of OxyS sRNA binds Hfq at its distal side. We also report two crystal structures of Hfq in complex with A-rich RNA fragments from this linker region. Both of these RNA fragments bind to the distal side of Hfq and adopt a different conformation compared with those previously reported for the (A-R-N)n tripartite recognition motif. Furthermore, using fluorescence polarization, electrophoresis mobility shift assays and in vivo translation assays, we found that an Hfq mutant, N48A, increases the binding affinity of OxyS for Hfq in vitro but is defective in the negative regulation of fhlA translation in vivo, suggesting that the normal function of OxyS depends on the details of the interaction with Hfq that may be related to the rapid recycling of Hfq in the cell. PMID:25670676

Quercetin-glutamic acid conjugate with a non-hydrolysable linker; a novel scaffold for multidrug resistance reversal agents through inhibition of P-glycoprotein.

PubMed

Kim, Mi Kyoung; Kim, Yunyoung; Choo, Hyunah; Chong, Youhoon

2017-02-01

Previously, we have reported remarkable effect of a quercetin-glutamic acid conjugate to reverse multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer agents through inhibition of P-glycoprotein (Pgp)-mediated drug efflux. Due to the hydrolysable nature, MDR-reversal activity of the quercetin conjugate was attributed to its hydrolysis product, quercetin. However, several lines of evidence demonstrated that the intact quercetin-glutamic acid conjugate has stronger MDR-reversal activity than quercetin. In order to evaluate this hypothesis and to identify a novel scaffold for MDR-reversal agents, we prepared quercetin conjugates with a glutamic acid attached at the 7-O position via a non-hydrolysable linker. Pgp inhibition assay, Pgp ATPase assay, and MDR-reversal activity assay were performed, and the non-hydrolysable quercetin conjugates showed significantly higher activities compared with those of quercetin. Unfortunately, the quercetin conjugates were not as effective as verapamil in Pgp-inhibition and thereby reversing MDR, but it is worth to note that the structurally modified quercetin conjugates with a non-cleavable linker showed significantly improved MDR-reversal activity compared with quercetin. Taken together, the quercetin conjugates with appropriate structural modifications were shown to have a potential to serve as a scaffold for the design of novel MDR-reversal agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling.

PubMed

Brdicka, Tomás; Imrich, Martin; Angelisová, Pavla; Brdicková, Nadezda; Horváth, Ondrej; Spicka, Jirí; Hilgert, Ivan; Lusková, Petra; Dráber, Petr; Novák, Petr; Engels, Niklas; Wienands, Jürgen; Simeoni, Luca; Osterreicher, Jan; Aguado, Enrique; Malissen, Marie; Schraven, Burkhart; Horejsí, Václav

2002-12-16

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.

Pyrene-nucleobase conjugates: synthesis, oligonucleotide binding and confocal bioimaging studies.

PubMed

Jabłoński, Artur; Fritz, Yannic; Wagenknecht, Hans-Achim; Czerwieniec, Rafał; Bernaś, Tytus; Trzybiński, Damian; Woźniak, Krzysztof; Kowalski, Konrad

2017-01-01

Fluorescent pyrene-linker-nucleobase (nucleobase = thymine, adenine) conjugates with carbonyl and hydroxy functionalities in the linker were synthesized and characterized. X-ray single-crystal structure analysis performed for the pyrene-C(O)CH 2 CH 2 -thymine ( 2 ) conjugate reveals dimers of molecules 2 stabilized by hydrogen bonds between the thymine moieties. The photochemical characterization showed structure-dependent fluorescence properties of the investigated compounds. The conjugates bearing a carbonyl function represent weak emitters as compared to compounds with a hydroxy function in the linker. The self-assembly properties of pyrene nucleobases were investigated in respect to their binding to single and double strand oligonucleotides in water and in buffer solution. In respect to the complementary oligothymidine T 10 template in water, compounds 3 and 5 both show a self-assembling behavior according to canonical base-base pairing. However, in buffer solution, derivative 5 was much more effective than 3 in binding to the T 10 template. Furthermore the adenine derivative 5 binds to the double-stranded (dA) 10 -T 10 template with a self-assembly ratio of 112%. Such a high value of a self-assembly ratio can be rationalized by a triple-helix-like binding, intercalation, or a mixture of both. Remarkably, compound 5 also shows dual staining pattern in living HeLa cells. Confocal microscopy confirmed that 5 predominantly stains mitochondria but it also accumulates in the nucleoli of the cells.

Synthesis, activity, and structure--activity relationship studies of novel cationic lipids for DNA transfer.

PubMed

Byk, G; Dubertret, C; Escriou, V; Frederic, M; Jaslin, G; Rangara, R; Pitard, B; Crouzet, J; Wils, P; Schwartz, B; Scherman, D

1998-01-15

We have designed and synthesized original cationic lipids for gene delivery. A synthetic method on solid support allowed easy access to unsymmetrically monofunctionalized polyamine building blocks of variable geometries. These polyamine building blocks were introduced into cationic lipids. To optimize the transfection efficiency in the novel series, we have carried out structure-activity relationship studies by introduction of variable-length lipids, of variable-length linkers between lipid and cationic moiety, and of substituted linkers. We introduce the concept of using the linkers within cationic lipids molecules as carriers of side groups harboring various functionalities (side chain entity), as assessed by the introduction of a library composed of cationic entities, additional lipid chains, targeting groups, and finally the molecular probes rhodamine and biotin for cellular traffic studies. The transfection activity of the products was assayed in vitro on Hela carcinoma, on NIH3T3, and on CV1 fibroblasts and in vivo on the Lewis Lung carcinoma model. Products from the series displayed high transfection activities. Results indicated that the introduction of a targeting side chain moiety into the cationic lipid is permitted. A primary physicochemical characterization of the DNA/lipid complexes was demonstrated with this leading compound. Selected products from the series are currently being developed for preclinical studies, and the labeled lipopolyamines can be used to study the intracellular traffic of DNA/cationic lipid complexes.

Synthetic surfactant- and cross-linker-free preparation of highly stable lipid-polymer hybrid nanoparticles as potential oral delivery vehicles.

PubMed

Wang, Taoran; Xue, Jingyi; Hu, Qiaobin; Zhou, Mingyong; Chang, Chao; Luo, Yangchao

2017-06-05

The toxicity associated with concentrated synthetic surfactants and the poor stability at gastrointestinal condition are two major constraints for practical applications of solid lipid nanoparticles (SLN) as oral delivery vehicles. In this study, a synthetic surfactant-free and cross-linker-free method was developed to fabricate effective, safe, and ultra-stable lipid-polymer hybrid nanoparticles (LPN). Bovine serum albumin (BSA) and dextran varying in molecular weights were first conjugated through Maillard reaction and the conjugates were exploited to emulsify solid lipid by a solvent diffusion and sonication method. The multilayer structure was formed by self-assembly of BSA-dextran micelles to envelope solid lipid via a pH- and heating-induced facile process with simultaneous surface deposition of pectin. The efficiency of different BSA-dextran conjugates was systematically studied to prepare LPN with the smallest size, the most homogeneous distribution and the greatest stability. The molecular interactions were characterized by Fourier transform infrared and fluorescence spectroscopies. Both nano spray drying and freeze-drying methods were tested to produce spherical and uniform pectin-coated LPN powders that were able to re-assemble nanoscale structure when redispersed in water. The results demonstrated the promise of a synthetic surfactant- and cross-linker-free technique to prepare highly stable pectin-coated LPN from all natural biomaterials as potential oral delivery vehicles.

Crucial role of dynamic linker histone binding and divalent ions for DNA accessibility and gene regulation revealed by mesoscale modeling of oligonucleosomes

PubMed Central

Collepardo-Guevara, Rosana; Schlick, Tamar

2012-01-01

Monte Carlo simulations of a mesoscale model of oligonucleosomes are analyzed to examine the role of dynamic-linker histone (LH) binding/unbinding in high monovalent salt with divalent ions, and to further interpret noted chromatin fiber softening by dynamic LH in monovalent salt conditions. We find that divalent ions produce a fiber stiffening effect that competes with, but does not overshadow, the dramatic softening triggered by dynamic-LH behavior. Indeed, we find that in typical in vivo conditions, dynamic-LH binding/unbinding reduces fiber stiffening dramatically (by a factor of almost 5, as measured by the elasticity modulus) compared with rigidly fixed LH, and also the force needed to initiate chromatin unfolding, making it consistent with those of molecular motors. Our data also show that, during unfolding, divalent ions together with LHs induce linker-DNA bending and DNA–DNA repulsion screening, which guarantee formation of heteromorphic superbeads-on-a-string structures that combine regions of loose and compact fiber independently of the characteristics of the LH–core bond. These structures might be important for gene regulation as they expose regions of the DNA selectively. Dynamic control of LH binding/unbinding, either globally or locally, in the presence of divalent ions, might constitute a mechanism for regulation of gene expression. PMID:22790986

A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

PubMed Central

Thie, Holger

2017-01-01

ABSTRACT Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs. PMID:28055297

A global view of structure–function relationships in the tautomerase superfamily

PubMed Central

Davidson, Rebecca; Baas, Bert-Jan; Akiva, Eyal; Holliday, Gemma L.; Polacco, Benjamin J.; LeVieux, Jake A.; Pullara, Collin R.; Zhang, Yan Jessie; Whitman, Christian P.

2018-01-01

The tautomerase superfamily (TSF) consists of more than 11,000 nonredundant sequences present throughout the biosphere. Characterized members have attracted much attention because of the unusual and key catalytic role of an N-terminal proline. These few characterized members catalyze a diverse range of chemical reactions, but the full scale of their chemical capabilities and biological functions remains unknown. To gain new insight into TSF structure–function relationships, we performed a global analysis of similarities across the entire superfamily and computed a sequence similarity network to guide classification into distinct subgroups. Our results indicate that TSF members are found in all domains of life, with most being present in bacteria. The eukaryotic members of the cis-3-chloroacrylic acid dehalogenase subgroup are limited to fungal species, whereas the macrophage migration inhibitory factor subgroup has wide eukaryotic representation (including mammals). Unexpectedly, we found that 346 TSF sequences lack Pro-1, of which 85% are present in the malonate semialdehyde decarboxylase subgroup. The computed network also enabled the identification of similarity paths, namely sequences that link functionally diverse subgroups and exhibit transitional structural features that may help explain reaction divergence. A structure-guided comparison of these linker proteins identified conserved transitions between them, and kinetic analysis paralleled these observations. Phylogenetic reconstruction of the linker set was consistent with these findings. Our results also suggest that contemporary TSF members may have evolved from a short 4-oxalocrotonate tautomerase–like ancestor followed by gene duplication and fusion. Our new linker-guided strategy can be used to enrich the discovery of sequence/structure/function transitions in other enzyme superfamilies. PMID:29184004

Structure and function of polyglycine hydrolases

USDA-ARS?s Scientific Manuscript database

Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave polyglycine linkers of targeted plant defense chitinases. Unlike typical endoproteases that cleave a specific peptide bond, these 640 amino acid glycoproteins selectively cleave one of multiple peptide bonds within polyglyci...

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

PubMed Central

Panjarian, Shoghag; Iacob, Roxana E.; Chen, Shugui; Wales, Thomas E.; Engen, John R.; Smithgall, Thomas E.

2013-01-01

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control. PMID:23303187

Linear Precision Glycomacromolecules with Varying Interligand Spacing and Linker Functionalities Binding to Concanavalin A and the Bacterial Lectin FimH.

PubMed

Igde, Sinaida; Röblitz, Susanna; Müller, Anne; Kolbe, Katharina; Boden, Sophia; Fessele, Claudia; Lindhorst, Thisbe K; Weber, Marcus; Hartmann, Laura

2017-12-01

A series of precision glycomacromolecules is prepared following previously established solid phase synthesis allowing for controlled variations of interligand spacing and the overall number of carbohydrate ligands. In addition, now also different linkers are installed between the carbohydrate ligand and the macromolecular scaffold. The lectin binding behavior of these glycomacromolecules is then evaluated in isothermal titration calorimetry (ITC) and kinITC experiments using the lectin Concanavalin A (Con A) in its dimeric and tetrameric form. The results indicate that both sterical and statistical effects impact lectin binding of precision glycomacromolecules. Moreover, ITC results show that highest affinity toward Con A can be achieved with an ethyl phenyl linker, which parallels earlier findings with the bacterial lectin FimH. In this way, a first set of glycomacromolecule structures is selected for testing in a bacterial adhesion-inhibition study. Here, the findings point to a one-sugar binding mode mainly affected by sterical restraints of the nonbinding parts of the respective glycomacromolecule. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

PubMed Central

Tan, Dan; Li, Qiang; Zhang, Mei-Jun; Liu, Chao; Ma, Chengying; Zhang, Pan; Ding, Yue-He; Fan, Sheng-Bo; Tao, Li; Yang, Bing; Li, Xiangke; Ma, Shoucai; Liu, Junjie; Feng, Boya; Liu, Xiaohui; Wang, Hong-Wei; He, Si-Min; Gao, Ning; Ye, Keqiong; Dong, Meng-Qiu; Lei, Xiaoguang

2016-01-01

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction. DOI: http://dx.doi.org/10.7554/eLife.12509.001 PMID:26952210

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal

PubMed Central

Lange, Allison; McLane, Laura M.; Mills, Ryan E.; Devine, Scott E.; Corbett, Anita H.

2010-01-01

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best-characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin-α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9-12 amino acid linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 amino acid linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 amino acid linker. Our studies reveal that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition PMID:20028483

Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

PubMed

Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

2017-11-15

BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels.

PubMed

Tuluc, Petronel; Benedetti, Bruno; Coste de Bagneaux, Pierre; Grabner, Manfred; Flucher, Bernhard E

2016-06-01

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3-S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3-S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3-S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3-S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3-S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively. © 2016 Tuluc et al.

Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels

PubMed Central

Tuluc, Petronel; Benedetti, Bruno; Coste de Bagneaux, Pierre; Grabner, Manfred

2016-01-01

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively. PMID:27185857

Rational assembly of nanoparticle superlattices with designed lattice symmetries

DOEpatents

Gang, Oleg; Lu, Fang; Tagawa, Miho

2017-09-05

A method for lattice design via multivalent linkers (LDML) is disclosed that introduces a rationally designed symmetry of connections between particles in order to achieve control over the morphology of their assembly. The method affords the inclusion of different programmable interactions within one linker that allow an assembly of different types of particles. The designed symmetry of connections is preferably provided utilizing DNA encoding. The linkers may include fabricated "patchy" particles, DNA scaffold constructs and Y-shaped DNA linkers, anisotropic particles, which are preferably functionalized with DNA, multimeric protein-DNA complexes, and particles with finite numbers of DNA linkers.

Consequences of cavity size and chemical environment on the adsorption properties of isoreticular metal-organic frameworks: an inverse gas chromatography study.

PubMed

Gutiérrez, Inés; Díaz, Eva; Vega, Aurelio; Ordóñez, Salvador

2013-01-25

The role of the structure of three isoreticular metal-organic frameworks (IRMOFs) on their adsorption behavior has been studied in this work, selecting different kinds of volatile organic compounds (VOCs) as adsorbates (alkanes, alkenes, cycloalkanes, aromatics and chlorinated). For this purpose, three samples (IRMOF-1, IRMOF-8 and IRMOF-10) with cubic structure and without functionalities on the organic linkers were synthesized. Adsorption capacities at infinite dilution were derived from the adsorption isotherms, whereas thermodynamic properties have been determined from chromatographic retention volume. The capacity and the strength of adsorption were strongly influenced by the adsorbate size. This effect is especially relevant for n-alkanes adsorption, indicating the key role of the cavity size on this phenomenon, and hence the importance of the IRMOF structural properties. A different behavior has been observed for the polar compounds, where an enhancement on the specificity of the adsorption with the π-electron rich regions was observed. This fact suggests the specific interaction of these molecules with the organic linkers of the IRMOFs. Copyright © 2012 Elsevier B.V. All rights reserved.

Mechanistic Studies of ε-Caprolactone Polymerization by (salen)AlOR Complexes and a Predictive Model for Cyclic Ester Polymerizations

PubMed Central

2016-01-01

Aluminum alkoxide complexes (2) of salen ligands with a three-carbon linker and para substituents having variable electron-withdrawing capabilities (X = NO2, Br, OMe) were prepared, and the kinetics of their ring-opening polymerization (ROP) of ε-caprolactone (CL) were investigated as a function of temperature, with the aim of drawing comparisons to similar systems with two-carbon linkers investigated previously (1). While 1 and 2 exhibit saturation kinetics and similar dependences of their ROP rates on substituents X (invariant Keq, similar Hammett ρ = +1.4(1) and 1.2(1) for k2, respectively), ROP by 2 was significantly faster than for 1. Theoretical calculations confirm that, while the reactant structures differ, the transition state geometries are quite similar, and by analyzing the energetics of the involved distortions accompanying the structural changes, a significant contribution to the basis for the rate differences was identified. Using this knowledge, a simplified computational method for evaluating ligand structural influences on cyclic ester ROP rates is proposed that may have utility for future catalyst design. PMID:26900488

Molecular mechanism of pharmacological activation of BK channels

PubMed Central

Gessner, Guido; Cui, Yong-Mei; Otani, Yuko; Ohwada, Tomohiko; Soom, Malle; Hoshi, Toshinori; Heinemann, Stefan H.

2012-01-01

Large-conductance voltage- and Ca2+-activated K+ (Slo1 BK) channels serve numerous cellular functions, and their dysregulation is implicated in various diseases. Drugs activating BK channels therefore bear substantial therapeutic potential, but their deployment has been hindered in part because the mode of action remains obscure. Here we provide mechanistic insight into how the dehydroabietic acid derivative Cym04 activates BK channels. As a representative of NS1619-like BK openers, Cym04 reversibly left-shifts the half-activation voltage of Slo1 BK channels. Using an established allosteric BK gating model, the Cym04 effect can be simulated by a shift of the voltage sensor and the ion conduction gate equilibria toward the activated and open state, respectively. BK activation by Cym04 occurs in a splice variant-specific manner; it does not occur in such Slo1 BK channels using an alternative neuronal exon 9, which codes for the linker connecting the transmembrane segment S6 and the cytosolic RCK1 domain—the S6/RCK linker. In addition, Cym04 does not affect Slo1 BK channels with a two-residue deletion within this linker. Mutagenesis and model-based gating analysis revealed that BK openers, such as Cym04 and NS1619 but not mallotoxin, activate BK channels by functionally interacting with the S6/RCK linker, mimicking site-specific shortening of this purported passive spring, which transmits force from the cytosolic gating ring structure to open the channel's gate. PMID:22331907

Non-T cell activation linker (NTAL) proteolytic cleavage as a terminator of activatory intracellular signals.

PubMed

Arbulo-Echevarria, Mikel M; Muñoz-Miranda, Juan Pedro; Caballero-García, Andrés; Poveda-Díaz, José L; Fernández-Ponce, Cecilia; Durán-Ruiz, M Carmen; Miazek, Arkadiusz; García-Cózar, Francisco; Aguado, Enrique

2016-08-01

Non-T cell activation linker is an adaptor protein that is tyrosine phosphorylated upon cross-linking of immune receptors expressed on B lymphocytes, NK cells, macrophages, basophils, or mast cells, allowing the recruitment of cytosolic mediators for downstream signaling pathways. Fas receptor acts mainly as a death receptor, and when cross-linked with Fas ligand, many proteins are proteolytically cleaved, including several signaling molecules in T and B cells. Fas receptor triggering also interferes with TCR intracellular signals, probably by means of proteolytic cleavage of several adaptor proteins. We have previously found that the adaptor linker for activation of T cells, evolutionarily related to non-T cell activation linker, is cleaved upon proapoptotic stimuli in T lymphocytes and thymocytes, in a tyrosine phosphorylation-dependent fashion. Here, we describe non-T cell activation linker proteolytic cleavage triggered in human B cells and monocytes by Fas cross-linking and staurosporine treatment. Non-T cell activation linker is cleaved, producing an N-terminal fragment of ∼22 kDa, and such cleavage is abrogated in the presence of caspase 8/granzyme B and caspase 3 inhibitors. Moreover, we have identified an aspartic acid residue at which non-T cell activation linker is cleaved, which similar to linker for activation of T cells, this aspartic acid residue is located close to tyrosine and serine residues, suggesting an interdependence of phosphorylation and proteolytic cleavage. Consistently, induction of non-T cell activation linker phosphorylation by pervanadate inhibits its cleavage. Interestingly, the truncated isoform of non-T cell activation linker, generated after cleavage, has a decreased signaling ability when compared with the full-length molecule. Altogether, our results suggest that cleavage of transmembrane adaptors constitutes a general mechanism for signal termination of immune receptors. © Society for Leukocyte Biology.

A dimethyl ketal-protected benzoin-based linker suitable for photolytic release of unprotected peptides.

PubMed

Chumachenko, Nataliya; Novikov, Yehor; Shoemaker, Richard K; Copley, Shelley D

2011-11-18

Photolabile 3',5'-dimethoxybenzoin-based linkers are advantageous for a variety of solid-phase synthetic procedures and manipulations of biomolecules because UV irradiation in aqueous media provides fast and essentially quantitative release of tethered molecules, while generating unreactive side products. Practical applications of previously reported linkers are compromised to some extent by the 1,3-dithiane protection of the benzoin carbonyl group and the lengthy synthesis. We have extended the group of photocleavable 3',5'-dimethoxybenzoin-based linkers by designing and synthesizing a linker in which the carbonyl group is protected as a dimethyl ketal. This protection is compatible with commonly used esterification and amide bond formation techniques, including the Fmoc/tBu strategy for solid phase peptide synthesis, is stable under mild acidic conditions, and can be quantitatively removed in <5 min by 3% TFA in dichloromethane. Irradiation of beads carrying peptides attached to the linker at 350 nm in aqueous or partially aqueous media affords >90% release after 30 min. The linker was synthesized from commercially available starting materials in five steps with an overall yield of 40% and without any column chromatography purification. Additionally, we developed a route to a dithiane-protected linker that requires only two steps and proceeds in 65% yield, a significant improvement over previous synthetic routes.

The linker connecting the two kringles plays a key role in prothrombin activation

PubMed Central

Pozzi, Nicola; Chen, Zhiwei; Pelc, Leslie A.; Shropshire, Daniel B.; Di Cera, Enrico

2014-01-01

The zymogen prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction that is accelerated >3,000-fold by cofactor Va. This physiologically important effect is paradigmatic of analogous cofactor-dependent reactions in the coagulation and complement cascades, but its structural determinants remain poorly understood. Prothrombin has three linkers connecting the N-terminal Gla domain to kringle-1 (Lnk1), the two kringles (Lnk2), and kringle-2 to the C-terminal protease domain (Lnk3). Recent developments indicate that the linkers, and particularly Lnk2, confer on the zymogen significant flexibility in solution and enable prothrombin to sample alternative conformations. The role of this flexibility in the context of prothrombin activation was tested with several deletions. Removal of Lnk2 in almost its entirety (ProTΔ146–167) drastically reduces the enhancement of thrombin generation by cofactor Va from >3,000-fold to 60-fold because of a significant increase in the rate of activation in the absence of cofactor. Deletion of Lnk2 mimics the action of cofactor Va and offers insights into how prothrombin is activated at the molecular level. The crystal structure of ProTΔ146–167 reveals a contorted architecture where the domains are not vertically stacked, kringle-1 comes within 9 Å of the protease domain, and the Gla-domain primed for membrane binding comes in contact with kringle-2. These findings broaden our molecular understanding of a key reaction of the blood coagulation cascade where cofactor Va enhances activation of prothrombin by factor Xa by compressing Lnk2 and morphing prothrombin into a conformation similar to the structure of ProTΔ146–167. PMID:24821807

Role of a Modulator in the Synthesis of Phase-Pure NU-1000.

PubMed

Webber, Thomas E; Liu, Wei-Guang; Desai, Sai Puneet; Lu, Connie C; Truhlar, Donald G; Penn, R Lee

2017-11-15

NU-1000 is a robust, mesoporous metal-organic framework (MOF) with hexazirconium nodes ([Zr 6 O 16 H 16 ] 8+ , referred to as oxo-Zr 6 nodes) that can be synthesized by combining a solution of ZrOCl 2 ·8H 2 O and a benzoic acid modulator in N,N-dimethylformamide with a solution of linker (1,3,6,8-tetrakis(p-benzoic acid)pyrene, referred to as H 4 TBAPy) and by aging at an elevated temperature. Typically, the resulting crystals are primarily composed of NU-1000 domains that crystallize with a more dense phase that shares structural similarity with NU-901, which is an MOF composed of the same linker molecules and nodes. Density differences between the two polymorphs arise from the differences in the node orientation: in NU-1000, the oxo-Zr 6 nodes rotate 120° from node to node, whereas in NU-901, all nodes are aligned in parallel. Considering this structural difference leads to the hypothesis that changing the modulator from benzoic acid to a larger and more rigid biphenyl-4-carboxylic acid might lead to a stronger steric interaction between the modulator coordinating on the oxo-Zr 6 node and misaligned nodes or linkers in the large pore and inhibit the growth of the more dense NU-901-like material, resulting in phase-pure NU-1000. Side-by-side reactions comparing the products of synthesis using benzoic acid or biphenyl-4-carboxylic acid as a modulator produce structurally heterogeneous crystals and phase-pure NU-1000 crystals. It can be concluded that the larger and more rigid biphenyl-4-carboxylate inhibits the incorporation of nodes with an alignment parallel to the neighboring nodes already residing in the crystal.

On the role, ecology, phylogeny, and structure of dual-family immunophilins.

PubMed

Barik, Sailen

2017-11-01

The novel class of dual-family immunophilins (henceforth abbreviated as DFI) represents naturally occurring chimera of classical FK506-binding protein (FKBP) and cyclophilin (CYN), connected by a flexible linker that may include a three-unit tetratricopeptide (TPR) repeat. Here, I report a comprehensive analysis of all current DFI sequences and their host organisms. DFIs are of two kinds: CFBP (cyclosporin- and FK506-binding protein) and FCBP (FK506- and cyclosporin-binding protein), found in eukaryotes. The CFBP type occurs in select bacteria that are mostly extremophiles, such as psychrophilic, thermophilic, halophilic, and sulfur-reducing. Essentially all DFI organisms are unicellular. I suggest that DFIs are specialized bifunctional chaperones that use their flexible interdomain linker to associate with large polypeptides or multisubunit megacomplexes to promote simultaneous folding or renaturation of two clients in proximity, essential in stressful and denaturing environments. Analysis of sequence homology and predicted 3D structures of the FKBP and CYN domains as well as the TPR linkers upheld the modular nature of the DFIs and revealed the uniqueness of their TPR domain. The CFBP and FCBP genes appear to have evolved in parallel pathways with no obvious single common ancestor. The occurrence of both types of DFI in multiple unrelated phylogenetic clades supported their selection in metabolic and environmental niche roles rather than a traditional taxonomic relationship. Nonetheless, organisms with these rare immunophilins may define an operational taxonomic unit (OTU) bound by the commonality of chaperone function.

Lanthanide-based coordination polymers assembled by a flexible multidentate linker: design, structure, photophysical properties, and dynamic solid-state behavior.

PubMed

Marchal, Claire; Filinchuk, Yaroslav; Chen, Xiao-Yan; Imbert, Daniel; Mazzanti, Marinella

2009-01-01

Four picolinate building blocks were implemented into the multidentate linker N,N',N'-tetrakis[(6-carboxypyridin-2-yl)methyl]butylenediamine (H(4)tpabn) with a linear flexible spacer to promote the assembly of lanthanide-based 1D coordination polymers. The role of the linker in directing the geometry of the final assembly is evidenced by the different results obtained in the presence of Htpabn(3-) and tpabn(4-) ions. The tpabn(4-) ion leads to the desired 1D polymer {[Nd(tpabn)]H(3)O x 6 H(2)O}(infinity) (12). The Htpabn(3-) ion leads to the assembly of Tb(III) and Er(III) ions into 1D zigzag chains of the general formula {[M(Htpabn)] x xH(2)O}(infinity) (M = Tb, x = 14 (1); M = Tb, x = 8 (11); M = Er, x = 14 (2); M = Er, x = 5.5 (4)), a 2D network is formed by the Eu(III) ion (i.e., {[Eu(Htpabn)] x 10 H(2)O}(infinity) (7)), and both supramolecular isomers (1D and 2D) are obtained by the Tb(III) ion. The high flexibility of the polymeric chains results in a dynamic behavior with a solvent-induced reversible structural transition. The Tb(III)- and Eu(III)-containing polymers display high-luminescence quantum yields (38 and 18%, respectively). A sizeable near-IR luminescence emission is observed for the Er(III)- and Nd(III)-containing polymers when lattice water molecules are removed.

Gel-forming reagents and uses thereof for preparing microarrays

DOEpatents

Golova, Julia; Chernov, Boris; Perov, Alexander

2010-11-09

New gel-forming reagents including monomers and cross-linkers, which can be applied to gel-drop microarray manufacturing by using co-polymerization approaches are disclosed. Compositions for the preparation of co-polymerization mixtures with new gel-forming monomers and cross-linker reagents are described herein. New co-polymerization compositions and cross-linkers with variable length linker groups between unsaturated C.dbd.C bonds that participate in the formation of gel networks are disclosed.

Improved sensitivity of a graphene FET biosensor using porphyrin linkers

NASA Astrophysics Data System (ADS)

Kawata, Takuya; Ono, Takao; Kanai, Yasushi; Ohno, Yasuhide; Maehashi, Kenzo; Inoue, Koichi; Matsumoto, Kazuhiko

2018-06-01

Graphene FET (G-FET) biosensors have considerable potential due to the superior characteristics of graphene. Realizing this potential requires judicious choice of the linker molecule connecting the target-specific receptor molecule to the graphene surface, yet there are few reports comparing linker molecules for G-FET biosensors. In this study, tetrakis(4-carboxyphenyl)porphyrin (TCPP) was used as a linker for surface modification of a G-FET and the properties of the device were compared to those of a G-FET device modified with the conventional linker 1-pyrenebutanoic acid succinimidyl ester (PBASE). TCPP modification resulted in a higher density of receptor immunoglobulin E (IgE) aptamer molecules on the G-FET. The detection limit of the target IgE was enhanced from 13 nM for the PBASE-modified G-FET to 2.2 nM for the TCPP-modified G-FET, suggesting that the TCPP linker is a powerful candidate for G-FET modification.

Use of designed sequences in protein structure recognition.

PubMed

Kumar, Gayatri; Mudgal, Richa; Srinivasan, Narayanaswamy; Sandhya, Sankaran

2018-05-09

Knowledge of the protein structure is a pre-requisite for improved understanding of molecular function. The gap in the sequence-structure space has increased in the post-genomic era. Grouping related protein sequences into families can aid in narrowing the gap. In the Pfam database, structure description is provided for part or full-length proteins of 7726 families. For the remaining 52% of the families, information on 3-D structure is not yet available. We use the computationally designed sequences that are intermediately related to two protein domain families, which are already known to share the same fold. These strategically designed sequences enable detection of distant relationships and here, we have employed them for the purpose of structure recognition of protein families of yet unknown structure. We first measured the success rate of our approach using a dataset of protein families of known fold and achieved a success rate of 88%. Next, for 1392 families of yet unknown structure, we made structural assignments for part/full length of the proteins. Fold association for 423 domains of unknown function (DUFs) are provided as a step towards functional annotation. The results indicate that knowledge-based filling of gaps in protein sequence space is a lucrative approach for structure recognition. Such sequences assist in traversal through protein sequence space and effectively function as 'linkers', where natural linkers between distant proteins are unavailable. This article was reviewed by Oliviero Carugo, Christine Orengo and Srikrishna Subramanian.

Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region.

PubMed

Costa, M G S; Silva, Y F; Batista, P R

2018-03-14

Microbial cellulosic degradation by cellulases has become a complementary approach for biofuel production. However, its efficiency is hindered by the recalcitrance of cellulose fibres. In this context, computational protein design methods may offer an efficient way to obtain variants with improved enzymatic activity. Cel9A-68 is a cellulase from Thermobifida fusca that is still active at high temperatures. In a previous work, we described a collective bending motion, which governs the overall cellulase dynamics. This movement promotes the approximation of its CBM and CD structural domains (that are connected by a flexible linker). We have identified two residues (G460 and P461) located at the linker that act as a hinge point. Herein, we applied a new level of protein design, focusing on the modulation of this collective motion to obtain cellulase variants with enhanced functional dynamics. We probed whether specific linker mutations would affect Cel9A-68 dynamics through computational simulations. We assumed that P461G and G460+ (with an extra glycine) constructs would present enhanced interdomain motions, while the G460P mutant would be rigid. From our results, the P461G mutation resulted in a broader exploration of the conformational space, as confirmed by clustering and free energy analyses. The WT enzyme was the most rigid system. However, G460P and P460+ explored distinct conformational states described by opposite directions of low-frequency normal modes; they sampled preferentially closed and open conformations, respectively. Overall, we highlight two significant findings: (i) all mutants explored larger conformational spaces than the WT; (ii) the selection of distinct conformational populations was intimately associated with the mutation considered. Thus, the engineering of Cel9A-68 motions through linker mutations may constitute an efficient way to improve cellulase activity, facilitating the disruption of cellulose fibres.

Eye patches: Protein assembly of index-gradient squid lenses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, J.; Townsend, J. P.; Dodson, T. C.

A parabolic relationship between lens radius and refractive index allows spherical lenses to avoid spherical aberration. We show that in squid, patchy colloidal physics resulted from an evolutionary radiation of globular S-crystallin proteins. Small-angle x-ray scattering experiments on lens tissue show colloidal gels of S-crystallins at all radial positions. Sparse lens materials form via low-valence linkages between disordered loops protruding from the protein surface. The loops are polydisperse and bind via a set of hydrogen bonds between disordered side chains. Peripheral lens regions with low particle valence form stable, volume-spanning gels at low density, whereas central regions with higher averagemore » valence gel at higher densities. The proteins demonstrate an evolved set of linkers for self-assembly of nanoparticles into volumetric materials.« less

Lecithin-linker formulations for self-emulsifying delivery of nutraceuticals.

PubMed

Chu, Jacquelene; Cheng, Yu-Ling; Rao, A Venketeshwer; Nouraei, Mehdi; Zarate-Muñoz, Silvia; Acosta, Edgar J

2014-08-25

Lecithin-linker microemulsions are formulations produced with soybean lecithin in combination with a highly lipophilic (lipophilic linker) and highly hydrophilic (hydrophilic linkers) surfactant-like additives. In this work, lecithin-linker systems were formulated to produce self-emulsifying delivery systems for β-carotene and β-sitosterol. The concentration of the lipophilic linker, sorbitan monooleate, was adjusted to minimize the formation of liquid crystals. The concentration of hydrophilic linkers, decaglyceryl caprylate/caprate and PEG-6-caprylic/capric glycerides, was gradually increased (scanned) until single phase clear microemulsions were obtained. For these scans, the oil (ethyl caprate) to water ratio was set to 1. The single phase, clear microemulsions were diluted with fed-state simulated intestinal fluid (FeSSIF) and produced stable emulsions, with drop sizes close to 200 nm. Using pseudo-ternary phase diagrams to evaluate the process of dilution of microemulsion preconcentrates (mixtures of oil, lecithin and linkers with little or no water) with FeSSIF, it was determined that self-emulsifying systems are obtained when the early stages of the dilution produce single phase microemulsions. If liquid crystals or multiple phase systems are obtained during those early stages, then the emulsification yields unstable emulsions with large drop sizes. An in vitro permeability study conducted using a Flow-Thru Dialyzer revealed that stable emulsions with drop sizes of 150-300 nm produce large and irreversible permeation of β-carotene to sheep intestine. On the other hand, unstable emulsions produced without the linker combination separated in the dialyzer chamber. Copyright © 2014 Elsevier B.V. All rights reserved.

Rational design of molecularly imprinted polymer: the choice of cross-linker.

PubMed

Muhammad, Turghun; Nur, Zohre; Piletska, Elena V; Yimit, Osmanjan; Piletsky, Sergey A

2012-06-07

The paper describes a rational approach for the selection of cross-linkers during the development of molecularly imprinted polymers (MIPs). As a model system for this research MIPs specific for the drug zidovudine (AZT) were designed and tested. Three cross-linkers trimethylolpropane trimethacrylate (TRIM), ethylene glycol dimethacrylate (EGDMA) and divinylbenzene (DVB) were studied. The analogue of zidovudine (AZT) ester (AZT-ES) was used as a dummy template. The imprinting factors for all of the polymers in the static adsorption experiments were calculated. The data on the AZT adsorption by control polymers (CP), which were prepared with different cross-linkers without a functional monomer, was also analyzed. DVB was found to be more inert towards zidovudine than EGDMA and TRIM, which was confirmed by both molecular modelling and adsorption experiments. It was demonstrated that DVB-based polymers had a higher imprinting factor (I = 1.85) compared with other tested cross-linked polymers. It was suggested that the selection of the cross-linker should be based on the strength of the interaction with the template: the cross-linker which displays lower binding of the template should be preferential because it generates MIPs with lower non-specific binding and a higher imprinting factor, and therefore specificity. Which cross-linker to use for the preparation of any particular MIP can be determined by analysis of the interactions between the cross-linker and template. This could be done either virtually using computational modelling or by template adsorption using a small library of polymers prepared using different cross-linkers.

Crystal engineering of versatile Hg(II) halides complexes of N,N,N‧,N‧-tetraalkyl substituted 3,5 pyridinedicarboxamides

NASA Astrophysics Data System (ADS)

Rana, Love Karan; Sharma, Sanyog; Hundal, Geeta

2018-02-01

Two new ligands N,N,N‧,N‧-tetraisopropyl/butyl-3,5-pyridinedicarboxamide (L3-L4) and six of their Hg(II)X2 complexes (where X = Cl-, Br- and I-), have been synthesized and characterized using single crystal X-ray diffraction and spectroscopic techniques. Complexes of L3 (1-3) with HgCl2/Br2/I2, have dimeric structure, with the ligand behaving as a 2-C linker. Complexes 4-6 are 1D coordination polymers with either 3- or 2-C, L4 linker and bridging halides. A delicate balance of anion, solvent, denticity and conformation of the ligands on the ensuing molecular and crystal structures has been delineated. Various non-covalent interactions, extending the dimensionality of the complexes are calculated, analyzed and discussed. A significant role of semi-localized LP···π non-covalent interactions in stabilizing the basic dimeric unit in the complexes, has been discerned.

Phase separation and the formation of the pyrenoid, a carbon-fixing organelle

NASA Astrophysics Data System (ADS)

Xu, Bin; Freeman Rosenzweig, Elizabeth; Mackinder, Luke; Jonikas, Martin; Wingreen, Ned S.

In the chloroplasts of most algae, the carbon-fixing enzyme Rubisco is concentrated in a non-membrane-bound structure called the pyrenoid, which enables more efficient carbon capture than that of most land plants. In contrast to the long-held assumptions of the field, the pyrenoid matrix is not a solid crystal, but behaves as a phase-separated, liquid-like organelle. In this system, the linker protein EPYC1 is thought to form multivalent specific bonds with Rubisco, and the formation of the pyrenoid occurs via the phase separation of these two associating proteins. Through analytical and numerical studies, we determine a phase diagram for this system. We also show how the length of the linker protein can affect the formation and dissolution of the pyrenoid in an unexpected manner. This new view of the pyrenoid matrix provides important insights into the structure, regulation, and inheritance of pyrenoid. More broadly, our findings give insights into fundamental principles of the architecture and inheritance of liquid-phase organelles.

Structural Basis of J Cochaperone Binding and Regulation of Hsp70

PubMed Central

Jiang, Jianwen; Maes, E. Guy; Taylor, Alex B; Wang, Liping; Hinck, Andrew P; Lafer, Eileen M; Sousa, Rui

2007-01-01

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) towards a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, while both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles. PMID:17996706

Role of H1 Linker Histones in Mammalian Development and Stem Cell Differentiation

PubMed Central

Pan, Chenyi; Fan, Yuhong

2016-01-01

H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome. PMID:26689747

Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation

NASA Astrophysics Data System (ADS)

Cheng, Jingdong; Yang, Huirong; Fang, Jian; Ma, Lixiang; Gong, Rui; Wang, Ping; Li, Ze; Xu, Yanhui

2015-05-01

DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 Å resolution. The interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1-USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1-USP7 interaction surface for therapeutic applications.

Macrocyclic bifunctional chelating agents

DOEpatents

Meares, Claude F.; DeNardo, Sally J.; Cole, William C.; Mol, Min K.

1987-01-01

A copper chelate conjugate which is stable in human serum. The conjugate includes the copper chelate of a cyclic tetraaza di-, tri-, or tetra-acetic acid, a linker attached at one linker end to a ring carbon of the chelate, and a biomolecule joined at the other end of the linker. The conjugate, or the linker-copper chelate compound used in forming the conjugate, are designed for use in diagnostic and therapeutic applications which involve Cu(II) localization via the systemic route.

Pharmaceutical differences between block copolymer self-assembled and cross-linked nanoassemblies as carriers for tunable drug release.

PubMed

Lee, Hyun Jin; Bae, Younsoo

2013-02-01

To identify the effects of cross-linkers and drug-binding linkers on physicochemical and biological properties of polymer nanoassembly drug carriers. Four types of polymer nanoassemblies were synthesized from poly(ethylene glycol)-poly(aspartate) [PEG-p(Asp)] block copolymers: self-assembled nanoassemblies (SNAs) and cross-linked nanoassemblies (CNAs) to each of which an anticancer drug doxorubicin (DOX) was loaded by either physical entrapment or chemical conjugation (through acid-sensitive hydrazone linkers). Drug loading in nanoassemblies was 27 ~ 56% by weight. The particle size of SNA changed after drug and drug-binding linker entrapment (20 ~ 100 nm), whereas CNAs remained 30 ~ 40 nm. Drug release rates were fine-tunable by using amide cross-linkers and hydrazone drug-binding linkers in combination. In vitro cytotoxicity assays using a human lung cancer A549 cell line revealed that DOX-loaded nanoassemblies were equally potent as free DOX with a wide range of drug release half-life (t(1/2) = 3.24 ~ 18.48 h, at pH 5.0), but 5 times less effective when t(1/2) = 44.52 h. Nanoassemblies that incorporate cross-linkers and drug-binding linkers in combination have pharmaceutical advantages such as uniform particle size, physicochemical stability, fine-tunable drug release rates, and maximum cytotoxicity of entrapped drug payloads.

Comparative Study of Ultrasonication-Induced and Naturally Self-Assembled Silk Fibroin-Wool Keratin Hydrogel Biomaterials

PubMed Central

Vu, Trang; Xue, Ye; Vuong, Trinh; Erbe, Matthew; Bennet, Christopher; Palazzo, Ben; Popielski, Lucas; Rodriguez, Nelson; Hu, Xiao

2016-01-01

This study reports the formation of biocompatible hydrogels using protein polymers from natural silk cocoon fibroins and sheep wool keratins. Silk fibroin protein contains β-sheet secondary structures, allowing for the formation of physical cross-linkers in the hydrogels. Comparative studies were performed on two groups of samples. In the first group, ultrasonication was used to induce a quick gelation of a protein aqueous solution, enhancing the ability of Bombyx mori silk fibroin chains to quickly entrap the wool keratin protein molecules homogenously. In the second group, silk/keratin mixtures were left at room temperature for days, resulting in naturally-assembled gelled solutions. It was found that silk/wool blended solutions can form hydrogels at different mixing ratios, with perfectly interconnected gel structure when the wool content was less than 30 weight percent (wt %) for the first group (ultrasonication), and 10 wt % for the second group (natural gel). Differential scanning calorimetry (DSC) and temperature modulated DSC (TMDSC) were used to confirm that the fibroin/keratin hydrogel system was well-blended without phase separation. Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structures of blended protein gels. It was found that intermolecular β-sheet contents significantly increase as the system contains more silk for both groups of samples, resulting in stable crystalline cross-linkers in the blended hydrogel structures. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to analyze the samples’ characteristic morphology on both micro- and nanoscales, which showed that ultrasonic waves can significantly enhance the cross-linker formation and avoid phase separation between silk and keratin molecules in the blended systems. With the ability to form cross-linkages non-chemically, these silk/wool hydrogels may be economically useful for various biomedical applications, thanks to the good biocompatibility of protein molecules and the various characteristics of hydrogel systems. PMID:27618011

Modular architecture of protein binding units for designing properties of cellulose nanomaterials.

PubMed

Malho, Jani-Markus; Arola, Suvi; Laaksonen, Päivi; Szilvay, Géza R; Ikkala, Olli; Linder, Markus B

2015-10-05

Molecular biomimetic models suggest that proteins in the soft matrix of nanocomposites have a multimodular architecture. Engineered proteins were used together with nanofibrillated cellulose (NFC) to show how this type of architecture leads to function. The proteins consist of two cellulose-binding modules (CBM) separated by 12-, 24-, or 48-mer linkers. Engineering the linkers has a considerable effects on the interaction between protein and NFC in both wet colloidal state and a dry film. The protein optionally incorporates a multimerizing hydrophobin (HFB) domain connected by another linker. The modular structure explains effects in the hydrated gel state, as well as the deformation of composite materials through stress distribution and crosslinking. Based on this work, strategies can be suggested for tuning the mechanical properties of materials through the coupling of protein modules and their interlinking architectures. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Photoluminescence spectroscopy of YVO4:Eu3+ nanoparticles with aromatic linker molecules: A precursor to biomedical functionalization

NASA Astrophysics Data System (ADS)

Senty, T. R.; Yalamanchi, M.; Zhang, Y.; Cushing, S. K.; Seehra, M. S.; Shi, X.; Bristow, A. D.

2014-04-01

Photoluminescence spectra of YVO4:Eu3+ nanoparticles are presented, with and without the attachment of organic molecules that are proposed for linking to biomolecules. YVO4:Eu3+ nanoparticles with 5% dopant concentration were synthesized via wet chemical synthesis. X-ray diffraction and transmission electron microscopy show the expected wakefieldite structure of tetragonal particles with an average size of 17 nm. Fourier-transform infrared spectroscopy determines that metal-carboxylate coordination is successful in replacing native metal-hydroxyl bonds with three organic linkers, namely, benzoic acid, 3-nitro 4-chloro-benzoic acid, and 3,4-dihydroxybenzoic acid, in separate treatments. UV-excitation photoluminescence spectra show that the position and intensity of the dominant 5D0 - 7F2 electric-dipole transition at 619 nm are unaffected by the benzoic acid and 3-nitro 4-chloro-benzoic acid treatments. Attachment of 3,4-dihydroxybenzoic acid produces an order-of-magnitude quenching in the photoluminescence, due to the presence of high-frequency vibrational modes in the linker. Ratios of the dominant electric- and magnetic-dipole transitions confirm infrared measurements, which indicate that the bulk crystal of the nanoparticle is unchanged by all three treatments.

Tunable gas adsorption in graphene oxide framework

NASA Astrophysics Data System (ADS)

Razmkhah, Mohammad; Moosavi, Fatemeh; Taghi Hamed Mosavian, Mohammad; Ahmadpour, Ali

2018-06-01

Effect of length of linker inter-space was studied on the adsorption capacity of CO2 by graphene oxide framework (GOF). Effect of linker inter-space of 14, 11, and 8 Å was studied here. The linker inter-space of 11 Å showed the highest CO2 adsorption capacity. A dual-site Langmuir model was observed for adsorption of CO2 and CH4 into the GOF. According to radial distribution function (RDF), facial and central atoms of linker are the dual-site predicted by Langmuir model. Two distinguishable sites of adsorption and parallel orientation of CO2 are the main reasons of high adsorption capacity in 11 Å linker inter-space. Gas-adsorbent affinity obtains the orientation of CO2 near the linker. The affinity in the 11 Å linker inter-space is the highest. Thus, it forces the CO2 to lay parallel and orient more localized than the other GOFs. In addition, CH4 resulted higher working capacity than CO2 in 14 Å. This occurs because of the change in gas-adsorbent affinity by changing pressure. An entrance adsorption occurs out of the pore of the GOF. This adsorption is not as stable as deep adsorption.

Mechanically tunable actin networks using programmable DNA based cross-linkers

NASA Astrophysics Data System (ADS)

Schnauss, Joerg; Lorenz, Jessica; Schuldt, Carsten; Kaes, Josef; Smith, David

Cells employ multiple cross-linkers with very different properties. Studies of the entire phase space, however, were infeasible since they were restricted to naturally occurring cross-linkers. These components cannot be controllably varied and differ in many parameters. We resolve this limitation by forming artificial actin cross-linkers, which can be controllably varied. The basic building block is DNA enabling a well-defined length variation. DNA can be attached to actin binding peptides with known binding affinities. We used bulk rheology to investigate mechanical properties of these networks. We were able to reproduce mechanical features of actin networks cross-linked by fascin by using a short version of our artificial complex with a high binding affinity. Additionally, we were able to resemble findings for the cross-linker alpha-actinin by employing a long cross-linker with a low binding affinity. Between these natural limits we investigated three different cross-linker lengths each with two different binding affinities. With these controlled variations we are able to precisely screen the phase space of cross-linked actin networks by changing only one specific parameter and not the entire set of properties as in the case of naturally occurring cross-linking complexes.

Effect of linkers on the αvβ3 integrin targeting efficiency of cyclic RGD-conjugates

NASA Astrophysics Data System (ADS)

Karmakar, Partha; Grabowska, Dorota; Sudlow, Gail; Ziabrev, Kostiantyn; Sanyal, Nibedita; Achilefu, Samuel

2018-02-01

Cyclic arginine-glycine-aspartic acid (cRGD) peptides are well known to target ανβ3 integrin expressed on cancer cells and neovasculature. Conjugation of these peptides with dyes, drugs, antibodies and other biomolecules through covalent linkers provides a facile way to deliver these products to tumor cells for targeted cancer therapy and diagnosis. Click chemistry and acid-amine couplings are widely used conjugation strategies. However, the effects of different linkers and the distance between the cRGD and the conjugates on the binding of cRGD ligand with ανβ3 has been underexplored. In this present study, we prepared cRGD-conjugates using different linkers and determined how they altered the tumor targeting efficiency in vitro and in vivo. The results demonstrate that different linkers significantly altered the pharmacokinetics of the cRGD conjugates and the tumor uptake kinetics. Unlike large antibodies, this preliminary finding shows that linkers used to attach drugs and fluorescent molecular probes to small peptides play a major role in the accuracy of tumor targeting and treatment outcomes. As a result, considerable attention should be paid to the nature of linkers used in the design of molecular probes and targeted therapeutics.

Crystal structure of zwitterionic bisimidazolium sulfonates

NASA Astrophysics Data System (ADS)

Kohmoto, Shigeo; Okuyama, Shinpei; Yokota, Nobuyuki; Takahashi, Masahiro; Kishikawa, Keiki; Masu, Hyuma; Azumaya, Isao

2012-05-01

Crystal structures of three zwitterionic bisimidazolium salts 1-3 in which imidazolium sulfonate moieties were connected with aromatic linkers, p-xylylene, 4,4'-dimethylenebiphenyl, and phenylene, respectively, were examined. The latter two were obtained as hydrates. An S-shaped molecular structure in which the sulfonate moiety was placed on the imidazolium ring was observed for 1. A helical array of hydrated water molecules was obtained for 2 while a linear array of hydrated water molecules was observed for 3.

Mixed-linker strategy for the construction of multifunctional metal–organic frameworks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Jun-Sheng; Yuan, Shuai; Wang, Qi

2017-01-01

Mixed-linker strategy is a promising way to construct multifunctional metal–organic frameworks (MOFs). In this review, we demonstrate the recent developments, discussions and challenges related to the preparation and applications of four types of mixed-linker MOF materials.

The unstructured linker arms of Mlh1-Pms1 are important for interactions with DNA during mismatch repair

PubMed Central

Plys, Aaron J.; Rogacheva, Maria V.; Greene, Eric C.; Alani, Eric

2012-01-01

DNA mismatch repair (MMR) models have proposed that MSH proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH proteins (primarily Mlh1-Pms1 in baker’s yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20 – 30 nanometers) unstructured arms that connect two terminal globular domains. These arms can vary between 100 to 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker’s yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR. PMID:22659005

Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger.

PubMed

Sauer, J; Christensen, T; Frandsen, T P; Mirgorodskaya, E; McGuire, K A; Driguez, H; Roepstorff, P; Sigurskjold, B W; Svensson, B

2001-08-07

Several variants of glucoamylase 1 (GA1) from Aspergillus niger were created in which the highly O-glycosylated peptide (aa 468--508) connecting the (alpha/alpha)(6)-barrel catalytic domain and the starch binding domain was substituted at the gene level by equivalent segments of glucoamylases from Hormoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae linker was elongated by proline-rich sequences as this linker itself apparently was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated polypeptide chain molecular masses for wild-type GA1, a nonnatural proline-rich linker variant, H. grisea, and R. oryzae linker variants were 65,784, 63,777, 63,912, and 65,614 Da, respectively, MALDI-TOF-MS gave values of 82,042, 73,800, 73,413, and 90,793 Da, respectively, where the latter value could partly be explained by an N-glycosylation site introduced near the linker C-terminus. The k(cat) and K(m) for hydrolysis of maltooligodextrins and soluble starch, and the rate of hydrolysis of barley starch granules were essentially the same for the variants as for wild-type GA1. beta-Cyclodextrin, acarbose, and two heterobidentate inhibitors were found by isothermal titration calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the starch binding site was maintained. The stability of GA1 linker variants toward GdnHCl and heat, however, was reduced compared to wild-type.

High-sensitivity analysis of naturally occurring sugar chains, using a novel fluorescent linker molecule.

PubMed

Sato, Masaki; Ito, Yuji; Arima, Naomichi; Baba, Masanori; Sobel, Michael; Wakao, Masahiro; Suda, Yasuo

2009-07-01

To analyse the binding of sugar chains to proteins, viruses and cells, the surface plasmon resonance (SPR) technique is very convenient and effective because it is a real-time, non-destructive detection system. Key to this method is linker compounds for immobilization of the sugar chains to the gold-coated chip for SPR. Also, well-designed fluorescent labelling reagents are essential when analysing the structure of trace amounts of sugar chains derived from natural sources, such as glycoproteins on the surface of specific cells. In this report, we developed a novel linker molecule, named 'f-mono', which has both of these properties: simple immobilization chemistry and a fluorescent label. Since the molecule contains a 2,5-diaminopyridyl group and a thioctic acid group, conjugation with sugar chains can be achieved using the well-established reductive amination reaction. This conjugate of sugar chain and fluorescent linker (fluorescent ligand-conjugate, FLC) has fluorescent properties (ex. 335 nm, em. 380 nm), and as little as 1 microg of FLC can be easily purified using HPLC with a fluorescent detector. MS and MS/MS analysis of the FLC is also possible. As a +2 Da larger MS peak ([M + H + 2](+) ion) was always associated with the theoretical MS peak ([M + H](+)) (due to the reduction of the thioctic acid moiety), the MS peaks of the FLC were easily found, even using unfractionated crude samples. Immobilization of the FLC onto gold-coated chips, and their subsequent SPR analyses were successively accomplished, as had been performed previously using non-fluorescent ligand conjugates.

The Role of H1 Linker Histone Subtypes in Preserving the Fidelity of Elaboration of Mesendodermal and Neuroectodermal Lineages during Embryonic Development

PubMed Central

Nguyen, Giang D.; Gokhan, Solen; Molero, Aldrin E.; Yang, Seung-Min; Kim, Byung-Ju; Skoultchi, Arthur I.; Mehler, Mark F.

2014-01-01

H1 linker histone proteins are essential for the structural and functional integrity of chromatin and for the fidelity of additional epigenetic modifications. Deletion of H1c, H1d and H1e in mice leads to embryonic lethality by mid-gestation with a broad spectrum of developmental alterations. To elucidate the cellular and molecular mechanisms underlying H1 linker histone developmental functions, we analyzed embryonic stem cells (ESCs) depleted of H1c, H1d and H1e subtypes (H1-KO ESCs) by utilizing established ESC differentiation paradigms. Our study revealed that although H1-KO ESCs continued to express core pluripotency genes and the embryonic stem cell markers, alkaline phosphatase and SSEA1, they exhibited enhanced cell death during embryoid body formation and during specification of mesendoderm and neuroectoderm. In addition, we demonstrated deregulation in the developmental programs of cardiomyocyte, hepatic and pancreatic lineage elaboration. Moreover, ectopic neurogenesis and cardiomyogenesis occurred during endoderm-derived pancreatic but not hepatic differentiation. Furthermore, neural differentiation paradigms revealed selective impairments in the specification and maturation of glutamatergic and dopaminergic neurons with accelerated maturation of glial lineages. These impairments were associated with deregulation in the expression profiles of pro-neural genes in dorsal and ventral forebrain-derived neural stem cell species. Taken together, these experimental observations suggest that H1 linker histone proteins are critical for the specification, maturation and fidelity of organ-specific cellular lineages derived from the three cardinal germ layers. PMID:24802750

Preparation of molecular imprinted polymers using bi-functional monomer and bi-crosslinker for solid-phase extraction of rutin.

PubMed

Zeng, Huan; Wang, Yuzhi; Liu, Xiaojie; Kong, Jinhuan; Nie, Chan

2012-05-15

Molecular imprinted polymers (MIPs) were prepared using rutin as the template, different reagents as the functional monomer and different reagents as the cross-linker by solution polymerization. Several parameters that would influence the performance of MIPs were investigated including the type of functional monomer (single or double) and cross-linker (single or double), and the molar ratio of the template, the functional monomer and the cross-linker. The optimum synthesis conditions of MIPs were found to be bi-monomers (acrylamide-co-2-vinyl pyridine, 3:1) and bi-crosslinker (ethylene glycol dimethacrylate-co-divinylbenzene, 3:1). The ratio of the template, the functional monomer and the cross-linker was found to be 1:6:20. MIPs synthesized under these conditions were filled into the cartridges as the adsorbents of solid-phase extraction (SPE). A competition test was conducted to authenticate the selectivity and the specificity of molecularly imprinted solid-phase extraction (MISPE) for rutin using the mixture solution of standard rutin and its structural analogs including quercetin, naringenin and kaempferol. Compared with purchased SPE including C(18), silica and PCX, MISPE showed better selectivity and enrichment property for rutin in the extracted solutions of Chinese medicinal plants than any others. The mean recoveries were 85.93% (RSD: 3.04%, n=3) for Saururus chinensis (Lour.) Bail and 88.61% (RSD: 3.36%, n=3) for Flos Sophorae, respectively, which indicated that the optimized rutin-MIPs possess the value of practical application. Copyright © 2012 Elsevier B.V. All rights reserved.

Formation mechanism of the secondary building unit in a chromium terephthalate metal-organic framework

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cantu Cantu, David; McGrail, B. Peter; Glezakou, Vassiliki Alexandra

2014-09-18

Based on density functional theory calculations and simulation, a detailed mechanism is presented on the formation of the secondary building unit (SBU) of MIL-101, a chromium terephthalate metal-organic framework (MOF). SBU formation is key to MOF nucleation, the rate-limiting step in the formation process of many MOFs. A series of reactions that lead to the formation of the SBU of MIL-101 is proposed in this work. Initial rate-limiting reactions form the metal cluster with three chromium (III) atoms linked to a central bridging oxygen. Terephthalate linkers play a key role as chromium (III) atoms are joined to linker carboxylate groupsmore » prior to the placement of the central bridging oxygen. Multiple linker addition reactions, which follow in different paths due to structural isomers, are limited by the removal of water molecules in the first chromium coordination shell. The least energy path is identified were all linkers on one face of the metal center plane are added first. A simple kinetic model based on transition state theory shows the rate of secondary building unit formation similar to the rate metal-organic framework nucleation. The authors are thankful to Dr. R. Rousseau for a critical reading of the manuscript. This research would not have been possible without the support of the Office of Fossil Energy, U.S. Department of Energy. This research was performed using EMSL, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and the PNNL Institutional Computing (PIC) program located at Pacific Northwest National Laboratory.« less

Linker-mediated assembly of gold nanoparticles into multimeric motifs

NASA Astrophysics Data System (ADS)

Sikora, Mateusz; Szymczak, Piotr; Thompson, Damien; Cieplak, Marek

2011-11-01

We present a theoretical description of linker-mediated self-assembly of gold nanoparticles (Au-NP). Using mesoscale simulations with a coarse-grained model for the Au NPs and dirhenium-based linker molecules, we investigate the conditions under which large clusters can grow and construct a phase diagram that identifies favorable growth conditions in terms of floating and bound linker concentrations. The findings can be considered as generic, as we expect other NP-linker systems to behave in a qualitatively similar way. In particular, we also discuss the case of antibody-functionalised Au NPs connected by the C-reactive proteins (CRPs). We extract some general rules for NP linking that may aid the production of size- and shape-specific NP clusters for technology applications.

The Use of Aryl Hydrazide Linkers for the Solid Phase Synthesis of Chemically Modified Peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woo, Y; Mitchell, A R; Camarero, J A

2006-11-03

Since Merrifield introduced the concept of solid phase synthesis in 1963 for the rapid preparation of peptides, a large variety of different supports and resin-linkers have been developed that improve the efficiency of peptide assembly and expand the myriad of synthetically feasible peptides. The aryl hydrazide is one of the most useful resin-linkers for the synthesis of chemically modified peptides. This linker is completely stable during Boc- and Fmoc-based solid phase synthesis and yet it can be cleaved under very mild oxidative conditions. The present article reviews the use of this valuable linker for the rapid and efficient synthesis ofmore » C-terminal modified peptides, head-to-tail cyclic peptides and lipidated peptides.« less

Cellobiohydrolase I enzymes

DOEpatents

Adney, William S; Himmel, Michael E; Decker, Stephen R; Knoshaug, Eric P; Nimlos, Mark R; Crowley, Michael F; Jeoh, Tina

2014-01-28

Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.

[Construction of cTnC-linker-TnI (P) Genes, Expression of Fusion Protein and Preparation of Lyophilized Protein].

PubMed

Song, Xiaoli; Liu, Xiaoyun; Cai, Lei; Wu, Jianwei; Wang, Jihua

2015-12-01

In order to construct and express human cardiac troponin C-linker-troponin I(P) [ cTnC-linker-TnI(P)] fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pCold I-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni²⁺ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo Finecare™ cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37 °C and 4 or more months at 25 °C and 4 °C. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.

4-N-Hydroxy-4-[1-(sulfonyl)piperidin-4-yl]-butyramides as HDAC inhibitors.

PubMed

Rossi, Cristina; Fincham, Christopher I; D'Andrea, Piero; Porcelloni, Marina; Ettorre, Alessandro; Mauro, Sandro; Bigioni, Mario; Binaschi, Monica; Maggi, Carlo A; Nardelli, Federica; Parlani, Massimo; Fattori, Daniela

2011-11-15

A series of N-substituted 4-alkylpiperidine hydroxamic acids, corresponding to the basic structure of histone deacetylase (HDAC) inhibitors (zinc binding moiety-linker-capping group) has been previously reported by our group. Linker length and aromatic capping group connection were systematically varied to find the optimal geometric parameters. A new series of submicromolar inhibitors was thus identified, which showed antiproliferative activity on HCT-116 colon carcinoma cells. We report here the second part of the strategy used in our research group to find a new class of HDAC inhibitors, namely the SAR study for the compounds bearing a sulfonyl group on the piperidine nitrogen. In the present work, we have considered both sulfonamides and sulfonyl ureas. Copyright © 2011 Elsevier Ltd. All rights reserved.

Structural analysis of poly-SUMO chain recognition by the RNF4-SIMs domain.

PubMed

Kung, Camy C-H; Naik, Mandar T; Wang, Szu-Huan; Shih, Hsiu-Ming; Chang, Che-Chang; Lin, Li-Ying; Chen, Chia-Lin; Ma, Che; Chang, Chi-Fon; Huang, Tai-Huang

2014-08-15

The E3 ubiquitin ligase RNF4 (RING finger protein 4) contains four tandem SIM [SUMO (small ubiquitin-like modifier)-interaction motif] repeats for selective interaction with poly-SUMO-modified proteins, which it targets for degradation. We employed a multi-faceted approach to characterize the structure of the RNF4-SIMs domain and the tetra-SUMO2 chain to elucidate the interaction between them. In solution, the SIM domain was intrinsically disordered and the linkers of the tetra-SUMO2 were highly flexible. Individual SIMs of the RNF4-SIMs domains bind to SUMO2 in the groove between the β2-strand and the α1-helix parallel to the β2-strand. SIM2 and SIM3 bound to SUMO with a high affinity and together constituted the recognition module necessary for SUMO binding. SIM4 alone bound to SUMO with low affinity; however, its contribution to tetra-SUMO2 binding avidity is comparable with that of SIM3 when in the RNF4-SIMs domain. The SAXS data of the tetra-SUMO2-RNF4-SIMs domain complex indicate that it exists as an ordered structure. The HADDOCK model showed that the tandem RNF4-SIMs domain bound antiparallel to the tetra-SUMO2 chain orientation and wrapped around the SUMO protamers in a superhelical turn without imposing steric hindrance on either molecule.

40 CFR 725.239 - Use of specific microorganisms in activities conducted outside a structure.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Bradyrhizobium japonicum. (2) Modification of traits. (i) The introduced genetic material must meet the criteria for poorly mobilizable listed in § 725.421(c). (ii) The introduced genetic material must consist only... sequences needed to move genetic material, including linkers, homopolymers, adaptors, transposons, insertion...

40 CFR 725.239 - Use of specific microorganisms in activities conducted outside a structure.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Bradyrhizobium japonicum. (2) Modification of traits. (i) The introduced genetic material must meet the criteria for poorly mobilizable listed in § 725.421(c). (ii) The introduced genetic material must consist only... sequences needed to move genetic material, including linkers, homopolymers, adaptors, transposons, insertion...

40 CFR 725.239 - Use of specific microorganisms in activities conducted outside a structure.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Bradyrhizobium japonicum. (2) Modification of traits. (i) The introduced genetic material must meet the criteria for poorly mobilizable listed in § 725.421(c). (ii) The introduced genetic material must consist only... sequences needed to move genetic material, including linkers, homopolymers, adaptors, transposons, insertion...

Different Interfacial Behaviors of Peptides Chemically Immobilized on Surfaces with Different Linker Lengths and via Different Termini

DTIC Science & Technology

2014-02-20

spectroscopy was applied to investigate such structures of peptides immobilized on self-assembled monolayers (SAMs). Here cysteine-modified antimicrobial ...modified antimicrobial peptide cecropin P1 (CP1) was chemically immobilized onto SAM with a maleimide terminal group. Two important characteristics...applied to investigate such structures of peptides immobilized on self-assembled monolayers (SAMs). Here cysteine-modified antimicrobial peptide cecropin

Analysis of the relationship between end-to-end distance and activity of single-chain antibody against colorectal carcinoma.

PubMed

Zhang, Jianhua; Liu, Shanhong; Shang, Zhigang; Shi, Li; Yun, Jun

2012-08-22

We investigated the relationship of End-to-end distance between VH and VL with different peptide linkers and the activity of single-chain antibodies by computer-aided simulation. First, we developed (G4S)n (where n = 1-9) as the linker to connect VH and VL, and estimated the 3D structure of single-chain Fv antibody (scFv) by homologous modeling. After molecular models were evaluated and optimized, the coordinate system of every protein was built and unified into one coordinate system, and End-to-end distances calculated using 3D space coordinates. After expression and purification of scFv-n with (G4S)n as n = 1, 3, 5, 7 or 9, the immunoreactivity of purified ND-1 scFv-n was determined by ELISA. A multi-factorial relationship model was employed to analyze the structural factors affecting scFv: rn=ABn-ABO2+CDn-CDO2+BCn-BCst2. The relationship between immunoreactivity and r-values revealed that fusion protein structure approached the desired state when the r-value = 3. The immunoreactivity declined as the r-value increased, but when the r-value exceeded a certain threshold, it stabilized. We used a linear relationship to analyze structural factors affecting scFv immunoreactivity.

The gating mechanism of the large mechanosensitive channel MscL

NASA Technical Reports Server (NTRS)

Sukharev, S.; Betanzos, M.; Chiang, C. S.; Guy, H. R.

2001-01-01

The mechanosensitive channel of large conductance, MscL, is a ubiquitous membrane-embedded valve involved in turgor regulation in bacteria. The crystal structure of MscL from Mycobacterium tuberculosis provides a starting point for analysing molecular mechanisms of tension-dependent channel gating. Here we develop structural models in which a cytoplasmic gate is formed by a bundle of five amino-terminal helices (S1), previously unresolved in the crystal structure. When membrane tension is applied, the transmembrane barrel expands and pulls the gate apart through the S1-M1 linker. We tested these models by substituting cysteines for residues predicted to be near each other only in either the closed or open conformation. Our results demonstrate that S1 segments form the bundle when the channel is closed, and crosslinking between S1 segments prevents opening. S1 segments interact with M2 when the channel is open, and crosslinking of S1 to M2 impedes channel closing. Gating is affected by the length of the S1-M1 linker in a manner consistent with the model, revealing critical spatial relationships between the domains that transmit force from the lipid bilayer to the channel gate.

Phylogeny-Based Systematization of Arabidopsis Proteins with Histone H1 Globular Domain1[OPEN

PubMed Central

Knizewski, Lukasz; Schmidt, Anja; Ginalski, Krzysztof

2017-01-01

H1 (or linker) histones are basic nuclear proteins that possess an evolutionarily conserved nucleosome-binding globular domain, GH1. They perform critical functions in determining the accessibility of chromatin DNA to trans-acting factors. In most metazoan species studied so far, linker histones are highly heterogenous, with numerous nonallelic variants cooccurring in the same cells. The phylogenetic relationships among these variants as well as their structural and functional properties have been relatively well established. This contrasts markedly with the rather limited knowledge concerning the phylogeny and structural and functional roles of an unusually diverse group of GH1-containing proteins in plants. The dearth of information and the lack of a coherent phylogeny-based nomenclature of these proteins can lead to misunderstandings regarding their identity and possible relationships, thereby hampering plant chromatin research. Based on published data and our in silico and high-throughput analyses, we propose a systematization and coherent nomenclature of GH1-containing proteins of Arabidopsis (Arabidopsis thaliana [L.] Heynh) that will be useful for both the identification and structural and functional characterization of homologous proteins from other plant species. PMID:28298478

Phylogeny-Based Systematization of Arabidopsis Proteins with Histone H1 Globular Domain.

PubMed

Kotliński, Maciej; Knizewski, Lukasz; Muszewska, Anna; Rutowicz, Kinga; Lirski, Maciej; Schmidt, Anja; Baroux, Célia; Ginalski, Krzysztof; Jerzmanowski, Andrzej

2017-05-01

H1 (or linker) histones are basic nuclear proteins that possess an evolutionarily conserved nucleosome-binding globular domain, GH1. They perform critical functions in determining the accessibility of chromatin DNA to trans-acting factors. In most metazoan species studied so far, linker histones are highly heterogenous, with numerous nonallelic variants cooccurring in the same cells. The phylogenetic relationships among these variants as well as their structural and functional properties have been relatively well established. This contrasts markedly with the rather limited knowledge concerning the phylogeny and structural and functional roles of an unusually diverse group of GH1-containing proteins in plants. The dearth of information and the lack of a coherent phylogeny-based nomenclature of these proteins can lead to misunderstandings regarding their identity and possible relationships, thereby hampering plant chromatin research. Based on published data and our in silico and high-throughput analyses, we propose a systematization and coherent nomenclature of GH1-containing proteins of Arabidopsis ( Arabidopsis thaliana [L.] Heynh) that will be useful for both the identification and structural and functional characterization of homologous proteins from other plant species. © 2017 American Society of Plant Biologists. All Rights Reserved.

The Formation Mechanism of Hydrogels.

PubMed

Lu, Liyan; Yuan, Shiliang; Wang, Jing; Shen, Yun; Deng, Shuwen; Xie, Luyang; Yang, Qixiang

2017-06-12

Hydrogels are degradable polymeric networks, in which cross-links play a vital role in structure formation and degradation. Cross-linking is a stabilization process in polymer chemistry that leads to the multi-dimensional extension of polymeric chains, resulting in network structures. By cross-linking, hydrogels are formed into stable structures that differ from their raw materials. Generally, hydrogels can be prepared from either synthetic or natural polymers. Based on the types of cross-link junctions, hydrogels can be categorized into two groups: the chemically cross-linked and the physically cross-linked. Chemically cross-linked gels have permanent junctions, in which covalent bonds are present between different polymer chains, thus leading to excellent mechanical strength. Although chemical cross-linking is a highly resourceful method for the formation of hydrogels, the cross-linkers used in hydrogel preparation should be extracted from the hydrogels before use, due to their reported toxicity, while, in physically cross-linked gels, dissolution is prevented by physical interactions, such as ionic interactions, hydrogen bonds or hydrophobic interactions. Physically cross-linked methods for the preparation of hydrogels are the alternate solution for cross-linker toxicity. Both methods will be discussed in this essay. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Synthesis and Structural Characterization of Magnesium Based Coordination Networks in Different Solvents

DOE Office of Scientific and Technical Information (OSTI.GOV)

D Banerjee; J Finkelstein; A Smirnov

2011-12-31

Three magnesium based metal-organic frameworks, Mg{sub 3}(3,5-PDC){sub 3}(DMF){sub 3} {center_dot} DMF [1], Mg(3,5-PDC)(H{sub 2}O) {center_dot} (H{sub 2}O) [3], and Mg{sub 4}(3,5-PDC){sub 4}(DMF){sub 2}(H{sub 2}O){sub 2} {center_dot} 2DMF {center_dot} 4.5H{sub 2}O [4], and a 2-D coordination polymer, [Mg(3,5-PDC)(H{sub 2}O){sub 2}] [2] [PDC = pyridinedicarboxylate], were synthesized using a combination of DMF, methanol, ethanol, and water. Compound 1 [space group P2{sub 1}/n, a = 12.3475(5) {angstrom}, b = 11.1929(5) {angstrom}, c = 28.6734(12) {angstrom}, {beta} = 98.8160(10){sup o}, V = 3916.0(3) {angstrom}{sup 3}] consists of a combination of isolated and corner-sharing magnesium octahedra connected by the organic linkers to form a 3-Dmore » network with a 12.2 {angstrom} x 4.6 {angstrom} 1-D channel. The channel contains coordinated and free DMF molecules. In compound 2 [space group C2/c, a = 9.964(5) {angstrom}, b = 12.0694(6) {angstrom}, c = 7.2763(4) {angstrom}, {beta} = 106.4970(6){sup o}, V = 836.70(6) {angstrom}{sup 3}], PDC connects isolated seven coordinated magnesium polyhedra into a layered structure. Compound 3 [space group P6{sub 1}22, a = 11.479(1) {angstrom}, c = 14.735(3) {angstrom}, V = 1681.7(4) {angstrom}{sup 3}] (previously reported) contains isolated magnesium octahedra connected by the organic linker with each other forming a 3D network. Compound 4 [space group P2{sub 1}/c, a = 13.7442(14) {angstrom}, b = 14.2887(15) {angstrom}, c = 14.1178(14) {angstrom}, {beta} = 104.912(2){sup o}, V = 2679.2(5) {angstrom}{sup 3}] also exhibits a 3D network based on isolated magnesium octahedra with square cavities containing both disordered DMF and water molecules. The structural topologies originate due to the variable coordination ability of solvent molecules with the metal center. Water molecules coordinate with the magnesium metal centers preferably over other polar solvents (DMF, methanol, ethanol) used to synthesize the coordination networks. Despite testing multiple desolvation routes, we were unable to measure BET surface areas greater than 51.9 m{sup 2}/g for compound 1.« less

Synthesis and Structural Characterization of Magnesium Based Coordination Networks in Different Solvents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Banerjee, Debasis; Finkelstein, Jeffrey; Smirnov, A.

2015-10-15

Three magnesium based metal-organic frameworks, Mg{sub 3}(3,5-PDC){sub 3}(DMF){sub 3} {center_dot} DMF [1], Mg(3,5-PDC)(H{sub 2}O) {center_dot} (H{sub 2}O) [3], and Mg4(3,5-PDC)4(DMF){sub 2}(H{sub 2}O){sub 2} {center_dot} 2DMF {center_dot} 4.5H{sub 2}O [4], and a 2-D coordination polymer, [Mg(3,5-PDC)(H{sub 2}O){sub 2}] [2] [PDC = pyridinedicarboxylate], were synthesized using a combination of DMF, methanol, ethanol, and water. Compound 1 [space group P2{sub 1}/n, a = 12.3475(5) {angstrom}, b = 11.1929(5) {angstrom}, c = 28.6734(12) {angstrom}, {beta} = 98.8160(10){sup o}, V = 3916.0(3) {angstrom}{sup 3}] consists of a combination of isolated and corner-sharing magnesium octahedra connected by the organic linkers to form a 3-D network withmore » a 12.2 {angstrom} x 4.6 {angstrom} 1-D channel. The channel contains coordinated and free DMF molecules. In compound 2 [space group C2/c, a = 9.964(5) {angstrom}, b = 12.0694(6) {angstrom}, c = 7.2763(4) {angstrom}, {beta} = 106.4970(6){sup o}, V = 836.70(6) {angstrom}{sup 3}], PDC connects isolated seven coordinated magnesium polyhedra into a layered structure. Compound 3 [space group P6{sub 1}22, a = 11.479(1) {angstrom}, c = 14.735(3) {angstrom}, V = 1681.7(4) {angstrom}{sup 3}] (previously reported) contains isolated magnesium octahedra connected by the organic linker with each other forming a 3D network. Compound 4 [space group P2{sub 1}/c, a = 13.7442(14) {angstrom}, b = 14.2887(15) {angstrom}, c = 14.1178(14) {angstrom}, {beta} = 104.912(2){sup o}, V = 2679.2(5) {angstrom}{sup 3}] also exhibits a 3D network based on isolated magnesium octahedra with square cavities containing both disordered DMF and water molecules. The structural topologies originate due to the variable coordination ability of solvent molecules with the metal center. Water molecules coordinate with the magnesium metal centers preferably over other polar solvents (DMF, methanol, ethanol) used to synthesize the coordination networks. Despite testing multiple desolvation routes, we were unable to measure BET surface areas greater than 51.9 m{sup 2}/g for compound 1.« less

Impact of linker engineering on the catalytic activity of metal–organic frameworks containing Pd(II)–bipyridine complexes

DOE PAGES

Li, Xinle; Van Zeeland, Ryan; Maligal-Ganesh, Raghu V.; ...

2016-08-09

A series of mixed-linker bipyridyl metal–organic framework (MOF)-supported palladium(II) catalysts were used to elucidate the electronic and steric effects of linker substitution on the activity of these catalysts in the context of Suzuki–Miyaura cross-coupling reactions. m-6,6'-Me 2bpy-MOF-PdCl 2 exhibited 110- and 496-fold enhancements in activity compared to nonfunctionalized m-bpy-MOF-PdCl 2 and m-4,4'-Me 2bpy-MOF-PdCl 2, respectively. Furthermore, this result clearly demonstrates that the stereoelectronic properties of metal-binding linker units are critical to the activity of single-site organometallic catalysts in MOFs and highlights the importance of linker engineering in the design and development of efficient MOF catalysts.

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases.

PubMed

Lohman, Jeremy R; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

2015-10-13

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.

Structural and evolutionary relationships of “AT-less” type I polyketide synthase ketosynthases

PubMed Central

Lohman, Jeremy R.; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

2015-01-01

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs. PMID:26420866

A structural comparison of 'real' and 'model' calmodulin clarified allosteric interactions regulating domain motion.

PubMed

Shimoyama, Hiromitsu

2018-05-07

Calmodulin (CaM) is a multifunctional calcium-binding protein, which regulates various biochemical processes. CaM acts via structural changes and complex forming with its target enzymes. CaM has two globular domains (N-lobe and C-lobe) connected by a long linker region. Upon calcium binding, the N-lobe and C-lobe undergo local conformational changes, after that, entire CaM wraps the target enzyme through a large conformational change. However, the regulation mechanism, such as allosteric interactions regulating the conformational changes, is still unclear. In order to clarify the allosteric interactions, in this study, experimentally obtained 'real' structures are compared to 'model' structures lacking the allosteric interactions. As the allosteric interactions would be absent in calcium-free CaM (apo-CaM), allostery-eliminated calcium-bound CaM (holo-CaM) models were constructed by combining the apo-CaM's linker and the holo-CaM's N- and C-lobe. Before the comparison, the 'real' and 'model' structures were clustered and cluster-cluster relationship was determined by a principal component analysis. The structures were compared based on the relationship, then, a distance map and a contact probability analysis clarified that the inter-domain motion is regulated by several groups of inter-domain contacting residue pairs. The analyses suggested that these residues cause inter-domain translation and rotation, and as a consequence, the motion encourage structural diversity. The resultant diversity would contribute to the functional versatility of CaM.

Structural and Functional Characterization of Reston Ebola Virus VP35 Interferon Inhibitory Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, Daisy W.; Shabman, Reed S.; Farahbakhsh, Mina

2010-09-21

Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address thismore » question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-{angstrom} crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 {angstrom}, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled with the multiple critical roles played by Ebola virus VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development.« less

Structural and functional characterization of Reston Ebola virus VP35 interferon inhibitory domain.

PubMed

Leung, Daisy W; Shabman, Reed S; Farahbakhsh, Mina; Prins, Kathleen C; Borek, Dominika M; Wang, Tianjiao; Mühlberger, Elke; Basler, Christopher F; Amarasinghe, Gaya K

2010-06-11

Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-A crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 A, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled with the multiple critical roles played by Ebola virus VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development. Copyright 2010 Elsevier Ltd. All rights reserved.

Structure of Pseudoknot PK26 Shows 3D Domain Swapping in an RNA

NASA Technical Reports Server (NTRS)

Lietzke, Susan E; Barnes, Cindy L.

1998-01-01

3D domain swapping provides a facile pathway for the evolution of oligomeric proteins and allosteric mechanisms and a means for using monomer-oligomer equilibria to regulate biological activity. The term "3D domain swapping" describes the exchange of identical domains between two protein monomers to create an oligomer. 3D domain swapping has, so far, only been recognized in proteins. In this study, the structure of the pseudoknot PK26 is reported and it is a clear example of 3D domain swapping in RNA. PK26 was chosen for study because RNA pseudoknots are required structures in several biological processes and they arise frequently in in vitro selection experiments directed against protein targets. PK26 specifically inhibits HIV-1 reverse transcriptase with nanomolar affinity. We have now determined the 3.1 A resolution crystal structure of PK26 and find that it forms a 3D domain swapped dimer. PK26 shows extensive base pairing between and within strands. Formation of the dimer requires the linker region between the pseudoknot folds to adopt a unique conformation that allows a base within a helical stem to skip one base in the stacking register. Rearrangement of the linker would permit a monomeric pseudoknot to form. This structure shows how RNA can use 3D domain swapping to build large scale oligomers like the putative hexamer in the packaging RNA of bacteriophage Phi29.

Modeling Protein Excited-state Structures from "Over-length" Chemical Cross-links.

PubMed

Ding, Yue-He; Gong, Zhou; Dong, Xu; Liu, Kan; Liu, Zhu; Liu, Chao; He, Si-Min; Dong, Meng-Qiu; Tang, Chun

2017-01-27

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones. Thus the "over-length" cross-links may arise from alternative excited-state conformation of the protein. Here we present a method and associated software DynaXL for visualizing the ensemble structures of multidomain proteins based on intramolecular cross-links identified by mass spectrometry with high confidence. Representing the cross-linkers and cross-linking reactions explicitly, we show that the protein excited-state structure can be modeled with as few as two over-length cross-links. We demonstrate the generality of our method with three systems: calmodulin, enzyme I, and glutamine-binding protein, and we show that these proteins alternate between different conformations for interacting with other proteins and ligands. Taken together, the over-length chemical cross-links contain valuable information about protein dynamics, and our findings here illustrate the relationship between dynamic domain movement and protein function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Design, Synthesis, and Pharmacokinetics of a Bone-Targeting Dual-Action Prodrug for the Treatment of Osteoporosis.

PubMed

Xie, Haibo; Chen, Gang; Young, Robert N

2017-08-24

A dual-action bone-targeting prodrug has been designed, synthesized, and evaluated for in vitro and in vivo metabolic stability, in vivo tissue distribution, and rates of release of the active constituents after binding to bones through the use of differentially double-labeled derivatives. The conjugate (general structure 7) embodies the merger of a very potent and proven anabolic selective agonist of the prostaglandin EP4 receptor, compound 5, and alendronic acid, a potent inhibitor of bone resorption, optimally linked through a differentially hydrolyzable linker unit, N-4-carboxymethylphenyl-methyloxycarbonyl-leucinyl-argininyl-para-aminophenylmethylalcohol (Leu-Arg-PABA). Optimized conjugate 16 was designed so that esterase activity will liberate 5 and cathepsin K cleavage of the Leu-Arg-PABA element will liberate alendronic acid. Studies with doubly radiolabeled 16 provide a proof-of-concept for the use of a cathepsin K cleavable peptide-linked conjugate for targeting of bisphosphonate prodrugs to bone and slow release liberation of the active constituents in vivo. Such conjugates are potential therapies for the treatment of bone disorders such as osteoporosis.

Arc is a flexible modular protein capable of reversible self-oligomerization

PubMed Central

Myrum, Craig; Baumann, Anne; Bustad, Helene J.; Flydal, Marte Innselset; Mariaule, Vincent; Alvira, Sara; Cuéllar, Jorge; Haavik, Jan; Soulé, Jonathan; Valpuesta, José Maria; Márquez, José Antonio; Martinez, Aurora; Bramham, Clive R.

2015-01-01

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes. PMID:25748042

Topography of the ISW2–nucleosome complex: insights into nucleosome spacing and chromatin remodeling

PubMed Central

Kagalwala, Mohamedi N; Glaus, Benjamin J; Dang, Weiwei; Zofall, Martin; Bartholomew, Blaine

2004-01-01

Linker DNA was found to be critical for the specific docking of ISW2 with nucleosomes as shown by mapping the physical contacts of ISW2 with nucleosomes at base-pair resolution. Hydroxyl radical footprinting revealed that ISW2 not only extensively interacts with the linker DNA, but also approaches the nucleosome from the side perpendicular to the axis of the DNA superhelix and contacts two disparate sites on the nucleosomal DNA from opposite sides of the superhelix. The topography of the ISW2–nucleosome was further delineated by finding which of the ISW2 subunits are proximal to specific sites within the linker and nucleosomal DNA regions by site-directed DNA photoaffinity labeling. Although ISW2 was shown to contact ∼63 bp of linker DNA, a minimum of 20 bp of linker DNA was required for stable binding of ISW2 to nucleosomes. The remaining ∼43 bp of flanking linker DNA promoted more efficient binding under competitive binding conditions and was functionally important for enhanced sliding of nucleosomes when ISW2 was significantly limiting. PMID:15131696

The electron injection rate in CdSe quantum dot sensitized solar cells: from a bifunctional linker and zinc oxide morphology.

PubMed

Ding, Wei-Lu; Peng, Xing-Liang; Sun, Zhu-Zhu; Li, Ze-Sheng

2017-11-09

Herein, we have investigated the effect of both the bifunctional linker (L1, L2, L3, and L4) and ZnO morphology (porous nanoparticles (NPs), nanowires (NWs), and nanotubes (NTs-A and NTs-Z)) on the electron injection in CdSe QD sensitized solar cells by first-principles simulation. Via calculating the partitioned interfaces formed by different components (linker/QDs and ZnO/linker), we found that the electronic states of QDs and every ZnO substrate are insensitive to any linker, while the frontier orbitals of L1-L4 (with increased delocalization) manifest a systematical negative-shift. Because of the lowest unoccupied molecular orbital (LUMO) of L1 compared to its counterparts aligned in the region of the virtual states of QDs or the substrate with a high density of states, it always yields a stronger electronic coupling with QDs and varied substrates. After characterization of the complete ZnO/linker/QD system, we found that the electron injection time (τ) vastly depends on both the linker and substrate. On the one hand, L1 bridged QDs and every substrate always achieve the shortest τ compared to their counterpart associated cases. On the other hand, NW supported systems always yield the shortest τ no matter what the linker is. Overall, the NW/L1/QD system achieves the fastest injection by ∼160 fs. This essentially stems from the shortest molecular length of L1 decreasing the distance between QDs and the substrate, subsequently improving the interfacial coupling. Meanwhile, the NW supported cases generate the less sensitive virtual states for both the QDs and NWs, ensuring a less variable interfacial coupling. These facts combined can provide understanding of the effects contributed from the linker and the oxide semiconductor morphology on charge transfer with the aim of choosing an appropriate component with fast directional electron injection.

cis-Apa: a practical linker for the microwave-assisted preparation of cyclic pseudopeptides via RCM cyclative cleavage.

PubMed

Baron, Alice; Verdié, Pascal; Martinez, Jean; Lamaty, Frédéric

2011-02-04

A new linker cis-5-aminopent-3-enoic acid (cis-Apa) was prepared for the synthesis of cyclic pseudopeptides by cyclization-cleavage by using ring-closing methatesis (RCM). We developed a new synthetic pathway for the preparation of the cis-Apa linker that was tested in the cyclization-cleavage process of different RGD peptide sequences. Different macrocyclic peptidomimetics were prepared by using this integrated microwave-assisted method, showing that the readily available cis-Apa amino acid is well adapted as a linker in the cyclization-cleavage process.

Designed inhibitors with hetero linkers for gastric proton pump H+,K+-ATPase: Steered molecular dynamics and metadynamics studies.

PubMed

Jana, Kalyanashis; Bandyopadhyay, Tusar; Ganguly, Bishwajit

2017-11-01

Acid suppressant SCH28080 and its derivatives reversibly reduce acid secretion activity of the H + ,K + -ATPase in a K + competitive manner. The results on homologation of the SCH28080 by varying the linker chain length suggested the improvement in efficacy. However, the pharmacokinetic studies reveal that the hydrophobic nature of the CH 2 linker units may not help it to function as a better acid suppressant. We have exploited the role of linker unit to enhance the efficacy of such reversible acid suppressant drug molecules using hetero linker, i.e., disulfide and peroxy linkers. The logarithm of partition coefficient defined for a drug molecule relates to the partition coefficient, which allows the optimum solubility characteristics to reach the active site. The logarithm of partition coefficient calculated for the designed inhibitors suggests that inhibitors would possibly reach the active site in sufficient concentration like in the case of SCH28080. The steered molecular dynamics studies have revealed that the Inhibitor-1 with disulfide linker unit is more stable at the active site due to greater noncovalent interactions compared to the SCH28080. Centre of mass distance analysis suggests that the Cysteine-813 amino acid residue selectively plays an important role in the inhibition of H + ,K + -ATPase for Inhibitor-1. Furthermore, the quantum chemical calculations with M11L/6-31+G(d,p) level of theory have been performed to account the noncovalent interactions responsible for the stabilization of inhibitor molecules in the active site gorge of the gastric proton pump at different time scale. The hydrogen bonding and hydrophobic interaction studies corroborate the center of mass distance analysis as well. Well-tempered metadynamics free energy surface and center of mass separation analysis for the Inhibitor-1 is in good agreement with the steered molecular dynamics results. The torsional angle of the linker units seems to be crucial for better efficacy of drug molecules. The torsional angle of linker units of SCH28080 (COCH 2 C) and of Inhibitor 1 (CSSC) prefers to lie within ∼60°-90° for a longer time during the simulations, whereas, the peroxy linker (COOC) of Inhibitor 2 prefers to adopt ∼120-160°. Therefore, it appears that the smaller torsion angle of linker units can achieve better interactions with the active site residues of H + ,K + -ATPase to inhibit the acid secretion activity. The reversible drug molecules with disulfide linker unit would be a promising candidate as proton pump antagonist to H + ,K + -ATPase. Copyright © 2017 Elsevier Inc. All rights reserved.

ATPase domain and interdomain linker play a key role in aggregation of mitochondrial Hsp70 chaperone Ssc1.

PubMed

Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

2010-02-12

The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1*

PubMed Central

Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai

2010-01-01

The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Δhep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer. PMID:20007714

Robust, Chiral, and Porous BINAP-Based Metal–Organic Frameworks for Highly Enantioselective Cyclization Reactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sawano, Takahiro; Thacker, Nathan C.; Lin, Zekai

2016-05-06

We report here the design of BINAP-based metal–organic frameworks and their postsynthetic metalation with Rh complexes to afford highly active and enantioselective single-site solid catalysts for the asymmetric cyclization reactions of 1,6-enynes. Robust, chiral, and porous Zr-MOFs of UiO topology, BINAP-MOF (I) or BINAP-dMOF (II), were prepared using purely BINAP-derived dicarboxylate linkers or by mixing BINAP-derived linkers with unfunctionalized dicarboxylate linkers, respectively. Upon metalation with Rh(nbd)2BF4 and [Rh(nbd)Cl]2/AgSbF6, the MOF precatalysts I·Rh(BF4) and I·Rh(SbF6) efficiently catalyzed highly enantioselective (up to 99% ee) reductive cyclization and Alder-ene cycloisomerization of 1,6-enynes, respectively. I·Rh catalysts afforded cyclization products at comparable enantiomeric excesses (ee’s)more » and 4–7 times higher catalytic activity than the homogeneous controls, likely a result of catalytic site isolation in the MOF which prevents bimolecular catalyst deactivation pathways. However, I·Rh is inactive in the more sterically encumbered Pauson–Khand reactions between 1,6-enynes and carbon monoxide. In contrast, with a more open structure, Rh-functionalized BINAP-dMOF, II·Rh, effectively catalyzed Pauson–Khand cyclization reactions between 1,6-enynes and carbon monoxide at 10 times higher activity than the homogeneous control. II·Rh was readily recovered and used three times in Pauson–Khand cyclization reactions without deterioration of yields or ee’s. Our work has expanded the scope of MOF-catalyzed asymmetric reactions and showed that the mixed linker strategy can effectively enlarge the open space around the catalytic active site to accommodate highly sterically demanding polycyclic metallocycle transition states/intermediates in asymmetric intramolecular cyclization reactions.« less

Cytochrome bc1-cy Fusion Complexes Reveal the Distance Constraints for Functional Electron Transfer Between Photosynthesis Components*

PubMed Central

Lee, Dong-Woo; Öztürk, Yavuz; Osyczka, Artur; Cooley, Jason W.; Daldal, Fevzi

2008-01-01

Photosynthetic (Ps) growth of purple non-sulfur bacteria such as Rhodobacter capsulatus depends on the cyclic electron transfer (ET) between the ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductases (cyt bc1 complex), and the photochemical reaction centers (RC), mediated by either a membrane-bound (cyt cy) or a freely diffusible (cyt c2) electron carrier. Previously, we constructed a functional cyt bc1-cy fusion complex that supported Ps growth solely relying on membrane-confined ET (Lee, D.-W., Ozturk, Y., Mamedova, A., Osyczka, A., Cooley, J. W., and Daldal, F. (2006) Biochim. Biophys. Acta1757 ,346 -35216781662). In this work, we further characterized this cyt bc1-cy fusion complex, and used its derivatives with shorter cyt cy linkers as “molecular rulers” to probe the distances separating the Ps components. Comparison of the physicochemical properties of both membrane-embedded and purified cyt bc1-cy fusion complexes established that these enzymes were matured and assembled properly. Light-activated, time-resolved kinetic spectroscopy analyses revealed that their variants with shorter cyt cy linkers exhibited fast, native-like ET rates to the RC via the cyt bc1. However, shortening the length of the cyt cy linker decreased drastically this electronic coupling between the cyt bc1-cy fusion complexes and the RC, thereby limiting Ps growth. The shortest and still functional cyt cy linker was about 45 amino acids long, showing that the minimal distance allowed between the cyt bc1-cy fusion complexes and the RC and their surrounding light harvesting proteins was very short. These findings support the notion that membrane-bound Ps components form large, active structural complexes that are “hardwired” for cyclic ET. PMID:18343816

Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains.

PubMed

de la Peña, Pilar; Domínguez, Pedro; Barros, Francisco

2018-03-01

Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4-S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4-S5 linker induces an increasing negative shift in activation voltage dependence, a reduced z g value and a more negative ΔG 0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4-S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4-S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.

Structural changes and cellular localization of resuscitation-promoting factor in environmental isolates of Micrococcus luteus.

PubMed

Koltunov, Viktoria; Greenblatt, Charles L; Goncharenko, Anna V; Demina, Galya R; Klein, Benjamin Y; Young, Michael; Kaprelyants, Arseny S

2010-02-01

Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in "spent culture medium." Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.

CryoEM structure of the spliceosome immediately after branching

PubMed Central

Galej, Wojciech P.; Wilkinson, Max E.; Fica, Sebastian M.; Oubridge, Chris; Newman, Andrew J.; Nagai, Kiyoshi

2016-01-01

Pre-mRNA splicing proceeds by two consecutive trans-esterification reactions via a lariat-intron intermediate. We present the 3.8Å cryoEM structure of the spliceosome immediately after lariat formation. The 5’-splice site is cleaved but remains close to the catalytic Mg2+ site in the U2/U6 snRNA triplex, and the 5’-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2’OH. The 5’-exon is held between the Prp8 N-terminal and Linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5’-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step one factors Yju2 and Cwc25 stabilise docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 RT and Linker domains and extends towards Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation. PMID:27459055

A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study.

PubMed

Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

2016-03-01

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science.

Recombinant human erythropoietin (rHuEPO): cross-linking with disuccinimidyl esters and identification of the interfacing domains in EPO.

PubMed Central

Haniu, M.; Narhi, L. O.; Arakawa, T.; Elliott, S.; Rohde, M. F.

1993-01-01

Several amino groups of recombinant human erythropoietin are selectively cross-linked by specific cross-linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate). Intramolecular cross-linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross-linkers, which possess an 11-12-A length of a spacer between two reacting groups. Intramolecularly cross-linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 A in the solution state. These interfacing amino groups include Lys 20-Lys 154, Lys 45-Lys 140, Lys 52-Lys 154, Lys 52-Lys 140, and Ala 1-Lys 116. A comparison of the cross-linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four alpha-helical bundles and a long stretch of beta-sheet structure are involved in the active protein. PMID:8401229

A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study

PubMed Central

Sadaf, Aiman; Mortensen, Jonas S.; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette

2015-01-01

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science. PMID:27110345

Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malaby, Andrew W.; Das, Sanchaita; Chakravarthy, Srinivas

Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization andmore » conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.« less

Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene.

PubMed

Chun, Jong-Yoon; Kim, Kyoung-Joong; Hwang, In-Taek; Kim, Yun-Jee; Lee, Dae-Hoon; Lee, In-Kyoung; Kim, Jong-Kee

2007-01-01

Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5'-segment that initiates stable priming, and a short 3'-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR.

Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts

PubMed Central

Maresca, Thomas J.; Freedman, Benjamin S.; Heald, Rebecca

2005-01-01

During cell division, condensation and resolution of chromosome arms and the assembly of a functional kinetochore at the centromere of each sister chromatid are essential steps for accurate segregation of the genome by the mitotic spindle, yet the contribution of individual chromatin proteins to these processes is poorly understood. We have investigated the role of embryonic linker histone H1 during mitosis in Xenopus laevis egg extracts. Immunodepletion of histone H1 caused the assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. Although functional kinetochores assembled, aligned, and exhibited poleward movement, long and tangled chromosome arms could not be segregated in anaphase. Histone H1 depletion did not significantly affect the recruitment of known structural or functional chromosomal components such as condensins or chromokinesins, suggesting that the loss of H1 affects chromosome architecture directly. Thus, our results indicate that linker histone H1 plays an important role in the structure and function of vertebrate chromosomes in mitosis. PMID:15967810

Metal-Organic Framework (MOF) Compounds: Photocatalysts for Redox Reactions and Solar Fuel Production.

PubMed

Dhakshinamoorthy, Amarajothi; Asiri, Abdullah M; García, Hermenegildo

2016-04-25

Metal-organic frameworks (MOFs) are crystalline porous materials formed from bi- or multipodal organic linkers and transition-metal nodes. Some MOFs have high structural stability, combined with large flexibility in design and post-synthetic modification. MOFs can be photoresponsive through light absorption by the organic linker or the metal oxide nodes. Photoexcitation of the light absorbing units in MOFs often generates a ligand-to-metal charge-separation state that can result in photocatalytic activity. In this Review we discuss the advantages and uniqueness that MOFs offer in photocatalysis. We present the best practices to determine photocatalytic activity in MOFs and for the deposition of co-catalysts. In particular we give examples showing the photocatalytic activity of MOFs in H2 evolution, CO2 reduction, photooxygenation, and photoreduction. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photoluminescence spectroscopy of YVO{sub 4}:Eu{sup 3+} nanoparticles with aromatic linker molecules: A precursor to biomedical functionalization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Senty, T. R.; Yalamanchi, M.; Cushing, S. K.

2014-04-28

Photoluminescence spectra of YVO{sub 4}:Eu{sup 3+} nanoparticles are presented, with and without the attachment of organic molecules that are proposed for linking to biomolecules. YVO{sub 4}:Eu{sup 3+} nanoparticles with 5% dopant concentration were synthesized via wet chemical synthesis. X-ray diffraction and transmission electron microscopy show the expected wakefieldite structure of tetragonal particles with an average size of 17 nm. Fourier-transform infrared spectroscopy determines that metal-carboxylate coordination is successful in replacing native metal-hydroxyl bonds with three organic linkers, namely, benzoic acid, 3-nitro 4-chloro-benzoic acid, and 3,4-dihydroxybenzoic acid, in separate treatments. UV-excitation photoluminescence spectra show that the position and intensity of the dominantmore » {sup 5}D{sub 0} – {sup 7}F{sub 2} electric-dipole transition at 619 nm are unaffected by the benzoic acid and 3-nitro 4-chloro-benzoic acid treatments. Attachment of 3,4-dihydroxybenzoic acid produces an order-of-magnitude quenching in the photoluminescence, due to the presence of high-frequency vibrational modes in the linker. Ratios of the dominant electric- and magnetic-dipole transitions confirm infrared measurements, which indicate that the bulk crystal of the nanoparticle is unchanged by all three treatments.« less

Auto-inhibition and phosphorylation-induced activation of PLC-γ isozymes

PubMed Central

Hajicek, Nicole; Charpentier, Thomas H.; Rush, Jeremy R.; Harden, T. Kendall; Sondek, John

2013-01-01

Multiple extracellular stimuli, such as growth factors and antigens, initiate signaling cascades through tyrosine phosphorylation and activation of phospholipase C (PLC)-γ isozymes. Like most other PLCs, PLC-γ1 is basally auto-inhibited by its X-Y linker, which separates the X-and Y-boxes of the catalytic core. The C-terminal SH2 (cSH2) domain within the X-Y linker is the critical determinant for auto-inhibition of phospholipase activity. Release of auto-inhibition requires an intramolecular interaction between the cSH2 domain and a phosphorylated tyrosine, Tyr783, also located within the X-Y linker. The molecular mechanisms that mediate auto-inhibition and phosphorylation-induced activation have not been defined. Here, we describe structures of the cSH2 domain both alone and bound to a PLC-γ1 peptide encompassing phosphorylated Tyr783. The cSH2 domain remains largely unaltered by peptide engagement. Point mutations in the cSH2 domain located at the interface with the peptide were sufficient to constitutively activate PLC-γ1 suggesting that peptide engagement directly interferes with the capacity of the cSH2 domain to block the lipase active site. This idea is supported by mutations in a complimentary surface of the catalytic core that also enhanced phospholipase activity. PMID:23777354

Fast H-DROP: A thirty times accelerated version of H-DROP for interactive SVM-based prediction of helical domain linkers

NASA Astrophysics Data System (ADS)

Richa, Tambi; Ide, Soichiro; Suzuki, Ryosuke; Ebina, Teppei; Kuroda, Yutaka

2017-02-01

Efficient and rapid prediction of domain regions from amino acid sequence information alone is often required for swift structural and functional characterization of large multi-domain proteins. Here we introduce Fast H-DROP, a thirty times accelerated version of our previously reported H-DROP (Helical Domain linker pRediction using OPtimal features), which is unique in specifically predicting helical domain linkers (boundaries). Fast H-DROP, analogously to H-DROP, uses optimum features selected from a set of 3000 ones by combining a random forest and a stepwise feature selection protocol. We reduced the computational time from 8.5 min per sequence in H-DROP to 14 s per sequence in Fast H-DROP on an 8 Xeon processor Linux server by using SWISS-PROT instead of Genbank non-redundant (nr) database for generating the PSSMs. The sensitivity and precision of Fast H-DROP assessed by cross-validation were 33.7 and 36.2%, which were merely 2% lower than that of H-DROP. The reduced computational time of Fast H-DROP, without affecting prediction performances, makes it more interactive and user-friendly. Fast H-DROP and H-DROP are freely available from http://domserv.lab.tuat.ac.jp/.

Computational modeling and functional analysis of Herpes simplex virus type-1 thymidine kinase and Escherichia coli cytosine deaminase fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jufeng; Wang, Zhanli; Wei, Fang

2007-08-17

Herpes simplex virus type-1 thymidine kinase (HSV-1TK) and Escherichia coli cytosine deaminase (CD) fusion protein was designed using InsightII software. The structural rationality of the fusion proteins incorporating a series of flexible linker peptide was analyzed, and a suitable linker peptide was chosen for further investigated. The recombinant plasmid containing the coding regions of HSV-1TK and CD cDNA connected by this linker peptide coding sequence was generated and subsequently transfected into the human embryonic kidney 293 cells (HEK293). The Western blotting indicated that the recombinant fusion protein existed as a dimer with a molecular weight of approximately 90 kDa. Themore » toxicity of the prodrug on the recombinant plasmid-transfected human lung cancer cell line NCIH460 was evaluated, which showed that TKglyCD-expressing cells conferred upon cells prodrug sensitivities equivalent to that observed for each enzyme independently. Most noteworthy, cytotoxicity could be enhanced by concurrently treating TKglyCD-expressing cells with prodrugs GCV and 5-FC. The results indicate that we have successfully constructed a HSV-1TKglyCD fusion gene which might have a potential application for cancer gene therapy.« less

Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

PubMed

Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

2005-04-01

After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

Modification of Titanium Substrates with Chimeric Peptides Comprising Antimicrobial and Titanium-Binding Motifs Connected by Linkers To Inhibit Biofilm Formation.

PubMed

Liu, Zihao; Ma, Shiqing; Duan, Shun; Xuliang, Deng; Sun, Yingchun; Zhang, Xi; Xu, Xinhua; Guan, Binbin; Wang, Chao; Hu, Meilin; Qi, Xingying; Zhang, Xu; Gao, Ping

2016-03-02

Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p < 0.05). Concentration-dependent adsorption is consistent with a single Langmuir model, whereas time-dependent adsorption is in line with a two-domain Langmuir model. Additionally, the chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis.

A Study into the Collision-induced Dissociation (CID) Behavior of Cross-Linked Peptides*

PubMed Central

Giese, Sven H.; Fischer, Lutz; Rappsilber, Juri

2016-01-01

Cross-linking/mass spectrometry resolves protein–protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers. PMID:26719564

Sequential Linker Installation: Precise Placement of Functional Groups in Multivariate Metal-Organic Frameworks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, S; Lu, WG; Chen, YP

2015-03-11

A unique strategy, sequential linker installation (SLI), has been developed to construct multivariate MOFs with functional groups precisely positioned. PCN-700, a Zr-MOF with eight-connected Zr6O4(OH)(8)(H2O)(4) clusters, has been judiciously designed; the Zr-6 clusters in this MOF are arranged in such a fashion that, by replacement of terminal OH-/H2O ligands, subsequent insertion of linear dicarboxylate linkers is achieved. We demonstrate that linkers with distinct lengths and functionalities can be sequentially installed into PCN-700. Single-crystal to single-crystal transformation is realized so that the positions of the subsequently installed linkers are pinpointed via single-crystal X-ray diffraction analyses. This methodology provides a powerful toolmore » to construct multivariate MOFs with precisely positioned functionalities in the desired proximity, which would otherwise be difficult to achieve.« less

Mechanically Robust 3D Nanostructure Chitosan-Based Hydrogels with Autonomic Self-Healing Properties.

PubMed

Karimi, Ali Reza; Khodadadi, Azam

2016-10-12

Fabrication of hydrogels based on chitosan (CS) with superb self-healing behavior and high mechanical and electrical properties has become a challenging and fascinating topic. Most of the conventional hydrogels lack these properties at the same time. Our objectives in this research were to synthesize, characterize, and evaluate the general properties of chitosan covalently cross-linked with zinc phthalocyanine tetra-aldehyde (ZnPcTa) framework. Our hope was to access an unprecedented self-healable three-dimensional (3D) nanostructure that would harvest the superior mechanical and electrical properties associated with chitosan. The properties of cross-linker such as the structure, steric effect, and rigidity of the molecule played important roles in determining the microstructure and properties of the resulting hydrogels. The tetra-functionalized phthalocyanines favor a dynamic Schiff-base linkage with chitosan to form a 3D porous nanostructure. Based on this strategy, the self-healing ability, as demonstrated by rheological recovery and macroscopic and microscopic observations, is introduced through dynamic covalent Schiff-base linkage between NH 2 groups in CS and benzaldehyde groups at cross-linker ends. The hydrogel was characterized using FT-IR, NMR, UV/vis, and rheological measurements. In addition, cryogenic scanning electron microscopy (cryo-SEM) was employed as a technique to visualize the internal morphology of the hydrogels. Study of the surface morphology of the hydrogel showed a 3D porous nanostructure with uniform morphology. Furthermore, incorporating the conductive nanofillers, such as carbon nanotubes (CNTs), into the structure can modulate the mechanical and electrical properties of the obtained hydrogels. Interestingly, these hydrogel nanocomposites proved to have very good film-forming properties, high modulus and strength, acceptable electrical conductivity, and excellent self-healing properties at neutral pH. Such properties can be finely tuned through variation of the cross-linker and CNT concentration, and as a result these structures are promising candidates for potential applications in various fields of research.

Studies on the chemical stability and functional group compatibility of the benzoin photolabile safety-catch linker using an analytical construct.

PubMed

Cano, Montserrat; Ladlow, Mark; Balasubramanian, Shankar

2002-01-01

A chemical stability study of the benzoin photolabile safety-catch linker (BPSC) has been carried out using a dual-linker analytical construct to establish its compatibility with a range of commonly employed solid-phase reaction conditions. As a result of this study, the dithiane-protected benzoin linker was shown to be reactive only toward strong acids and fluoride nucleophile. Furthermore, a scan of diverse functional groups thought to be unstable toward the safety-catch removal conditions has also been carried out. These data should provide assistance in future utilization of the BPSC for syntheses.

Constitutive Smad linker phosphorylation in melanoma: a mechanism of resistance to transforming growth factor-β-mediated growth inhibition.

PubMed

Cohen-Solal, Karine A; Merrigan, Kim T; Chan, Joseph L-K; Goydos, James S; Chen, Wenjin; Foran, David J; Liu, Fang; Lasfar, Ahmed; Reiss, Michael

2011-06-01

Melanoma cells are resistant to transforming growth factor-β (TGFβ)-induced cell-cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFβ. Melanoma cells in culture and tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15(INK4B) and p21(WAF1) , as compared with cells transfected with wild-type (WT) Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared with WT Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFβ-mediated growth inhibition. 2011 John Wiley & Sons A/S.

Constitutive Smad linker phosphorylation in melanoma: A mechanism of resistance to Transforming Growth Factor-β-mediated growth inhibition

PubMed Central

Cohen-Solal, Karine A.; Merrigan, Kim T.; Chan, Joseph L.-K.; Goydos, James S.; Chen, Wenjin; Foran, David J.; Liu, Fang; Lasfar, Ahmed; Reiss, Michael

2011-01-01

SUMMARY Melanoma cells are resistant to Transforming Growth Factor-β (TGFβ)-induced cell cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFβ. Melanoma cells in culture and in tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15INK4B and p21WAF1, as compared with cells transfected with wild-type Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared to wild-type Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFβ-mediated growth inhibition. PMID:21477078

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lohman, Jeremy; Ma, Ming; Osipiuk, Jerzy

2015-10-13

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representationmore » of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.« less

Insertion of inter-domain linkers improves expression and bioactivity of Zygote arrest (Zar) fusion proteins.

PubMed

Cook, Jonathan M; Charlesworth, Amanda

2017-04-01

Developmentally important proteins that are crucial for fertilization and embryogenesis are synthesized through highly regulated translation of maternal mRNA. The Zygote arrest proteins, Zar1 and Zar2, are crucial for embryogenesis and have been implicated in binding mRNA and repressing mRNA translation. To investigate Zar1 and Zar2, the full-length proteins had been fused to glutathione-S-transferase (GST) or MS2 protein tags with minimal inter-domain linkers derived from multiple cloning sites; however, these fusion proteins expressed poorly and/or lacked robust function. Here, we tested the effect of inserting additional linkers between the fusion domains. Three linkers were tested, each 17 amino acids long with different physical and chemical properties: flexible hydrophilic, rigid extended or rigid helical. In the presence of any of the three linkers, GST-Zar1 and GST-Zar2 had fewer breakdown products. Moreover, in the presence of any of the linkers, MS2-Zar1 was expressed to higher levels, and in dual luciferase tethered assays, both MS2-Zar1 and MS2-Zar2 repressed luciferase translation to a greater extent. These data suggest that for Zar fusion proteins, increasing the length of linkers, regardless of their physical or chemical properties, improves stability, expression and bioactivity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Role of the external NH2 linker on the conformation of surface immobilized single strand DNA probes and their SERS detection

NASA Astrophysics Data System (ADS)

He, Lijie; Langlet, Michel; Stambouli, Valerie

2017-03-01

The conformation and topological properties of DNA single strand probe molecules attached on solid surfaces are important, notably for the performances of devices such as biosensors. Commonly, the DNA probes are tethered to the surface using external linkers such as NH2. In this study, the role and influence of this amino-linker on the immobilization way and conformation of DNA probes on Ag nanoparticle surface is emphasized using Surface Enhanced Raman Spectroscopy (SERS). We compare the SERS spectra and their reproducibility in the case of two groups of DNA polybase probes which are polyA, polyC, polyT, and polyG. In the first group, the polybases exhibit an external NH2 functional linker while in the second group the polybases are NH2-free. The results show that the reproducibility of SERS spectra is enhanced in the case of the first group. It leads us to propose two models of polybase conformation on Ag surface according to the presence or the absence of the external NH2 linker. In the presence of the NH2 external linker, the latter would act as a major anchoring point. As a result, the polybases are much ordered with a less random orientation than in the case of NH2-free polybases. Consequently, in view of further in situ hybridization for biosensing applications, it is strongly recommended to use NH2 linker functionalized DNA probes.

Role of the S4-S5 linker in CNG channel activation.

PubMed

Kusch, Jana; Zimmer, Thomas; Holschuh, Jascha; Biskup, Christoph; Schulz, Eckhard; Nache, Vasilica; Benndorf, Klaus

2010-10-20

Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus that is located at the intracellular side. The molecular events translating the ligand binding to the pore opening are still unknown. We investigated the role of the S4-S5 linker in the activation process by quantifying its interaction with other intracellular regions. To this end, we constructed chimeric channels in which the N-terminus, the S4-S5 linker, the C-linker, and the CNBD of the retinal CNGA1 subunit were systematically replaced by the respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by thermodynamic double-mutant cycle analysis. We observed that all four intracellular regions, including the relatively short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits, as derived from the Hill coefficient. The interaction energies reveal an interaction of the S4-S5 linker with both the N-terminus and the C-linker, but no interaction with the CNBD. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A Self-Assembling Protein Hydrogel Technology for Enzyme Incorporation onto Electrodes in Biofuel Cells

DTIC Science & Technology

2015-10-26

amount of the product J and the unreacted N/C in the same lane. Figure 2. Crystal structure of Tip1-Tip1lig ( PDB code: 3IDW). (A...stability (able to retain its trimeric quaternary structure in solutions after boiling in SDS buffer). We reasoned that its very strong...109, 10 is a flexible polyanionic linker and was incorporated as the midblock for water retention. Mixing of the two protein block copolymers

Solvothermal growth of a ruthenium metal-organic framework featuring HKUST-1 structure type as thin films on oxide surfaces.

PubMed

Kozachuk, Olesia; Yusenko, Kirill; Noei, Heshmat; Wang, Yuemin; Walleck, Stephan; Glaser, Thorsten; Fischer, Roland A

2011-08-14

Phase-pure crystalline thin films of a mixed-valence Ru(2)(II,III) metal-organic framework with 1,3,5-benzenetricarboxylate (btc) as a linker were solvothermally grown on amorphous alumina and silica surfaces. Based on the Rietveld refinement, the structure of Ru-MOF was assigned to be analogous to [Cu(3)(btc)(2)] (HKUST-1). This journal is © The Royal Society of Chemistry 2011

Coordination-Driven Syntheses of Compact Supramolecular Metallacycles toward Extended Metallo-organic Stacked Supramolecular Assemblies.

PubMed

Lescop, Christophe

2017-04-18

One important concept associated with supramolecular chemistry is supramolecular self-assembly, which deals with the way discrete individual components interact via intermolecular interactions in order to build, upon their spontaneous association, high order functional assemblies. The accumulation of these very simple and localized noncovalent interactions (such as H-bonding, dipole-dipole, hydrophobic/hydrophilic, van der Waals, π-π, π-CH, etc.) is ubiquitous in the complexity of natural systems (such as DNA, proteins, membranes, micelles, etc.). It can also be transposed to the directed synthesis of intricate artificial scaffolds, which have anticipated geometries and properties. Among the synthetic strategies based on this concept, coordination-driven supramolecular chemistry uses the robust, reversible, and directional metal-to-ligand coordinative bond to build discrete metallo-supramolecular architectures. Within the last two decades, coordination-driven supramolecular chemistry has proved to be one of the most powerful contemporary synthetic approaches and has provided a significant number of increasingly complex supramolecular assemblies, which have predetermined sizes and geometries. While much focus has been devoted to architectures bearing internal cavities for host-guest chemistry or to generate specific reactivity, particular attention can also be paid to compact supramolecular assemblies given that their specific structures are characterized by peculiar synthetic guiding rules as well as by alternative long-range self-assembling properties. This Account describes how a preassembled Cu I bimetallic clip bearing short intermetallic distances can be used as a U-shaped molecular clip to give general and versatile access to a large variety of original compact supramolecular metallacycles. When this Cu I precursor is reacted with various cyano-capped ditopic linkers that have increasing lengths and complexities, specific effects guiding the selective and straightforward syntheses of such compact supramolecular objects are highlighted. Whereas a subtle compromise between the length of the ditopic linkers and the steric bulk of the molecular clip appears to be a purely stereogeometric preliminary parameter to master, lateral interlinker interactions (π-π stacking interactions or aurophilic interactions depending on the nature of the internal cores of the linkers) can circumvent these constraints regardless of the length of the linkers and allow the selective formation of new compact supramolecular structures. Generally, such derivatives presented a strong tendency to self-assemble in the solid state due to inter-supramolecule interactions. This approach thus opens a new door toward molecular materials having an attractive solid state structure for potential applications related to charge carrier mobility and luminescence properties. These compact supramolecular assemblies can therefore be considered as original secondary binding units directing the predictive preparation of such extended networks. The on-purpose design of original building blocks bearing specific cores allowed the formation of new compact supramolecular metallacycles such as "U-shaped" π-stacked assemblies or "pseudodouble paracyclophanes". Similarly, the control of the secondary structure of one-dimensional coordination polymers alternating π-stacked compact supramolecular metallacycles was also conducted. The results that are discussed in this Account illustrate how the rational design of both preassembled polymetallic precursors bearing short intermetallic distances and ditopic linkers able to induce cumulative lateral weak interactions can implement the general synthetic guiding rules of coordination driven supramolecular chemistry. This opens perspectives to use such compact supramolecular assemblies as secondary building blocks for the design of long-range organized functional molecular materials that have predictable architectures and targeted properties.

Mixed-linker UiO-66: structure-property relationships revealed by a combination of high-resolution powder X-ray diffraction and density functional theory calculations.

PubMed

Taddei, Marco; Tiana, Davide; Casati, Nicola; van Bokhoven, Jeroen A; Smit, Berend; Ranocchiari, Marco

2017-01-04

The use of mixed-linker metal-organic frameworks (MIXMOFs) is one of the most effective strategies to modulate the physical-chemical properties of MOFs without affecting the overall crystal structure. In many instances, MIXMOFs have been recognized as solid solutions, with random distribution of ligands, in agreement with the empirical rule known as Vegard's law. In this work, we have undertaken a study combining high-resolution powder X-ray diffraction (HR-PXRD) and density functional theory (DFT) calculations with the aim of understanding the reasons why UiO-66-based amino- and bromo-functionalized MIXMOFs (MIXUiO-66) undergo cell expansion obeying Vegard's law and how this behaviour is related to their physical-chemical properties. DFT calculations predict that the unit cell in amino-functionalized UiO-66 experiences only minor expansion as a result of steric effects, whereas major modification to the electronic features of the framework leads to weaker metal-linker interaction and consequently to the loss of stability at higher degrees of functionalization. For bromo-functionalized UiO-66, steric repulsion due to the size of bromine yields a large cell expansion, but the electronic features remain very similar to pristine UiO-66, preserving the stability of the framework upon functionalization. MIXUiO-66 obtained by either direct synthesis or by post-synthetic exchange shows Vegard-like behaviour, suggesting that both preparation methods yield solid solutions, but the thermal stability and the textural properties of the post-synthetic exchanged materials do not display a clear dependence on the chemical composition, as observed for the MOFs obtained by direct synthesis.

MOF Crystal Chemistry Paving the Way to Gas Storage Needs: Aluminum-Based soc-MOF for CH4, O2, and CO2 Storage.

PubMed

Alezi, Dalal; Belmabkhout, Youssef; Suyetin, Mikhail; Bhatt, Prashant M; Weseliński, Łukasz J; Solovyeva, Vera; Adil, Karim; Spanopoulos, Ioannis; Trikalitis, Pantelis N; Emwas, Abdul-Hamid; Eddaoudi, Mohamed

2015-10-21

The molecular building block approach was employed effectively to construct a series of novel isoreticular, highly porous and stable, aluminum-based metal-organic frameworks with soc topology. From this platform, three compounds were experimentally isolated and fully characterized: namely, the parent Al-soc-MOF-1 and its naphthalene and anthracene analogues. Al-soc-MOF-1 exhibits outstanding gravimetric methane uptake (total and working capacity). It is shown experimentally, for the first time, that the Al-soc-MOF platform can address the challenging Department of Energy dual target of 0.5 g/g (gravimetric) and 264 cm(3) (STP)/cm(3) (volumetric) methane storage. Furthermore, Al-soc-MOF exhibited the highest total gravimetric and volumetric uptake for carbon dioxide and the utmost total and deliverable uptake for oxygen at relatively high pressures among all microporous MOFs. In order to correlate the MOF pore structure and functionality to the gas storage properties, to better understand the structure-property relationship, we performed a molecular simulation study and evaluated the methane storage performance of the Al-soc-MOF platform using diverse organic linkers. It was found that shortening the parent Al-soc-MOF-1 linker resulted in a noticeable enhancement in the working volumetric capacity at specific temperatures and pressures with amply conserved gravimetric uptake/working capacity. In contrast, further expansion of the organic linker (branches and/or core) led to isostructural Al-soc-MOFs with enhanced gravimetric uptake but noticeably lower volumetric capacity. The collective experimental and simulation studies indicated that the parent Al-soc-MOF-1 exhibits the best compromise between the volumetric and gravimetric total and working uptakes under a wide range of pressure and temperature conditions.

Two polymeric nickel(II) complexes with aromatic benzene-1,2,4,5-tetracarboxylate and pyridine-2,5-dicarboxylate linkers.

PubMed

Atria, Ana María; Corsini, Gino; González, Lissette; Garland, Maria Teresa; Baggio, Ricardo

2009-07-01

(Mu-benzene-1,2,4,5-tetracarboxylato-kappa(2)O(1):O(4))bis[aquabis(2,2-methylpropane-1,3-diamine-kappa(2)N,N')nickel(II)] methanol disolvate tetrahydrate, [Ni(2)(C(10)H(2)O(8))(C(5)H(14)N(2))(4)(H(2)O)(2)].2CH(4)O.4H(2)O, (I), is dinuclear, with elemental units built up around an inversion centre halving the benzene-1,2,4,5-tetracarboxylate (btc) anion, which bridges two symmetry-related Ni(II) cations. The octahedral Ni polyhedron is completed by two chelating 2,2-methylpropane-1,3-diamine (dmpda) groups and a terminal aqua ligand. Two methanol and four water solvent molecules are involved in a number of N-H...O and O-H...O hydrogen bonds which define a strongly bound two-dimensional supramolecular structure. The structure of catena-poly[[[bis(2,2-methylpropane-1,3-diamine-kappa(2)N,N')nickel(II)]-mu-pyridine-2,5-dicarboxylato-kappa(3)O(5):N,O(2)-[(2,2-methylpropane-1,3-diamine-kappa(2)N,N')nickel(II)]-mu-pyridine-2,5-dicarboxylato-kappa(3)N,O(2):O(5)] octahydrate], {[Ni(2)(C(7)H(3)NO(4))(2)(C(5)H(14)N(2))(3)].8H(2)O}(n), (II), is polymeric, forming twisted chains around three independent Ni centres, two of which lie on inversion centres and the third in a general position. There are three chelating dmpda ligands (one disordered over two equally populated positions), which are each attached to a different cation, and two pyridine-2,5-dicarboxylate (pdc) anions, both chelating the Ni centre in general positions through an -O-C-C-N- loop, while acting as bridges to the remaining two centrosymmetric Ni atoms. There are, in addition, eight noncoordinated water molecules in the structure, some of which are disordered.

Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches.

PubMed

Saadi, Mahdiye; Karkhah, Ahmad; Nouri, Hamid Reza

2017-07-01

Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7-L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7-L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Centrins in unicellular organisms: functional diversity and specialization.

PubMed

Zhang, Yu; He, Cynthia Y

2012-07-01

Centrins (also known as caltractins) are conserved, EF hand-containing proteins ubiquitously found in eukaryotes. Similar to calmodulins, the calcium-binding EF hands in centrins fold into two structurally similar domains separated by an alpha-helical linker region, shaping like a dumbbell. The small size (15-22 kDa) and domain organization of centrins and their functional diversity/specialization make them an ideal system to study protein structure-function relationship. Here, we review the work on centrins with a focus on their structures and functions characterized in unicellular organisms.

Controlled molecular self-assembly of complex three-dimensional structures in soft materials

PubMed Central

Huang, Changjin; Quinn, David; Suresh, Subra

2018-01-01

Many applications in tissue engineering, flexible electronics, and soft robotics call for approaches that are capable of producing complex 3D architectures in soft materials. Here we present a method using molecular self-assembly to generate hydrogel-based 3D architectures that resembles the appealing features of the bottom-up process in morphogenesis of living tissues. Our strategy effectively utilizes the three essential components dictating living tissue morphogenesis to produce complex 3D architectures: modulation of local chemistry, material transport, and mechanics, which can be engineered by controlling the local distribution of polymerization inhibitor (i.e., oxygen), diffusion of monomers/cross-linkers through the porous structures of cross-linked polymer network, and mechanical constraints, respectively. We show that oxygen plays a role in hydrogel polymerization which is mechanistically similar to the role of growth factors in tissue growth, and the continued growth of hydrogel enabled by diffusion of monomers/cross-linkers into the porous hydrogel similar to the mechanisms of tissue growth enabled by material transport. The capability and versatility of our strategy are demonstrated through biomimetics of tissue morphogenesis for both plants and animals, and its application to generate other complex 3D architectures. Our technique opens avenues to studying many growth phenomena found in nature and generating complex 3D structures to benefit diverse applications. PMID:29255037

Phosphate uptake studies of cross-linked chitosan bead materials.

PubMed

Mahaninia, Mohammad H; Wilson, Lee D

2017-01-01

A systematic experimental study is reported that provides a molecular based understanding of cross-linked chitosan beads and their adsorption properties in aqueous solution containing phosphate dianion (HPO 4 2- ) species. Synthetically modified chitosan using epichlorohydrin and glutaraldehyde cross-linkers result in surface modified beads with variable hydrophile-lipophile character and tunable HPO 4 2- uptake properties. The kinetic and thermodynamic adsorption properties of cross-linked chitosan beads with HPO 4 2- species were studied in aqueous solution. Complementary structure and physicochemical characterization of chitosan beads via potentiometry, Raman spectroscopy, DSC, and dye adsorption measurements was carried out to establish structure-property relationships. The maximum uptake (Q m ) of bead systems with HPO 4 2- at equilibrium was 52.1mgg -1 ; whereas, kinetic uptake results for chitosan bead/phosphate systems are relatively rapid (0.111-0.113min -1 ) with an intraparticle diffusion rate-limiting step. The adsorption process follows a multi-step pathway involving inner- and outer-sphere complexes with significant changes in hydration. Phosphate uptake strongly depends on the composition and type of cross-linker used for preparation of chitosan beads. The adsorption isotherms and structural characterization of bead systems illustrate the role of surface charge, hydrophile-lipophile balance, adsorption site accessibility, and hydration properties of the chitosan bead surface. Copyright © 2016 Elsevier Inc. All rights reserved.

Combinatorial Synthesis of Structurally Diverse Triazole-Bridged Flavonoid Dimers and Trimers.

PubMed

Sum, Tze Han; Sum, Tze Jing; Galloway, Warren R J D; Collins, Súil; Twigg, David G; Hollfelder, Florian; Spring, David R

2016-09-16

Flavonoids are a large family of compounds associated with a broad range of biologically useful properties. In recent years, synthetic compounds that contain two flavonoid units linked together have attracted attention in drug discovery and development projects. Numerous flavonoid dimer systems, incorporating a range of monomers attached via different linkers, have been reported to exhibit interesting bioactivities. From a medicinal chemistry perspective, the 1,2,3-triazole ring system has been identified as a particularly attractive linker moiety in dimeric derivatives (owing to several favourable attributes including proven biological relevance and metabolic stability) and triazole-bridged flavonoid dimers possessing anticancer and antimalarial activities have recently been reported. However, there are relatively few examples of libraries of triazole-bridged flavonoid dimers and the diversity of flavonoid subunits present within these is typically limited. Thus, this compound type arguably remains underexplored within drug discovery. Herein, we report a modular strategy for the synthesis of novel and biologically interesting triazole-bridged flavonoid heterodimers and also very rare heterotrimers from readily available starting materials. Application of this strategy has enabled step-efficient and systematic access to a library of structurally diverse compounds of this sort, with a variety of monomer units belonging to six different structural subclasses of flavonoid successfully incorporated.

Determinants of FIV and HIV Vif sensitivity of feline APOBEC3 restriction factors.

PubMed

Zhang, Zeli; Gu, Qinyong; Jaguva Vasudevan, Ananda Ayyappan; Hain, Anika; Kloke, Björn-Philipp; Hasheminasab, Sascha; Mulnaes, Daniel; Sato, Kei; Cichutek, Klaus; Häussinger, Dieter; Bravo, Ignacio G; Smits, Sander H J; Gohlke, Holger; Münk, Carsten

2016-07-01

Feline immunodeficiency virus (FIV) is a global pathogen of Felidae species and a model system for Human immunodeficiency virus (HIV)-induced AIDS. In felids such as the domestic cat (Felis catus), APOBEC3 (A3) genes encode for single-domain A3Z2s, A3Z3 and double-domain A3Z2Z3 anti-viral cytidine deaminases. The feline A3Z2Z3 is expressed following read-through transcription and alternative splicing, introducing a previously untranslated exon in frame, encoding a domain insertion called linker. Only A3Z3 and A3Z2Z3 inhibit Vif-deficient FIV. Feline A3s also are restriction factors for HIV and Simian immunodeficiency viruses (SIV). Surprisingly, HIV-2/SIV Vifs can counteract feline A3Z2Z3. To identify residues in feline A3s that Vifs need for interaction and degradation, chimeric human-feline A3s were tested. Here we describe the molecular direct interaction of feline A3s with Vif proteins from cat FIV and present the first structural A3 model locating these interaction regions. In the Z3 domain we have identified residues involved in binding of FIV Vif, and their mutation blocked Vif-induced A3Z3 degradation. We further identified additional essential residues for FIV Vif interaction in the A3Z2 domain, allowing the generation of FIV Vif resistant A3Z2Z3. Mutated feline A3s also showed resistance to the Vif of a lion-specific FIV, indicating an evolutionary conserved Vif-A3 binding. Comparative modelling of feline A3Z2Z3 suggests that the residues interacting with FIV Vif have, unlike Vif-interacting residues in human A3s, a unique location at the domain interface of Z2 and Z3 and that the linker forms a homeobox-like domain protruding of the Z2Z3 core. HIV-2/SIV Vifs efficiently degrade feline A3Z2Z3 by possible targeting the linker stretch connecting both Z-domains. Here we identified in feline A3s residues important for binding of FIV Vif and a unique protein domain insertion (linker). To understand Vif evolution, a structural model of the feline A3 was developed. Our results show that HIV Vif binds human A3s differently than FIV Vif feline A3s. The linker insertion is suggested to form a homeo-box domain, which is unique to A3s of cats and related species, and not found in human and mouse A3s. Together, these findings indicate a specific and different A3 evolution in cats and human.

Dynamic interactions of proteins in complex networks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Appella, E.; Anderson, C.

2009-10-01

Recent advances in techniques such as NMR and EPR spectroscopy have enabled the elucidation of how proteins undergo structural changes to act in concert in complex networks. The three minireviews in this series highlight current findings and the capabilities of new methodologies for unraveling the dynamic changes controlling diverse cellular functions. They represent a sampling of the cutting-edge research presented at the 17th Meeting of Methods in Protein Structure Analysis, MPSA2008, in Sapporo, Japan, 26-29 August, 2008 (http://www.iapsap.bnl.gov). The first minireview, by Christensen and Klevit, reports on a structure-based yeast two-hybrid method for identifying E2 ubiquitin-conjugating enzymes that interact withmore » the E3 BRCA1/BARD1 heterodimer ligase to generate either mono- or polyubiquitinated products. This method demonstrated for the first time that the BRCA1/BARD1 E3 can interact with 10 different E2 enzymes. Interestingly, the interaction with multiple E2 enzymes displayed unique ubiquitin-transfer properties, a feature expected to be common among other RING and U-box E3s. Further characterization of new E3 ligases and the E2 enzymes that interact with them will greatly enhance our understanding of ubiquitin transfer and facilitate studies of roles of ubiquitin and ubiquitin-like proteins in protein processing and trafficking. Stein et al., in the second minireview, describe recent progress in defining the binding specificity of different peptide-binding domains. The authors clearly point out that transient peptide interactions mediated by both post-translational modifications and disordered regions ensure a high level of specificity. They postulate that a regulatory code may dictate the number of combinations of domains and post-translational modifications needed to achieve the required level of interaction specificity. Moreover, recognition alone is not enough to obtain a stable complex, especially in a complex cellular environment. Increasing evidence indicates that disordered domains can acquire structural features that modulate the binding and strength of the signaling cascade. Whereas the first two minireviews describe ways in which protein interactions are modulated, the third, by Tompa, focuses on the importance of protein disorder in a subset of amyloid proteins. It is apparent that within this group, part of the polypeptide chain remains disordered during amyloid formation. Moreover, the disordered segments have different amino acid composition and physicochemical characteristics, which suggests that they may play a role in amyloid stability. The disordered region may serve as a linker to connect the ordered core and a globular domain, maintaining the stability and structure of the globular domain and minimizing protein refolding upon amyloid formation. As techniques in protein chemistry advance, we are learning more and more about the mechanisms that regulate and are regulated by protein interactions. The three minireviews in this series offer a glimpse of the complex dynamics fundamental to protein-protein interactions. In the future, we expect that the knowledge gained will help to augment our ability to control complex pathologies and treat diverse diseases states.« less

Molecular design of light-harvesting photosensitizers: effect of varied linker conjugation on interfacial electron transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Jianbing; Swierk, John R.; Hedstrom, Svante

2016-06-30

Here, interfacial electron transfer dynamics of a series of photosensitizers bound to TiO 2 via linkers of varying conjugation strength are explored by spectroscopic and computational techniques. Injection and recombination depend on the extent of conjugation in the linker, where the LUMO delocalization determines the injection dynamics but both the HOMO and HOMO–1 are involved in recombination.

Co-evolution of chitinases from maize and other cereals with secreted proteases from Pleosporineae fungi

USDA-ARS?s Scientific Manuscript database

Plant class IV chitinases are composed of a carboxy-terminal chitinase domain that is attached, through a linker sequence, to a small amino-terminal domain that can be thought of as a structured peptide. While both the peptide-like domain and the chitinase domain share sequence homology throughout m...

Plasmonic core-satellite assemblies with high stability and yield (Conference Presentation)

NASA Astrophysics Data System (ADS)

Huang, Li-Ching; Lin, Tien-Hsin; Liu, Zhi-Yan; Chen, Jyun-Hao; Wang, Yi-Chen; Chen, Shiuan-Yeh

2016-09-01

Plasmonic structures are attractive due to their optical properties in the near-field and far-field. In the near-field, the enhanced field they generated strongly interacts with materials in proximity to the surface and even produces the quantum hybrid states in the strong coupling regime. In the far-field, the larger scattering cross section of plasmonic particles provides better contrast for tissue imaging. In addition, the strong absorption can generate substantial amount of heat for cancer cell elimination. These optical properties are usually engineered through tuning the size and morphology of individual nanoparticles by various chemical synthesis methods. The alternative way is to use coupled structure based on existing particles. The molecule-linked structure is a common way for 3D plasmonic materials. However, to produce a stable coupled structure in the solution phase is challenging. The formation of linkage between linker molecules is usually time-consuming and at low efficiency. Increasing the concentration of linker molecules may raise the reaction speed but also result in the random aggregation of particles. In this work, a polyelectrolyte coating is used to connect spherical nanoparticles of different sizes to form core-satellite assemblies (CSA). The coupled core-satellite structure is formed almost immediately after the solutions of two particles are mixed. The output efficiency is nearly 100%. The CSA is robust under the additional silica shell coating and strong laser illumination. The stability of this CSA is confirmed by the Raman spectra and this assembly can potentially be used as Raman tags.

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit*

PubMed Central

Suwa, Yoshiaki; Gu, Jianyou; Baranovskiy, Andrey G.; Babayeva, Nigar D.; Pavlov, Youri I.; Tahirov, Tahir H.

2015-01-01

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Å2. Importantly, in the structure of the p180C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes. PMID:25847248

Structural and mechanistic insights into nuclear transport and delivery of the critical pluripotency factor Oct4 to DNA.

PubMed

Okuyama, Takahide; Yamagishi, Ryosuke; Shimada, Jiro; Ikeda, Masaaki; Maruoka, Yayoi; Kaneko, Hiroki

2018-02-01

Oct4 is a master regulator of the induction and maintenance of cellular pluripotency, and has crucial roles in early stages of differentiation. It is the only factor that cannot be substituted by other members of the same protein family to induce pluripotency. However, although Oct4 nuclear transport and delivery to target DNA are critical events for reprogramming to pluripotency, little is known about the molecular mechanism. Oct4 is imported to the nucleus by the classical nuclear transport mechanism, which requires importin α as an adaptor to bind the nuclear localization signal (NLS). Although there are structures of complexes of the NLS of transcription factors (TFs) in complex with importin α, there are no structures available for complexes involving intact TFs. We have therefore modeled the structure of the complex of the whole Oct4 POU domain and importin α2 using protein-protein docking and molecular dynamics. The model explains how the Ebola virus VP24 protein has a negative effect on the nuclear import of STAT1 by importin α but not on Oct4, and how Nup 50 facilitates cargo release from importin α. The model demonstrates the structural differences between the Oct4 importin α bound and DNA bound crystal states. We propose that the 'expanded linker' between the two DNA-binding domains of Oct4 is an intrinsically disordered region and that its conformational changes have a key role in the recognition/binding to both DNA and importin α. Moreover, we propose that this structural change enables efficient delivery to DNA after release from importin α.

Dielectrophoretic trapping of multilayer DNA origami nanostructures and DNA origami-induced local destruction of silicon dioxide.

PubMed

Shen, Boxuan; Linko, Veikko; Dietz, Hendrik; Toppari, J Jussi

2015-01-01

DNA origami is a widely used method for fabrication of custom-shaped nanostructures. However, to utilize such structures, one needs to controllably position them on nanoscale. Here we demonstrate how different types of 3D scaffolded multilayer origamis can be accurately anchored to lithographically fabricated nanoelectrodes on a silicon dioxide substrate by DEP. Straight brick-like origami structures, constructed both in square (SQL) and honeycomb lattices, as well as curved "C"-shaped and angular "L"-shaped origamis were trapped with nanoscale precision and single-structure accuracy. We show that the positioning and immobilization of all these structures can be realized with or without thiol-linkers. In general, structural deformations of the origami during the DEP trapping are highly dependent on the shape and the construction of the structure. The SQL brick turned out to be the most robust structure under the high DEP forces, and accordingly, its single-structure trapping yield was also highest. In addition, the electrical conductivity of single immobilized plain brick-like structures was characterized. The electrical measurements revealed that the conductivity is negligible (insulating behavior). However, we observed that the trapping process of the SQL brick equipped with thiol-linkers tended to induce an etched "nanocanyon" in the silicon dioxide substrate. The nanocanyon was formed exactly between the electrodes, that is, at the location of the DEP-trapped origami. The results show that the demonstrated DEP-trapping technique can be readily exploited in assembling and arranging complex multilayered origami geometries. In addition, DNA origamis could be utilized in DEP-assisted deformation of the substrates onto which they are attached. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Propagation of Spin Information at the Supramolecular Scale through Heteroaromatic Linkers

NASA Astrophysics Data System (ADS)

Bellini, V.; Lorusso, G.; Candini, A.; Wernsdorfer, W.; Faust, T. B.; Timco, G. A.; Winpenny, R. E. P.; Affronte, M.

2011-06-01

We report an in-depth study on how spin information propagates at supramolecular scale through a family of heteroaromatic linkers. By density-functional theory calculations, we rationalize the behavior of a series of Cr7Ni dimers for which we are able to systematically change the aromatic linker thus tuning the strength of the magnetic interaction, as experimentally shown by low temperature micro-SQUID and specific heat measurements. We also predict a cos⁡2 dependence of the magnetic coupling on the twisting angle between the aromatic cycles in bicyclic linkers, a mechanism parallel to charge transport on similar systems [L. Venkataraman , Nature (London)NATUAS0028-0836 442, 904 (2006)10.1038/nature05037].

A "methyl extension" strategy for polyketide natural product linker site validation and its application to dictyostatin.

PubMed

Ho, Stephen; Sackett, Dan L; Leighton, James L

2015-11-11

An approach to the validation of linker strategies for polyketide natural products with few or no obvious handles for linker attachment, and its application to dictyostatin, are described. Analogues in which the C(6)- and C(12)-methyl groups were replaced by 4-azidobutyl groups were prepared and shown to retain the low nanomolar potency of dictyostatin. Further, conjugation of the C(6) analogue with a cyclooctyne resulted in only minor attenuations in potency. Together, these results shed light on the binding of dictyostatin to β-tubulin, establish a validated linker strategy for dictyostatin, and set the stage for the synthesis and study of dictyostatin conjugates.

Incorporation of Pyrazine and Bipyridine Linkers with High-Spin Fe(II) and Co(II) in a Metal–Organic Framework

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawamura, Airi; Greenwood, Arin R.; Filatov, Alexander S.

2017-02-27

A series of isoreticular metal organic frameworks (MOFs) of the formula M(BDC)(L) (M = Fe(II) or Co(II), BDC = 1,4-benzenedicarboxylate, L = pyrazine (pyz) or 4,4'-bipyridine (bipy)) has been synthesized and characterized by N-2 gas uptake Measurements, single crystal and powder X-ray diffraction, magnetometry, X-ray absorption spectroscopy, and Mossbauer spectroscopy. These studies indicate the formation of a permanently porous solid with high-spin Fe(II) and Co(II) centers that are weakly coupled, consistent with first-principles density functional theory calculations. This family of materials represents unusual examples of paramagnetic metal centers coordinated by linkers capable of mediating magnetic or electronic coupling in amore » porous framework. While only weak interactions are observed, the rigid 3D framework of the MOF dramatically impacts the properties of these materials when compared with close structural analogues.« less

Ammonia Adsorption and Co-adsorption with Water in HKUST-1: Spectroscopic Evidence for Cooperative Interactions

DOE PAGES

Nijem, Nour; Fürsich, Katrin; Bluhm, Hendrik; ...

2015-10-09

Ammonia interactions and competition with water at the interface of nanoporous metal organic framework thin films of HKUST-1 (Cu 3Btc 2 , Btc = 1,3,5-benzenedicarboxylate) are investigated with ambient pressure X-ray photoelectron spectroscopy (APXPS). In the absence of water, ammonia adsorption at the Cu 2+ metal center weakens the metal-linker bond of the framework. In the presence of water, due to the higher binding energy (adsorption strength) of ammonia compared to water, ammonia replaces water at the unsaturated Cu 2+ metal centers. The water molecules remaining in the pores are stabilized by hydrogen bonding to ammonia. Hydrogen bonding between themore » water and ammonia strengthens the metal-ammonia interaction due to cooperative interactions. Cooperative interactions result in a reduction in the metal center oxidation state facilitating linker replacement by other species explaining the previously reported structure degradation.« less

Ammonia Adsorption and Co-adsorption with Water in HKUST-1: Spectroscopic Evidence for Cooperative Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nijem, Nour; Fürsich, Katrin; Bluhm, Hendrik

Ammonia interactions and competition with water at the interface of nanoporous metal organic framework thin films of HKUST-1 (Cu 3Btc 2 , Btc = 1,3,5-benzenedicarboxylate) are investigated with ambient pressure X-ray photoelectron spectroscopy (APXPS). In the absence of water, ammonia adsorption at the Cu 2+ metal center weakens the metal-linker bond of the framework. In the presence of water, due to the higher binding energy (adsorption strength) of ammonia compared to water, ammonia replaces water at the unsaturated Cu 2+ metal centers. The water molecules remaining in the pores are stabilized by hydrogen bonding to ammonia. Hydrogen bonding between themore » water and ammonia strengthens the metal-ammonia interaction due to cooperative interactions. Cooperative interactions result in a reduction in the metal center oxidation state facilitating linker replacement by other species explaining the previously reported structure degradation.« less

Effects of the capping ligands, linkers and oxide surface on the electron injection mechanism of copper sulfide quantum dot-sensitized solar cells.

PubMed

Suárez, Javier Amaya; Plata, Jose J; Márquez, Antonio M; Sanz, Javier Fdez

2017-06-07

Quantum dot-sensitized solar cells, QDSCs, are a clean and effective alternative to fossil fuels to reduce CO 2 emissions. However, the different components that constitute the QDSCs and the difficulty of isolating experimentally their effects on the performance of the whole system slow down the development of more efficient devices. In this work, DFT calculations are combined with a bottom-up approach to differentiate the effect of each component on the electronic structure and absorption spectra. First, Cu 2 S QDs were built including a U parameter to effectively describe the localization of electrons. The effect of capping agents is addressed using ligands with different electron-donating/withdrawing groups. The role of linkers and their adsorption on the oxide surface are also examined. Finally, we propose a main indirect electron injection mechanism based on the position of the peaks of the spectra.

Design of monodisperse and well-defined polypeptide-based polyvalent inhibitors of anthrax toxin.

PubMed

Patke, Sanket; Boggara, Mohan; Maheshwari, Ronak; Srivastava, Sunit K; Arha, Manish; Douaisi, Marc; Martin, Jacob T; Harvey, Ian B; Brier, Matthew; Rosen, Tania; Mogridge, Jeremy; Kane, Ravi S

2014-07-28

The design of polyvalent molecules, presenting multiple copies of a specific ligand, represents a promising strategy to inhibit pathogens and toxins. The ability to control independently the valency and the spacing between ligands would be valuable for elucidating structure-activity relationships and for designing potent polyvalent molecules. To that end, we designed monodisperse polypeptide-based polyvalent inhibitors of anthrax toxin in which multiple copies of an inhibitory toxin-binding peptide were separated by flexible peptide linkers. By tuning the valency and linker length, we designed polyvalent inhibitors that were over four orders of magnitude more potent than the corresponding monovalent ligands. This strategy for the rational design of monodisperse polyvalent molecules may not only be broadly applicable for the inhibition of toxins and pathogens, but also for controlling the nanoscale organization of cellular receptors to regulate signaling and the fate of stem cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extremely stretchable thermosensitive hydrogels by introducing slide-ring polyrotaxane cross-linkers and ionic groups into the polymer network.

PubMed

Bin Imran, Abu; Esaki, Kenta; Gotoh, Hiroaki; Seki, Takahiro; Ito, Kohzo; Sakai, Yasuhiro; Takeoka, Yukikazu

2014-10-08

Stimuli-sensitive hydrogels changing their volumes and shapes in response to various stimulations have potential applications in multiple fields. However, these hydrogels have not yet been commercialized due to some problems that need to be overcome. One of the most significant problems is that conventional stimuli-sensitive hydrogels are usually brittle. Here we prepare extremely stretchable thermosensitive hydrogels with good toughness by using polyrotaxane derivatives composed of α-cyclodextrin and polyethylene glycol as cross-linkers and introducing ionic groups into the polymer network. The ionic groups help the polyrotaxane cross-linkers to become well extended in the polymer network. The resulting hydrogels are surprisingly stretchable and tough because the cross-linked α-cyclodextrin molecules can move along the polyethylene glycol chains. In addition, the polyrotaxane cross-linkers can be used with a variety of vinyl monomers; the mechanical properties of the wide variety of polymer gels can be improved by using these cross-linkers.

Extremely stretchable thermosensitive hydrogels by introducing slide-ring polyrotaxane cross-linkers and ionic groups into the polymer network

PubMed Central

Bin Imran, Abu; Esaki, Kenta; Gotoh, Hiroaki; Seki, Takahiro; Ito, Kohzo; Sakai, Yasuhiro; Takeoka, Yukikazu

2014-01-01

Stimuli-sensitive hydrogels changing their volumes and shapes in response to various stimulations have potential applications in multiple fields. However, these hydrogels have not yet been commercialized due to some problems that need to be overcome. One of the most significant problems is that conventional stimuli-sensitive hydrogels are usually brittle. Here we prepare extremely stretchable thermosensitive hydrogels with good toughness by using polyrotaxane derivatives composed of α-cyclodextrin and polyethylene glycol as cross-linkers and introducing ionic groups into the polymer network. The ionic groups help the polyrotaxane cross-linkers to become well extended in the polymer network. The resulting hydrogels are surprisingly stretchable and tough because the cross-linked α-cyclodextrin molecules can move along the polyethylene glycol chains. In addition, the polyrotaxane cross-linkers can be used with a variety of vinyl monomers; the mechanical properties of the wide variety of polymer gels can be improved by using these cross-linkers. PMID:25296246

Zinc chelation with hydroxamate in histone deacetylases modulated by water access to the linker binding channel.

PubMed

Wu, Ruibo; Lu, Zhenyu; Cao, Zexing; Zhang, Yingkai

2011-04-27

It is of significant biological interest and medical importance to develop class- and isoform-selective histone deacetylase (HDAC) modulators. The impact of the linker component on HDAC inhibition specificity has been revealed but is not understood. Using Born-Oppenheimer ab initio QM/MM MD simulations, a state-of-the-art approach to simulating metallo-enzymes, we have found that the hydroxamic acid remains to be protonated upon its binding to HDAC8, and thus disproved the mechanistic hypothesis that the distinct zinc-hydroxamate chelation modes between two HDAC subclasses come from different protonation states of the hydroxamic acid. Instead, our simulations suggest a novel mechanism in which the chelation mode of hydroxamate with the zinc ion in HDACs is modulated by water access to the linker binding channel. This new insight into the interplay between the linker binding and the zinc chelation emphasizes its importance and gives guidance regarding linker design for the development of new class-IIa-specific HDAC inhibitors.

Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells.

PubMed

Gao, Yunfeng; Foo, Yong Hwee; Winardhi, Ricksen S; Tang, Qingnan; Yan, Jie; Kenney, Linda J

2017-11-21

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.

The measles virus phosphoprotein interacts with the linker domain of STAT1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devaux, Patricia, E-mail: devaux.patricia@mayo.edu; Priniski, Lauren; Cattaneo, Roberto

2013-09-15

The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainlymore » to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. - Highlights: • Residue in the linker and SH2 domains of STAT1 are important for MV-P interaction. • Residue in the linker and SH2 domains of STAT1 are important for STAT1 phosphorylation. • Residues interferring with both functions have similar location on STAT1. • The viral P and V proteins may operate in concert to inhibit the JAK/STAT pathway.« less

On artifacts in single-molecule force spectroscopy

PubMed Central

Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

2015-01-01

In typical force spectroscopy experiments, a small biomolecule is attached to a soft polymer linker that is pulled with a relatively large bead or cantilever. At constant force, the total extension stochastically changes between two (or more) values, indicating that the biomolecule undergoes transitions between two (or several) conformational states. In this paper, we consider the influence of the dynamics of the linker and mesoscopic pulling device on the force-dependent rate of the conformational transition extracted from the time dependence of the total extension, and the distribution of rupture forces in force-clamp and force-ramp experiments, respectively. For these different experiments, we derive analytic expressions for the observables that account for the mechanical response and dynamics of the pulling device and linker. Possible artifacts arise when the characteristic times of the pulling device and linker become comparable to, or slower than, the lifetimes of the metastable conformational states, and when the highly anharmonic regime of stretched linkers is probed at high forces. We also revisit the problem of relating force-clamp and force-ramp experiments, and identify a linker and loading rate-dependent correction to the rates extracted from the latter. The theory provides a framework for both the design and the quantitative analysis of force spectroscopy experiments by highlighting, and correcting for, factors that complicate their interpretation. PMID:26540730

A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis.

PubMed

Nury, Catherine; Redeker, Virginie; Dautrey, Sébastien; Romieu, Anthony; van der Rest, Guillaume; Renard, Pierre-Yves; Melki, Ronald; Chamot-Rooke, Julia

2015-02-03

The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and N-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson's disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein-protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes.

Cross-linkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis.

PubMed

Descovich, Carlos Patino; Cortes, Daniel B; Ryan, Sean; Nash, Jazmine; Zhang, Li; Maddox, Paul S; Nedelec, Francois; Maddox, Amy Shaub

2018-03-01

Cell shape changes such as cytokinesis are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in nonmuscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal cross-linking by myosins and nonmotor cross-linkers, are thought to promote contractility. Here we examined how the abundance of motor and nonmotor cross-linkers affects the speed of cytokinetic furrowing. We built a minimal model to simulate contractile dynamics in the Caenorhabditis elegans zygote cytokinetic ring. This model predicted that intermediate levels of nonmotor cross-linkers are ideal for contractility; in vivo, intermediate levels of the scaffold protein anillin allowed maximal contraction speed. Our model also demonstrated a nonlinear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of nonmuscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, cytokinetic ring closure is promoted by moderate levels of both motor and nonmotor cross-linkers but attenuated by an over-abundance of motor and nonmotor cross-linkers. Together, our findings extend the growing appreciation for the roles of cross-linkers in cytokinesis and reveal that they not only drive but also brake cytoskeletal remodeling. © 2018 Descovich, Cortes, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma.

PubMed

Abushahba, Walid; Olabisi, Oyenike O; Jeong, Byeong-Seon; Boregowda, Rajeev K; Wen, Yu; Liu, Fang; Goydos, James S; Lasfar, Ahmed; Cohen-Solal, Karine A

2012-01-01

Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas.

Non-Canonical Smads Phosphorylation Induced by the Glutamate Release Inhibitor, Riluzole, through GSK3 Activation in Melanoma

PubMed Central

Jeong, Byeong-Seon; Boregowda, Rajeev K.; Wen, Yu; Liu, Fang; Goydos, James S.; Lasfar, Ahmed; Cohen-Solal, Karine A.

2012-01-01

Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas. PMID:23077590

Structural link between giant molybdenum oxide based ions and derived Keggin structure: modular assemblies based on the [BW11O39]9- ion and pentagonal {M'M5} units (M' = W; M = Mo,W).

PubMed

Leclerc-Laronze, Nathalie; Marrot, Jérôme; Thouvenot, René; Cadot, Emmanuel

2009-01-01

Linked to the Pentagon: The addition of molybdate to [HBW(11)O(39)](8-) ions leads to the formation of mixed pentagonal units {W(Mo(5))} and {W(WMo(4))} trapped as linkers in the resulting modular assemblies, thus establishing the first link between the conventional Keggin ion derivatives and the giant molybdenum oxide and keplerate ions.

Anisotropic Dye Adsorption and Anhydrous Proton Conductivity in Smectic Liquid Crystal Networks: The Role of Cross-Link Density, Order, and Orientation.

PubMed

Liang, Ting; van Kuringen, Huub P C; Mulder, Dirk J; Tan, Shuai; Wu, Yong; Borneman, Zandrie; Nijmeijer, Kitty; Schenning, Albertus P H J

2017-10-11

In this work, the decisive role of rigidity, orientation, and order in the smectic liquid crystalline network on the anisotropic proton and adsorbent properties is reported. The rigidity in the hydrogen-bonded polymer network has been altered by changing the cross-link density, the order by using different mesophases (smectic, nematic, and isotropic phases), whereas the orientation of the mesogens was controlled by alignment layers. Adding more cross-linkers improved the integrity of the polymer films. For the proton conduction, an optimum was found in the amount of cross-linker and the smectic organization results in the highest anhydrous proton conduction. The polymer films show anisotropic proton conductivity with a 54 times higher conductivity in the direction perpendicular to the molecular director. After a base treatment of the smectic liquid crystalline network, a nanoporous polymer film is obtained that also shows anisotropic adsorption of dye molecules and again straight smectic pores are favored over disordered pores in nematic and isotropic networks. The highly cross-linked films show size-selective adsorption of dyes. Low cross-linked materials do not show this difference due to swelling, which decreases the order and creates openings in the two-dimensional polymer layers. The latter is, however, beneficial for fast adsorption kinetics.

Architectural plasticity of AMPK revealed by electron microscopy and X-ray crystallography

PubMed Central

Ouyang, Yan; Zhu, Li; Li, Yifang; Guo, Miaomiao; Liu, Yang; Cheng, Jin; Zhao, Jing; Wu, Yi

2016-01-01

Mammalian AMP-activated protein kinase (AMPK) acts as an important sensor of cellular energy homeostasis related with AMP/ADP to ATP ratio. The overall architecture of AMPK has been determined in either homotrimer or monomer form by electron microscopy (EM) and X-ray crystallography successively. Accordingly proposed models have consistently revealed a key role of the α subunit linker in sensing adenosine nucleoside binding on the γ subunit and mediating allosteric regulation of kinase domain (KD) activity, whereas there are vital differences in orienting N-terminus of α subunit and locating carbohydrate-binding module (CBM) of β subunit. Given that Mg2+, an indispensable cofactor of AMPK was present in the EM sample preparation buffer however absent when forming crystals, here we carried out further reconstructions without Mg2+ to expectably inspect if this ion may contribute to this difference. However, no essential alteration has been found in this study compared to our early work. Further analyses indicate that the intra-molecular movement of the KD and CBM are most likely due to the flexible linkage of the disordered linkers with the rest portion as well as a contribution from the plasticity in the inter-molecular assembly mode, which might ulteriorly reveal an architectural complication of AMPK. PMID:27063142

Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?

PubMed Central

Gil-Moreno, Selene; Jiménez-Martí, Elena; Palacios, Òscar; Zerbe, Oliver; Dallinger, Reinhard; Capdevila, Mercè; Atrian, Sílvia

2015-01-01

Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metal ions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion. PMID:26703589

End-Group Effects on the Properties of PEG-co-PGA Hydrogels

PubMed Central

Bencherif, Sidi A.; Srinivasan, Abiraman; Sheehan, Jeffrey A.; Walker, Lynn M.; Gayathri, Chakicherla; Gil, Roberto; Hollinger, Jeffrey O.; Matyjaszewski, Krzysztof; Washburn, Newell R.

2009-01-01

A series of resorbable poly(ethylene glycol)-co-poly(glycolic acid) macromonomers have been synthesized with the chemistries from three different photopolymerizable end-groups (acrylates, methacrylates, and urethane methacrylates). The aim of the study is to examine the effects of the chemistry of the cross-linker group on the properties of photocross-linkable hydrogels. PEG-co-PGA (4KG5) hydrogels were prepared by photopolymerization with high vinyl group conversion as confirmed by 1H NMR spectroscopy using DOSY 1D pulse sequence. Our study reveals that the nature of end-groups in a moderately amphiphilic polymer can adjust the distribution and size of the micellar configuration in water leading to changes in the macroscopic structure of hydrogels. By varying the chemistry of the cross-linker group (diacrylates; DA, dimethacrylates; DM, and urethane dimethacrylates; UDM), we determined that the hydrophobocity of a single core polymer consisting of poly(glycolic acid) could be fine-tuned leading to significant variations in the mechanical, swelling, and degradation properties of the gels. In addition, the effects of cross-linker chemistry on cytotoxicity and proliferation were examined. Cytotoxicity assays showed that all the three types of hydrogels (4KG5 DA, DM, and UDM) were biocompatible and the introduction of RGD ligand enhanced cell adhesion. However, differences in gel properties and stability differentially affected the spreading and proliferation of myoblast C2C12 cells. PMID:19328754

Structure-based design of oxygen-linked macrocyclic kinase inhibitors: discovery of SB1518 and SB1578, potent inhibitors of Janus kinase 2 (JAK2) and Fms-like tyrosine kinase-3 (FLT3)

NASA Astrophysics Data System (ADS)

Poulsen, Anders; William, Anthony; Blanchard, Stéphanie; Lee, Angeline; Nagaraj, Harish; Wang, Haishan; Teo, Eeling; Tan, Evelyn; Goh, Kee Chuan; Dymock, Brian

2012-04-01

Macrocycles from our Aurora project were screened in a kinase panel and were found to be active on other kinase targets, mainly JAKs, FLT3 and CDKs. Subsequently these compounds became leads in our JAK2 project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. This residue is conserved in most CDKs resulting in potent pan CDK inhibition. One of the main project objectives was to achieve JAK2 potency with 100-fold selectivity against CDKs. Macrocycles with an ether linker have potent JAK2 activity with the ether oxygen forming a hydrogen bond to Ser936. A hydrogen bond to the equivalent residues of JAK3 and most CDKs cannot be formed resulting in good selectivity for JAK2 over JAK3 and CDKs. Further optimization of the macrocyclic linker and side chain increased JAK2 and FLT3 activity as well as improving DMPK properties. The selective JAK2/FLT3 inhibitor 11 (Pacritinib, SB1518) has successfully finished phase 2 clinical trials for myelofibrosis and lymphoma. Another selective JAK2/FLT3 inhibitor, 33 (SB1578), has entered phase 1 clinical development for the non-oncology indication rheumatoid arthritis.

Mode changes associated with oil droplet movement in solutions of gemini cationic surfactants.

PubMed

Banno, Taisuke; Miura, Shingo; Kuroha, Rie; Toyota, Taro

2013-06-25

Micrometer-sized self-propelled oil droplets in nonequilibrium systems have attracted much attention, since they form stable emulsions composed of oil, water, and surfactant which represent a primitive type of inanimate chemical machinery. In this work, we examined means of controlling the movement of oil droplets by studying the dynamics of n-heptyloxybenzaldehyde droplets in phosphate buffers containing alkanediyl-α,ω-bis(N-dodecyl-N,N-dimethylammonium bromide) (nG12) with either tetramethylene (4G12), octaethylene (8G12), or dodecamethylene (12G12) chains in the linker moiety. Significant differences in droplet dynamics were observed to be induced by changes in the linker structure of these gemini cationic surfactants. In a phosphate buffer containing 30 mM 4G12, self-propelled motion of droplets concurrent with the formation of molecular aggregates on their surfaces was observed, whereas the fusion of oil droplets was evident in both 8G12 and 12G12 solutions. We also determined that the surface activities and the extent of molecular self-assembly of the surfactants in phosphate buffer were strongly influenced by the alkyl chain length in the linker moiety. We therefore conclude that the surface activities of the gemini cationic surfactant have important effects on the oil-water interfacial tension of oil droplets and the formation of molecular aggregates and that both of these factors induce the unique movement of the droplets.

{Mo96La8} eggshell ring and self-assembly to {Mo132} Keplerate through Mo-blue intermediate, involved in UV-photolysis of [Mo7O24](6-)/carboxylic acid system at pH 4.

PubMed

Yamase, Toshihiro; Kumagai, Shun; Prokop, Petra V; Ishikawa, Eri; Tomsa, Adrian-Raul

2010-10-18

The prolonged UV-photolysis of aqueous solutions containing [Mo(7)O(24)](6-) and C(2)H(5)CO(2)H (as electron donor) at pH 3.9-4.1 generates the carboxylate-coordinated {Mo(132)} Keplerate (1a) isolated as a formamidinium/ammonium-mixed salt, [HC(NH(2))(2)](26)(NH(4))(28)[Mo(V)(60)Mo(VI)(72)O(372)(H(2)O)(48)(C(2)H(5)CO(2))(36)((i)C(3)H(7)CO(2))(6)]·16H(2)O (1), through the Mo-blue intermediate (2). The coordination of 2 to La(3+) gives rise to the formation of the chain structure of the C(2)-symmetric {Mo(96)La(8)} eggshell rings, formulated by H(22)[Mo(V)(20)Mo(VI)(76)O(301)(H(2)O)(29){La(H(2)O)(6)}(2)]{La(H(2)O)(5)}(6)]·54.5H(2)O (3). The eggshell-ring geometry results from the insertion of [Mo(VI)(2)O(7)(H(2)O)](2-) (spacer) into the equator outer ring of the wheel-shaped Mo-blue, and 10 {(Mo(VI))(Mo(VI)(5))} pentagonal subunits alternately above and below the equator outer ring are connected by eight La(3+) and two {Mo(VI)(2)} linkers within two inner rings. The neighboring eggshell rings are linked through two Mo-O-Mo bonds formed by dehydrative condensation between the {Mo(VI)(2)} linkers to result in the chain structure. Together with the results of the elemental analysis and IR, electronic absorption, (13)C NMR, and ESI-MS spectra for 2, the ring profile analysis of 3 let us identify 2 with a carbolylate-coordinated Mo-blue ring of high nuclearity. The Mo(VI)→Mo(V) photoreductive change of 2 to the 60-electron reduced Keplerate in the presence of C(2)H(5)CO(2)H involves both degradation of the outer ring and splitting of the binuclear linkers, which leads to the formation of [(Mo(VI))Mo(VI)(5)O(21)(H(2)O)(4)(carboxylate)](7-) pentagonal subunits and [Mo(V)(2)O(4)(carboxylate)](+)/[Mo(V)O(2)(carboxylate)(1/2)](0.5+)-mixed linkers for 1.

Elongated and substituted triazine-based tricarboxylic acid linkers for MOFs.

PubMed

Klinkebiel, Arne; Beyer, Ole; Malawko, Barbara; Lüning, Ulrich

2016-01-01

New triazine-based tricarboxylic acid linkers were prepared as elongated relatives of triazinetribenzoic acid (TATB). Additionally, functional groups (NO 2 , NH 2 , OMe, OH) were introduced for potential post-synthetic modification (PSM) of MOFs. Functionalized tris(4-bromoaryl)triazine "cores" ( 3a , 3b ) were obtained by unsymmetric trimerization mixing one equivalent of an acid chloride (OMe or NO 2 substituted) with two equivalents of an unsubstituted nitrile. Triple Suzuki coupling of the cores 3 with suitable phenyl- and biphenylboronic acid derivatives provided elongated tricarboxylic acid linkers as carboxylic acids 17 and 20 or their esters 16 and 19 . Reduction of the nitro group and cleavage of the methoxy group gave the respective amino and hydroxy-substituted triazine linkers.

Elongated and substituted triazine-based tricarboxylic acid linkers for MOFs

PubMed Central

Klinkebiel, Arne; Beyer, Ole; Malawko, Barbara

2016-01-01

New triazine-based tricarboxylic acid linkers were prepared as elongated relatives of triazinetribenzoic acid (TATB). Additionally, functional groups (NO2, NH2, OMe, OH) were introduced for potential post-synthetic modification (PSM) of MOFs. Functionalized tris(4-bromoaryl)triazine “cores” (3a,3b) were obtained by unsymmetric trimerization mixing one equivalent of an acid chloride (OMe or NO2 substituted) with two equivalents of an unsubstituted nitrile. Triple Suzuki coupling of the cores 3 with suitable phenyl- and biphenylboronic acid derivatives provided elongated tricarboxylic acid linkers as carboxylic acids 17 and 20 or their esters 16 and 19. Reduction of the nitro group and cleavage of the methoxy group gave the respective amino and hydroxy-substituted triazine linkers. PMID:28144293

Cystathionine β-Synthase (CBS) Domains 1 and 2 Fulfill Different Roles in Ionic Strength Sensing of the ATP-binding Cassette (ABC) Transporter OpuA*

PubMed Central

Karasawa, Akira; Erkens, Guus B.; Berntsson, Ronnie P.-A.; Otten, Renee; Schuurman-Wolters, Gea K.; Mulder, Frans A. A.; Poolman, Bert

2011-01-01

The cystathionine β-synthase module of OpuA in conjunction with an anionic membrane surface acts as a sensor of internal ionic strength, which allows the protein to respond to osmotic stress. We now show by chemical modification and cross-linking studies that CBS2-CBS2 interface residues are critical for transport activity and/or ionic regulation of transport, whereas CBS1 serves no functional role. We establish that Cys residues in CBS1, CBS2, and the nucleotide-binding domain are more accessible for cross-linking at high than low ionic strength, indicating that these domains undergo conformational changes when transiting between the active and inactive state. Structural analyses suggest that the cystathionine β-synthase module is largely unstructured. Moreover, we could substitute CBS1 by a linker and preserve ionic regulation of transport. These data suggest that CBS1 serves as a linker and the structured CBS2-CBS2 interface forms a hinge point for ionic strength-dependent rearrangements that are transmitted to the nucleotide-binding domain and thereby affect translocation activity. PMID:21878634

DOE Office of Scientific and Technical Information (OSTI.GOV)

Banerjee, Debasis; Borkowski, Lauren A.; Kim, Sun Jin

Two lithium-based metal-organic frameworks, Li{sub 2}(C{sub 14}H{sub 8}O{sub 4}) [Li{sub 2}(4,4'-BPDC) [1]; ULMOF-2, UL = ultralight; BPDC = biphenyldicarboxylate]; space group P2{sub 1}/c, a = 12.758(2) {angstrom}, b = 5.142(4) {angstrom}, c = 8.00(2) {angstrom}, {beta} = 97.23{sup o}, V = 520.6(14) {angstrom}{sup 3} and Li{sub 2}(C{sub 14}H{sub 8}O{sub 6}S) [Li{sub 2}(4,4'-SDB) [2]; ULMOF-3, UL = ultralight; SDB = sulfonyldibenzoate], space group P2{sub 1}/n, a = 5.5480(11) {angstrom}, b = 23.450(5) {angstrom}, c = 10.320(2) {angstrom}, {beta} = 96.47(3){sup o}, V = 1334.1(5) {angstrom}3, were synthesized. Compounds 1 and 2 were synthesized by solvothermal methods and were characterized using singlemore » crystal X-ray diffraction. Structure 1 consists of layers of two-dimensional antifluorite related LiO motif connected by BPDC linkers, whereas structure 2 is constructed by a combination of tetrameric lithium polyhedral clusters connected by the sulfonyldibenzoate linker. The frameworks are stable up to 575 and 500 C, respectively, under N{sub 2} atmosphere.« less

Structural studies and molecular dynamics simulations suggest a processive mechanism of exolytic lytic transglycosylase from Campylobacter jejuni.

PubMed

Vijayaraghavan, Jagamya; Kumar, Vijay; Krishnan, Nikhil P; Kaufhold, Ross T; Zeng, Ximin; Lin, Jun; van den Akker, Focco

2018-01-01

The bacterial soluble lytic transglycosylase (LT) breaks down the peptidoglycan (PG) layer during processes such as cell division. We present here crystal structures of the soluble LT Cj0843 from Campylobacter jejuni with and without bulgecin A inhibitor in the active site. Cj0843 has a doughnut shape similar but not identical to that of E. coli SLT70. The C-terminal catalytic domain is preceded by an L-domain, a large helical U-domain, a flexible linker, and a small N-terminal NU-domain. The flexible linker allows the NU-domain to reach over and complete the circular shape, using residues conserved in the Epsilonproteobacteria LT family. The inner surface of the Cj0843 doughnut is mostly positively charged including a pocket that has 8 Arg/Lys residues. Molecular dynamics simulations with PG strands revealed a potential functional role for this pocket in anchoring the negatively charged terminal tetrapeptide of the PG during several steps in the reaction including homing and aligning the PG strand for exolytic cleavage, and subsequent ratcheting of the PG strand to enhance processivity in degrading PG strands.

Interleukin 1 β-induced SMAD2/3 linker modifications are TAK1 dependent and delay TGFβ signaling in primary human mesenchymal stem cells.

PubMed

van den Akker, Guus G; van Beuningen, Henk M; Vitters, Elly L; Koenders, Marije I; van de Loo, Fons A; van Lent, Peter L; Blaney Davidson, Esmeralda N; van der Kraan, Peter M

2017-12-01

Chondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFβ) signaling. TGFβ binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 β (IL1β) on SMAD2/3 signaling dynamics and post-translational modifications. Co-stimulation of MSC with TGFβ and IL1β did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA 12 -luciferase reporter construct, was enhanced by co-stimulation of TGFβ and IL1β compared to TGFβ alone. Furthermore, IL1β stimulation induced CAGA 12 -luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFβ signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFβ, IL1β or TGFβ and IL1β stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2-4h was inhibited. A detailed time course analysis of IL1β-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFβ. Co-stimulation with both factors resulted in a similar kinetic profile as TGFβ alone. Nevertheless, IL1β did subtly alter TGFβ-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFβ target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1β on TGFβ signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1β-induced SMAD2 linker modifications and CAGA 12 -luciferase activity. TGFβ and IL1β signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFβ target genes were repressed by IL1β independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1β-induced TGFβ signal modulation. Copyright © 2017 Elsevier Inc. All rights reserved.

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

PubMed Central

2013-01-01

Background Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture. Results Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins. Conclusions The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production. PMID:24341331

Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools.

PubMed

Mandacaru, Samuel C; do Vale, Luis H F; Vahidi, Siavash; Xiao, Yiming; Skinner, Owen S; Ricart, Carlos A O; Kelleher, Neil L; de Sousa, Marcelo Valle; Konermann, Lars

2017-03-21

Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue α-helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high-resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. We also investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrate that the native complexes predominantly exist in a (MRJP1 4 apisimin 4 ) stoichiometry. Hydrogen/deuterium exchange MS reveals that MRJP1 within these complexes is extensively disordered in the range of residues 20-265. Marginally stable secondary structure (likely antiparallel β-sheet) exists around residues 266-432. These weakly structured regions interchange with conformers that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a "dimer of dimers" quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316 VLFFGLV 322 . Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP1 2 apisimin 1 ) stoichiometry. The information uncovered in this work will help pave the way toward a better understanding of the unique physiological role played by MRJP1 during queen differentiation.

Small C-terminal domain phosphatases dephosphorylate the regulatory linker regions of Smad2 and Smad3 to enhance transforming growth factor-beta signaling.

PubMed

Wrighton, Katharine H; Willis, Danielle; Long, Jianyin; Liu, Fang; Lin, Xia; Feng, Xin-Hua

2006-12-15

Transforming growth factor-beta (TGF-beta) controls a diverse set of cellular processes, and its canonical signaling is mediated via TGF-beta-induced phosphorylation of receptor-activated Smads (2 and 3) at the C-terminal SXS motif. We recently discovered that PPM1A can dephosphorylate Smad2/3 at the C-terminal SXS motif, implicating a critical role for phosphatases in regulating TGF-beta signaling. Smad2/3 activity is also regulated by phosphorylation in the linker region (and N terminus) by a variety of intracellular kinases, making it a critical platform for cross-talk between TGF-beta and other signaling pathways. Using a functional genomic approach, we identified the small C-terminal domain phosphatase 1 (SCP1) as a specific phosphatase for Smad2/3 dephosphorylation in the linker and N terminus. A catalytically inactive SCP1 mutant (dnSCP1) had no effect on Smad2/3 phosphorylation in vitro or in vivo. Of the other FCP/SCP family members SCP2 and SCP3, but not FCP1, could also dephosphorylate Smad2/3 in the linker/N terminus. Depletion of SCP1/2/3 enhanced Smad2/3 linker phosphorylation. SCP1 increased TGF-beta-induced transcriptional activity in agreement with the idea that phosphorylation in the Smad2/3 linker must be removed for a full transcriptional response. SCP1 overexpression also counteracts the inhibitory effect of epidermal growth factor on TGF-beta-induced p15 expression. Taken together, this work identifies the first example of a Smad2/3 linker phosphatase(s) and reveals an important new substrate for SCPs.

DOMMINO 2.0: integrating structurally resolved protein-, RNA-, and DNA-mediated macromolecular interactions

PubMed Central

Kuang, Xingyan; Dhroso, Andi; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

2016-01-01

Macromolecular interactions are formed between proteins, DNA and RNA molecules. Being a principle building block in macromolecular assemblies and pathways, the interactions underlie most of cellular functions. Malfunctioning of macromolecular interactions is also linked to a number of diseases. Structural knowledge of the macromolecular interaction allows one to understand the interaction’s mechanism, determine its functional implications and characterize the effects of genetic variations, such as single nucleotide polymorphisms, on the interaction. Unfortunately, until now the interactions mediated by different types of macromolecules, e.g. protein–protein interactions or protein–DNA interactions, are collected into individual and unrelated structural databases. This presents a significant obstacle in the analysis of macromolecular interactions. For instance, the homogeneous structural interaction databases prevent scientists from studying structural interactions of different types but occurring in the same macromolecular complex. Here, we introduce DOMMINO 2.0, a structural Database Of Macro-Molecular INteractiOns. Compared to DOMMINO 1.0, a comprehensive database on protein-protein interactions, DOMMINO 2.0 includes the interactions between all three basic types of macromolecules extracted from PDB files. DOMMINO 2.0 is automatically updated on a weekly basis. It currently includes ∼1 040 000 interactions between two polypeptide subunits (e.g. domains, peptides, termini and interdomain linkers), ∼43 000 RNA-mediated interactions, and ∼12 000 DNA-mediated interactions. All protein structures in the database are annotated using SCOP and SUPERFAMILY family annotation. As a result, protein-mediated interactions involving protein domains, interdomain linkers, C- and N- termini, and peptides are identified. Our database provides an intuitive web interface, allowing one to investigate interactions at three different resolution levels: whole subunit network, binary interaction and interaction interface. Database URL: http://dommino.org PMID:26827237

The roles of RIIbeta linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of Type IIbeta Protein Kinase A. A small angle X-ray and neutron scattering study

DOE PAGES

Blumenthal, Donald K.; Copps, Jeffrey; Smith-Nguyen, Eric V.; ...

2014-08-11

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. Moreover, the PKA holoenzyme is a tetramer (R 2:C 2), with a regulatory subunit homodimer (R 2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the typemore » IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1–280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. These results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA.« less

The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

PubMed

Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

2014-10-10

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular weight dependency of polyrotaxane-cross-linked polymer gel extensibility.

PubMed

Ohmori, Kana; Abu Bin, Imran; Seki, Takahiro; Liu, Chang; Mayumi, Koichi; Ito, Kohzo; Takeoka, Yukikazu

2016-12-11

This work investigates the influence of the molecular weight of polyrotaxane (PR) cross-linkers on the extensibility of polymer gels. The polymer gels, which were prepared using PR cross-linkers of three different molecular weights but the same number of cross-linking points per unit volume of gel, have almost the same Young's modulus. By contrast, the extensibility and rupture strength of the polymer gels are substantially increased with increasing molecular weight of the PR cross-linker.

Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group.

PubMed

Petrova, Natalya S; Chernikov, Ivan V; Meschaninova, Mariya I; Dovydenko, Iiya S; Venyaminova, Aliya G; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

2012-03-01

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5'-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.

Disruption of the IS6-AID linker affects voltage-gated calcium channel inactivation and facilitation.

PubMed

Findeisen, Felix; Minor, Daniel L

2009-03-01

Two processes dominate voltage-gated calcium channel (Ca(V)) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6-alpha-interaction domain (AID) linker provides a rigid connection between the pore and Ca(V)beta/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate Ca(V)1.2 (L-type) and Ca(V)2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt Ca(V)beta/I-II association sharply decelerate CDI and reduce a second Ca(2+)/CaM/Ca(V)alpha(1)-C-terminal-mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.

Disruption of the IS6-AID Linker Affects Voltage-gated Calcium Channel Inactivation and Facilitation

PubMed Central

Findeisen, Felix

2009-01-01

Two processes dominate voltage-gated calcium channel (CaV) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The CaVβ/CaVα1-I-II loop and Ca2+/calmodulin (CaM)/CaVα1–C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6–α-interaction domain (AID) linker provides a rigid connection between the pore and CaVβ/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate CaV1.2 (L-type) and CaV2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt CaVβ/I-II association sharply decelerate CDI and reduce a second Ca2+/CaM/CaVα1–C-terminal–mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, CaVβ and the IS6-AID linker, are essential for calcium-dependent modulation, and that both CaVβ-dependent and CaM-dependent components couple to the pore by a common mechanism requiring CaVβ and an intact IS6-AID linker. PMID:19237593

Computational modeling and in-vitro/in-silico correlation of phospholipid-based prodrugs for targeted drug delivery in inflammatory bowel disease

NASA Astrophysics Data System (ADS)

Dahan, Arik; Markovic, Milica; Keinan, Shahar; Kurnikov, Igor; Aponick, Aaron; Zimmermann, Ellen M.; Ben-Shabat, Shimon

2017-11-01

Targeting drugs to the inflamed intestinal tissue(s) represents a major advancement in the treatment of inflammatory bowel disease (IBD). In this work we present a powerful in-silico modeling approach to guide the molecular design of novel prodrugs targeting the enzyme PLA2, which is overexpressed in the inflamed tissues of IBD patients. The prodrug consists of the drug moiety bound to the sn-2 position of phospholipid (PL) through a carbonic linker, aiming to allow PLA2 to release the free drug. The linker length dictates the affinity of the PL-drug conjugate to PLA2, and the optimal linker will enable maximal PLA2-mediated activation. Thermodynamic integration and Weighted Histogram Analysis Method (WHAM)/Umbrella Sampling method were used to compute the changes in PLA2 transition state binding free energy of the prodrug molecule (ΔΔGtr) associated with decreasing/increasing linker length. The simulations revealed that 6-carbons linker is the optimal one, whereas shorter or longer linkers resulted in decreased PLA2-mediated activation. These in-silico results were shown to be in excellent correlation with experimental in-vitro data. Overall, this modern computational approach enables optimization of the molecular design of novel prodrugs, which may allow targeting the free drug specifically to the diseased intestinal tissue of IBD patients.

A unique mid-sequence linker used to multimerize the lipid-phosphatidylserine (PS) binding peptide-peptoid hybrid PPS1.

PubMed

Shukla, Satya Prakash; Manarang, Joseph C; Udugamasooriya, D Gomika

2017-09-08

Ligand multimerizations enhance the binding affinity towards cell surface biomarkers through their avidity effects. Typical linkers connect individual monomeric ligand moieties from one end (e.g., C- or N-terminus of a peptide) and exclusively target protein receptors. The lipid phosphatidylserine (PS) is normally present on the cytoplasmic side of the eukaryotic cell membrane, but in tumors and tumor endothelial cells, this negatively charged PS flips to the outer layer. We recently reported a PS binding peptide-peptoid hybrid (PPS1) that has distinct positively charged and hydrophobic residue-containing regions. The PPS1 monomer is inactive, and upon C-terminal dimerization (PPS1D1), it triggers cytotoxicity. In the current study, a unique series of PPS1 multimeric derivatives were synthesized by switching the linker from the C-terminus to an internal position. The unimportant fourth residue (N-lys) from the C-terminus was utilized to build the linker. The synthesis strategy was developed employing variations of (I) the linker size, (II) the number of positively charged residues, and (III) the number of hydrophobic regions. Cytotoxicity of these new derivatives on HCC4017 lung cancer cells showed that a minimum of two hydrophobic regions was important to retain the activity and that the shortest linker length was optimal for activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Periodic perturbations in Shaker K+ channel gating kinetics by deletions in the S3–S4 linker

PubMed Central

Gonzalez, Carlos; Rosenman, Eduardo; Bezanilla, Francisco; Alvarez, Osvaldo; Latorre, Ramon

2001-01-01

Upon depolarization positive charges contained in the transmembrane segment S4 of voltage-dependent channels are displaced from the cytoplasmic to the external milieu. This charge movement leads to channel opening. In Shaker K+ channels four positively charged arginines in the S4 domain are transferred from the internal to the external side of the channel during activation. The distance traveled by the S4 segment during activation is unknown, but large movements should be constrained by the S3–S4 linker. Constructing deletion mutants, we show that the activation time constant and the midpoint of the voltage activation curve of the Shaker K+ channel macroscopic currents becomes a periodic function of the S3–S4 linker length for linkers shorter than 7 aa residues. The periodicity is that typical of α-helices. Moreover, a linker containing only 3 aa is enough to recover the wild-type phenotype. The deletion method revealed the importance of the S3–S4 linker in determining the channel gating kinetics and indicated that the α-helical nature of S4 extends toward its N terminus. These results support the notion that a small displacement of the S4 segment suffices to displace the four gating charges involved in channel opening. PMID:11493701

Theoretical design of a new chimeric protein for the treatment of breast cancer

PubMed Central

Soleimani, Meysam; Mahnam, Karim; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

2016-01-01

p28 and NRC peptides are two anticancer peptides with various mechanisms have shown to be effective against breast cancer. Therefore, it seems that construction of a chimeric protein containing the two peptides might cause synergistic cytotoxic effects. However, since the two peptides bear opposite charges, production of a chimeric protein in which the two moieties do not intervene each other is difficult. In this study, our goal was to find a suitable peptide linker for the new chimeric protein in a manner that none of the peptides intervene the other’s function. We selected some linkers with different characteristics and lengths and created a small library of the chimeric proteins harboring these linkers. Homology modeling and molecular dynamic simulation revealed that (PA)5P and (EAAAK)3 linkers can separate the p28 and NRC peptides effectively. Thus, the chimeric protein linked with (PA)5P or (EAAAK)3 linkers might show synergistic and stronger anticancer effects than the separate peptide moieties because they could exert their cytotoxic effects freely which is not influenced by the other part. PMID:27499788

Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets.

PubMed

Quick, Quincy A

2018-01-26

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents.

Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets

PubMed Central

Quick, Quincy A.

2018-01-01

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents. PMID:29373494

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

PubMed

Yeliseev, Alexei; Zoubak, Lioudmila; Schmidt, Thomas G M

2017-03-01

Human cannabinoid receptor CB 2 belongs to the class A of G protein-coupled receptor (GPCR). CB 2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB 2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB 2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB 2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB 2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB 2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB 2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies. Published by Elsevier Inc.

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1*

PubMed Central

Musayev, Faik N.; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W.; Escalante, Carlos R.

2015-01-01

Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains. PMID:26370092

Tuning spin-spin interactions in radical dendrimers.

PubMed

Vidal-Gancedo, José; Lloveras, Vega; Liko, Flonja; Pinto, Luiz F; Muñoz-Gómez, Jose L

2018-05-10

Two generations of phosphorous dendrimers were synthesized and fully functionalized with TEMPO radicals via acrylamido or imino group linkers to evaluate the impact of the linker substitution on the radical-radical interactions. A drastic change in the way that the radicals interacted among them was observed by EPR and CV studies: while radicals in Gn-imino-TEMPO dendrimers presented a strong spin-spin interaction, in the Gn-acrylamido-TEMPO ones they acted mainly as independent radicals. This shows that these interactions could be tuned by the solely substitution of the radical linker, opening the perspective of controlling and modulating the extension of these interactions depending on each application. The chemical properties of the linker strongly influence the spin-spin exchange between pendant radicals. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Linker length dependent binding of a focal adhesion kinase derived peptide to the Src SH3-SH2 domains.

PubMed

Lindfors, Hanna E; Venkata, Bharat Somireddy; Drijfhout, Jan W; Ubbink, Marcellus

2011-02-18

The interaction between a peptide encompassing the SH3 and SH2 binding motifs of focal adhesion kinase (FAK) and the Src SH3-SH2 domains has been investigated with NMR spectroscopy and calorimetry. The binding to both motifs is anti-cooperative. Reduction of the long linker connecting the motifs does not lead to cooperativity. Short linkers that do not allow simultaneous intramolecular binding of the peptide to both motifs cause peptide-mediated dimerisation, even with a linker of only three amino acids. The role of the SH3 binding motif is discussed in view of the independent nature of the SH interactions. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker.

PubMed

Kawashima, Satoshi; Ikehata, Hiroki; Tada, Chihiro; Ogino, Tomohiro; Kakizaki, Hiromi; Ikeda, Mana; Fukushima, Hideto; Matsumiya, Masahiro

2016-01-20

Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa) and SmeChiB (56 kDa), were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2) are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (β/α)₈-fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.

Formaldehyde cross-linking and structural proteomics: Bridging the gap.

PubMed

Srinivasa, Savita; Ding, Xuan; Kast, Juergen

2015-11-01

Proteins are dynamic entities constantly moving and altering their structures based on their functions and interactions inside and outside the cell. Formaldehyde cross-linking combined with mass spectrometry can accurately capture interactions of these rapidly changing biomolecules while maintaining their physiological surroundings. Even with its numerous established uses in biology and compatibility with mass spectrometry, formaldehyde has not yet been applied in structural proteomics. However, formaldehyde cross-linking is moving toward analyzing tertiary structure, which conventional cross-linkers have already accomplished. The purpose of this review is to describe the potential of formaldehyde cross-linking in structural proteomics by highlighting its applications, characteristics and current status in the field. Copyright © 2015 Elsevier Inc. All rights reserved.

2008 Annual Report

DTIC Science & Technology

2008-01-01

TS(GGGGS)4AAA 25-aa flexible linker used extensively in recombinant antibody fragments L37 TSAG(EAAAK)6AAA 37-aa α-helical linker used for...used between the first two pathway enzymes and the L37 linker between the last two enzymes, thus making MgsA-L16- DkgA/FucO- L37 -GldA fusions. After...interestingly there is also an increase in the protein levels of these samples, which both appear in the soluble and insoluble fractions. Likewise the L37

Controlled molecular self-assembly of complex three-dimensional structures in soft materials.

PubMed

Huang, Changjin; Quinn, David; Suresh, Subra; Hsia, K Jimmy

2018-01-02

Many applications in tissue engineering, flexible electronics, and soft robotics call for approaches that are capable of producing complex 3D architectures in soft materials. Here we present a method using molecular self-assembly to generate hydrogel-based 3D architectures that resembles the appealing features of the bottom-up process in morphogenesis of living tissues. Our strategy effectively utilizes the three essential components dictating living tissue morphogenesis to produce complex 3D architectures: modulation of local chemistry, material transport, and mechanics, which can be engineered by controlling the local distribution of polymerization inhibitor (i.e., oxygen), diffusion of monomers/cross-linkers through the porous structures of cross-linked polymer network, and mechanical constraints, respectively. We show that oxygen plays a role in hydrogel polymerization which is mechanistically similar to the role of growth factors in tissue growth, and the continued growth of hydrogel enabled by diffusion of monomers/cross-linkers into the porous hydrogel similar to the mechanisms of tissue growth enabled by material transport. The capability and versatility of our strategy are demonstrated through biomimetics of tissue morphogenesis for both plants and animals, and its application to generate other complex 3D architectures. Our technique opens avenues to studying many growth phenomena found in nature and generating complex 3D structures to benefit diverse applications. Copyright © 2017 the Author(s). Published by PNAS.

Two separate interfaces between the voltage sensor and pore are required for the function of voltage-dependent K(+) channels.

PubMed

Lee, Seok-Yong; Banerjee, Anirban; MacKinnon, Roderick

2009-03-03

Voltage-dependent K(+) (Kv) channels gate open in response to the membrane voltage. To further our understanding of how cell membrane voltage regulates the opening of a Kv channel, we have studied the protein interfaces that attach the voltage-sensor domains to the pore. In the crystal structure, three physical interfaces exist. Only two of these consist of amino acids that are co-evolved across the interface between voltage sensor and pore according to statistical coupling analysis of 360 Kv channel sequences. A first co-evolved interface is formed by the S4-S5 linkers (one from each of four voltage sensors), which form a cuff surrounding the S6-lined pore opening at the intracellular surface. The crystal structure and published mutational studies support the hypothesis that the S4-S5 linkers convert voltage-sensor motions directly into gate opening and closing. A second co-evolved interface forms a small contact surface between S1 of the voltage sensor and the pore helix near the extracellular surface. We demonstrate through mutagenesis that this interface is necessary for the function and/or structure of two different Kv channels. This second interface is well positioned to act as a second anchor point between the voltage sensor and the pore, thus allowing efficient transmission of conformational changes to the pore's gate.

The Histone Database: an integrated resource for histones and histone fold-containing proteins

PubMed Central

Mariño-Ramírez, Leonardo; Levine, Kevin M.; Morales, Mario; Zhang, Suiyuan; Moreland, R. Travis; Baxevanis, Andreas D.; Landsman, David

2011-01-01

Eukaryotic chromatin is composed of DNA and protein components—core histones—that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins. Database URL: The Histone Sequence Database is freely available and can be accessed at http://research.nhgri.nih.gov/histones/. PMID:22025671

Structurally simplified biphenyl combretastatin A4 derivatives retain in vitro anti-cancer activity dependent on mitotic arrest

PubMed Central

Tarade, Daniel; Ma, Dennis; Pignanelli, Christopher; Mansour, Fadi; Simard, Daniel; van den Berg, Sean; Gauld, James; McNulty, James; Pandey, Siyaram

2017-01-01

The cis-stilbene, combretastatin A4 (CA4), is a potent microtubule targeting and vascular damaging agent. Despite promising results at the pre-clinical level and extensive clinical evaluation, CA4 has yet to be approved for therapeutic use. One impediment to the development of CA4 is an inherent conformational instability about the ethylene linker, which joins two aromatic rings. We have previously published preliminary data regarding structurally simplified biphenyl derivatives of CA4, lacking an ethylene linker, which retain anti-proliferative and pro-apoptotic activity, albeit at higher doses. Our current study provides a more comprehensive evaluation regarding the anti-proliferative and pro-apoptotic properties of biphenyl CA4 derivatives in both 2D and 3D cancerous and non-cancerous cell models. Computational analysis has revealed that cytotoxicity of CA4 and biphenyl analogues correlates with predicted tubulin affinity. Additional mechanistic evaluation of the biphenyl derivatives found that their anti-cancer activity is dependent on prolonged mitotic arrest, in a similar manner to CA4. Lastly, we have shown that cancer cells deficient in the extrinsic pathway of apoptosis experience delayed cell death following treatment with CA4 or analogues. Biphenyl derivatives of CA4 represent structurally simplified analogues of CA4, which retain a similar mechanism of action. The biphenyl analogues warrant in vivo examination to evaluate their potential as vascular damaging agents. PMID:28253265

How ligands improve the hydrothermal stability and affect the adsorption in the IRMOF family.

PubMed

Bellarosa, Luca; Gutiérrez-Sevillano, Juan J; Calero, Sofía; López, Núria

2013-10-28

Metal-Organic Frameworks are considered to be the next generation of sorbents both because of their synthetic versatility and high selectivity potential. In the first generation (IRMOF), the main drawback for commercial implementation is the lack of hydrothermal stability. Even if several studies have been conducted to elucidate the reasons behind their structural weakness in humid environments, how apparently small changes in the stoichiometry of the building units affect the stability of the lattice is still poorly understood. Using density functional theory and ab initio molecular dynamics we investigated the reason behind the different behaviour of several substituted IRMOF-1 structures. We show that hydrophilic variations in the organic linkers work as new basins of attraction for the incoming water molecules, thus depleting the water content at the metal center. To confirm this, we performed Monte Carlo simulations to provide insights into the adsorption energies and check the effectiveness of the adsorption sites in the substituted structures for a variety of polar and non-polar molecules. The results show that linker modification affects molecular adsorption and can improve the overall stability of the lattice redirecting water to the new sites in the case of hydrophilic units. Three key parameters have been singled out to rationalize this behaviour, and used to predict the favoured adsorption sites in the case of gas mixtures.

Structure-based design of Aurora A & B inhibitors

NASA Astrophysics Data System (ADS)

Poulsen, Anders; William, Anthony; Lee, Angeline; Blanchard, Stéphanie; Teo, Eeling; Deng, Weiping; Tu, Noah; Tan, Evelyn; Sun, Eric; Goh, Kay Lin; Ong, Wai Chung; Ng, Chee Pang; Goh, Kee Chuan; Bonday, Zahid

2008-12-01

The Aurora family of serine/threonine kinases are mitotic regulators involved in centrosome duplication, formation of the bipolar mitotic spindle and the alignment of the chromosomes along the spindle. These proteins are frequently overexpressed in tumor cells as compared to normal cells and are therefore potential therapeutic oncology targets. An Aurora A high throughput screen revealed a promising sub-micromolar indazole-benzimidazole lead. Modification of the benzimidazole portion of the lead to a C2 linker with a phenyl ring was proposed to achieve novelty. Docking revealed that a conjugated linker was optimal and the resulting compounds were equipotent with the lead. Further structure-guided optimization of substituents on the 5 & 6 position of the indazole led to single digit nanomolar potency. The homology between the Aurora A & Aurora B kinase domains is 71% but their binding sites only differ at residues 212 & 217 (Aurora A numbering). However interactions with only the latter residue may be used for obtaining selectivity. An analysis of published Aurora A and Aurora B X-ray structures reveals subtle differences in the shape of the binding sites. This was exploited by introduction of appropriately sized substituents in the 4 & 6 position of the indazole leading to Aurora B selective inhibitors. Finally we calculate the conformational energy penalty of the putative bioactive conformation of our inhibitors and show that this property correlates well with the Aurora A binding affinity.

Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env.

PubMed

Georgiev, Ivelin S; Joyce, M Gordon; Yang, Yongping; Sastry, Mallika; Zhang, Baoshan; Baxa, Ulrich; Chen, Rita E; Druz, Aliaksandr; Lees, Christopher R; Narpala, Sandeep; Schön, Arne; Van Galen, Joseph; Chuang, Gwo-Yu; Gorman, Jason; Harned, Adam; Pancera, Marie; Stewart-Jones, Guillaume B E; Cheng, Cheng; Freire, Ernesto; McDermott, Adrian B; Mascola, John R; Kwong, Peter D

2015-05-01

Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained. The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve structural and antigenic mimicry of mature Env spikes on virions. Here we show that replacement of the cleavage site between gp120 and gp41 in a lead soluble gp140 construct, BG505.SOSIP, with flexible linkers can result in molecules that do not require cleavage to fold efficiently into the mature closed state. Our results provide insights into the impact of cleavage on HIV-1 Env folding. In some contexts such as genetic immunization, optimized cleavage-independent soluble gp140 constructs may have utility over the parental BG505.SOSIP, as they would not require furin cleavage to achieve mimicry of mature Env spikes on virions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cerium-based metal organic frameworks with UiO-66 architecture: synthesis, properties and redox catalytic activity.

PubMed

Lammert, Martin; Wharmby, Michael T; Smolders, Simon; Bueken, Bart; Lieb, Alexandra; Lomachenko, Kirill A; Vos, Dirk De; Stock, Norbert

2015-08-14

A series of nine Ce(iv)-based metal organic frameworks with the UiO-66 structure containing linker molecules of different sizes and functionalities were obtained under mild synthesis conditions and short reaction times. Thermal and chemical stabilities were determined and a Ce-UiO-66-BDC/TEMPO system was successfully employed for the aerobic oxidation of benzyl alcohol.

Identifying a New Mechanism of HIV Core Formation | Center for Cancer Research

Cancer.gov

During the maturation of human immunodeficiency virus 1 (HIV-1), viral particles transition from a noninfectious form to an infectious one, and this conversion requires the cleavage of the HIV-1 Gag polyprotein. Gag is made up of three structural proteins—matrix (MA), capsid (CA), and nucleocapsid (NC)—connected by linkers. MA anchors Gag in the membrane, CA surrounds the

Molecular dynamics simulation unveils the conformational flexibility of the interdomain linker in the bacterial transcriptional regulator GabR from Bacillus subtilis bound to pyridoxal 5’-phosphate

PubMed Central

Narzi, Daniele; Guidoni, Leonardo

2017-01-01

GabR from Bacillus subtilis is a transcriptional regulator belonging to the MocR subfamily of the GntR regulators. The structure of the MocR regulators is characterized by the presence of two domains: i) a N-terminal domain, about 60 residue long, possessing the winged-Helix-Turn-Helix (wHTH) architecture with DNA recognition and binding capability; ii) a C-terminal domain (about 350 residue) folded as the pyridoxal 5’-phosphate (PLP) dependent aspartate aminotransferase (AAT) with dimerization and effector binding functions. The two domains are linked to each other by a peptide bridge. Although structural and functional characterization of MocRs is proceeding at a fast pace, virtually nothing is know about the molecular changes induced by the effector binding and on how these modifications influence the properties of the regulator. An extensive molecular dynamics simulation on the crystallographic structure of the homodimeric B. subtilis GabR has been undertaken with the aim to envisage the role and the importance of conformational flexibility in the action of GabR. Molecular dynamics has been calculated for the apo (without PLP) and holo (with PLP bound) forms of the GabR. A comparison between the molecular dynamics trajectories calculated for the two GabR forms suggested that one of the wHTH domain detaches from the AAT-like domain in the GabR PLP-bound form. The most evident conformational change in the holo PLP-bound form is represented by the rotation and the subsequent detachment from the subunit surface of one of the wHTH domains. The movement is mediated by a rearrangement of the linker connecting the AAT domain possibly triggered by the presence of the negative charge of the PLP cofactor. This is the second most significant conformational modification. The C-terminal section of the linker docks into the “active site” pocket and establish stabilizing contacts consisting of hydrogen-bonds, salt-bridges and hydrophobic interactions. PMID:29253008

Design of molecular beacons as signaling probes for adenosine triphosphate detection in cancer cells based on chemiluminescence resonance energy transfer.

PubMed

Zhang, Shusheng; Yan, Yameng; Bi, Sai

2009-11-01

In the present study, binary and triplex DNA molecular beacons, as signaling probes based on a luminol-H(2)O(2)-horseradish peroxidase (HRP)-fluorescein chemiluminescence resonance energy transfer (CRET) system and structure-switching aptamers for highly sensitive detection of small molecules, are developed using adenosine triphosphate (ATP) as a model analyte to demonstrate the generality of the strategy. This CRET process occurs from donor luminol to acceptor fluorescein, which is oxidized by H(2)O(2) and catalyzed by HRP. DNA aptamer for ATP is first attached on the surface of magnetic nanoparticles (MNPs). The cDNA linker has an extension that hybridizes with two other DNAs (LumAuNP-DNA and F-DNA) or three other DNAs (HRP-DNA, LumAuNP-DNA, and F-DNA) to fabricate CRET-BMBP-MNP or CRET-TMBP-MNP conjugates that provide the CRET signals. Thus, in the absence of ATP, when the MNPs are removed from the solution, they also take with them the linker DNA and the CRET signal probes, and no CRET signal can be detected. However, when ATP is introduced, a competition for the ATP aptamer between ATP and the cDNA linker occurs. As a result, CRET-BMBP and CRET-TMBP are forced to dissociate from the MNP surface based on the structure switching of the aptamer. The CRET signals are proportional to the concentration of ATP. In order to accelerate the rate of the aptamer structure-switching process, an invader DNA is introduced into the proposed strategy. The present CRET system provides a low detection limit of 1.1 x 10(-7) and 3.2 x 10(-7) M for ATP detection by BMBP and TMBP, respectively, which also exhibits a good selectivity for ATP detection. Sample assays of ATP in K562 leukemia cells and 4T1 breast cancer cells confirm the reliability and practicality of the protocol, which reveal a good prospect of this platform for biological sample analysis.

A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities*

PubMed Central

Lim, Siew Pheng; Koh, Jolene Hong Kiew; Seh, Cheah Chen; Liew, Chong Wai; Davidson, Andrew D.; Chua, Leng Shiew; Chandrasekaran, Ramya; Cornvik, Tobias C.; Shi, Pei-Yong; Lescar, Julien

2013-01-01

The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263–271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1–4 proteins and improves their de novo polymerase initiation activities by enhancing the turnover of the RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication. We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265–900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272–900) reported previously. This new orthorhombic crystal form (space group P21212) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed “preinitiation” conformation similar to the one that was captured previously in space group C2221 with one molecule per asymmetric unit. The structure reveals that residues 269–271 interact with the RdRp domain and suggests that residues 263–268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp. PMID:24025331

Luminescent MOFs comprising mixed tritopic linkers and Cd(II)/Zn(II) nodes for selective detection of organic nitro compounds and iodine capture

NASA Astrophysics Data System (ADS)

Rachuri, Yadagiri; Bisht, Kamal Kumar; Parmar, Bhavesh; Suresh, Eringathodi

2015-03-01

Two CPs {[Cd3(BTC)2(TIB)2(H2O)4].(H2O)2}n (1) and {[Zn3(BTC)2(TIB)2].(H2O)6}n (2) composed of tripodal linkers BTC (1,3,5-benzenetricarboxylate) and TIB (1,3,5-tris(imidazol-1-ylmethyl)benzene) were synthesized via solvothermal route and structurally characterized. Single crystal structural analysis reveals 1 possesses a novel 3D framework structure, whereas 2 represents a previously established compound. Owing to the d10 configuration of metal nodes and robust 3D frameworks, 1 and 2 exhibit excellent fluorescence properties which have been exploited to sense organic nitro compounds in vapor phase. Compound 1 demonstrates selective sensing of nitromethane over structurally similar methanol with ca. 70 and 43% fluorescence quenching in case of former and later. Similarly, 58% fluorescence quenching was observed in case of nitrobenzene over the structurally resembling toluene for which 30% quenching was observed. Compound 2 did not show any preference for nitro compounds and exhibited comparable fluorescence quenching when exposed to the vapors of nitro or other geometrically resembling organic molecules. Furthermore, adsorption experiments revealed that 1 and 2 can uptake 2.74 and 14.14 wt% molecular iodine respectively in vapor phase which can be released in organic solvents such as hexane and acetonitrile. The maximal iodine uptake in case of 1 and 2 corresponds to 0.15 and 0.80 molecules of iodine per formula unit of respective frameworks. Comprehensive structural description, thermal stability and luminescence behavior for both CPs has also been presented.

Cross-linked polyelectrolyte for direct methanol fuel cells applications based on a novel sulfonated cross-linker

NASA Astrophysics Data System (ADS)

Li, Mingyu; Zhang, Gang; Xu, Shuai; Zhao, Chengji; Han, Miaomiao; Zhang, Liyuan; Jiang, Hao; Liu, Zhongguo; Na, Hui

2014-06-01

A novel type of cross-linked proton exchange membrane of lower methanol permeation and high proton conductivity is prepared, based on a newly synthesized sulfonated cross-linker: carboxyl terminated benzimidazole trimer bearing sulfonic acid groups (s-BI). Compared to membranes cross-linked with non-sulfonated cross-linker (BI), SPEEK/s-BI-n membranes show higher IEC values and proton conductivities. Meanwhile, oxidative stability and mechanical property of SPEEK/s-BI-n membranes are obviously improved. Among SPEEK/s-BI-n membranes, SPEEK/s-BI-2 exhibits high proton conductivity, low swelling ratio (0.122 S cm-1 and 15.2% at 60 °C, respectively) and low methanol permeability coefficient. These results imply that the cross-linked membranes prepared with the newly sulfonated cross-linker are promising for the direct methanol fuel cells (DMFCs) application.

Thermoelectric efficiency of single-molecule junctions with long molecular linkers.

PubMed

Zimbovskaya, Natalya A

2018-06-18

We report results of theoretical studies of thermoelectric efficiency of single-molecule junctions with long molecular linkers. The linker is simulated by a chain of identical sites described using a tight-binding model. It is shown that thermoelectric figure of merit ZT strongly depends on the bridge length, being controlled by the lineshape of electron transmission function within the tunnel energy range corresponding to HOMO/LUMO transport channel. Using the adopted model we demonstrate that ZT may significantly increase as the linker lengthens, and that gateway states on the bridge (if any) may noticeably affect the length-dependent ZT. Temperature dependences of ZT for various bridge lengths are analyzed. It is shown that broad minima emerge in ZT versus temperature curves whose positions are controlled by the bridge lengths. © 2018 IOP Publishing Ltd.

Unveiling the Effects of Linker Substitution in Suzuki Coupling with Palladium Nanoparticles in Metal–Organic Frameworks [Unveiling the Effects of Linker Substitution in Suzuki Coupling Reaction with Palladium Nanoparticles in Metal–Organic Frameworks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Xinle; Zhang, Biying; Van Zeeland, Ryan

The establishment of structure–property relationships in heterogeneous catalysis is of prime importance but remains a formidable challenge. Metal–organic frameworks (MOFs) featuring excellent chemical tunability are emerging as an auspicious platform for the atomic-level control of heterogeneous catalysis. Herein, we encapsulate palladium nanoparticles (Pd NPs) in a series of isoreticular mixed-linker MOFs, and the obtained MOF-Pd NPs catalysts were used to unveil the electronic and steric effects of linker substitution on the activity of these catalysts in the Suzuki–Miyaura cross-coupling reactions. Significantly, m-6,6'-Me2bpy-MOF-Pd exhibits a remarkable enhancement in the activity compared to non-functionalized m-bpy-MOF-Pd and m-4,4'-Me 2bpy-MOF-Pd. This study unambiguously demonstratesmore » that the stereoelectronic properties of linker units are crucial to the catalytic activity of nanoparticles encapsulated in MOFs. More interestingly, the trend of activity change is consistent with our previous work on catalytic sites generated in situ from Pd(II) coordinated in MOFs bearing the same functional groups, which suggests that both MOF-Pd NPs and MOF-Pd(II) catalysts generate similar active centers during Suzuki–Miyaura coupling reactions. Lastly, this work paves a new avenue to the fabrication of advanced and tunable MOF-based catalysts through rational linker engineering.« less

Unveiling the Effects of Linker Substitution in Suzuki Coupling with Palladium Nanoparticles in Metal–Organic Frameworks [Unveiling the Effects of Linker Substitution in Suzuki Coupling Reaction with Palladium Nanoparticles in Metal–Organic Frameworks

DOE PAGES

Li, Xinle; Zhang, Biying; Van Zeeland, Ryan; ...

2018-01-18

The establishment of structure–property relationships in heterogeneous catalysis is of prime importance but remains a formidable challenge. Metal–organic frameworks (MOFs) featuring excellent chemical tunability are emerging as an auspicious platform for the atomic-level control of heterogeneous catalysis. Herein, we encapsulate palladium nanoparticles (Pd NPs) in a series of isoreticular mixed-linker MOFs, and the obtained MOF-Pd NPs catalysts were used to unveil the electronic and steric effects of linker substitution on the activity of these catalysts in the Suzuki–Miyaura cross-coupling reactions. Significantly, m-6,6'-Me2bpy-MOF-Pd exhibits a remarkable enhancement in the activity compared to non-functionalized m-bpy-MOF-Pd and m-4,4'-Me 2bpy-MOF-Pd. This study unambiguously demonstratesmore » that the stereoelectronic properties of linker units are crucial to the catalytic activity of nanoparticles encapsulated in MOFs. More interestingly, the trend of activity change is consistent with our previous work on catalytic sites generated in situ from Pd(II) coordinated in MOFs bearing the same functional groups, which suggests that both MOF-Pd NPs and MOF-Pd(II) catalysts generate similar active centers during Suzuki–Miyaura coupling reactions. Lastly, this work paves a new avenue to the fabrication of advanced and tunable MOF-based catalysts through rational linker engineering.« less

Trithiocarbonates: exploration of a new head group for HDAC inhibitors.

PubMed

Dehmel, Florian; Ciossek, Thomas; Maier, Thomas; Weinbrenner, Steffen; Schmidt, Beate; Zoche, Martin; Beckers, Thomas

2007-09-01

Inhibition of histone deacetylases class I/II enzymes is a new, promising approach for cancer therapy. In the present study, we disclose a new structural class of HDAC inhibitors with the trithiocarbonate motif. A clear structure-activity-relationship was obtained for the cap-linker motif and the putative Zn(2+) complexing head group. Selected analogs display potent inhibition of HDAC enzymatic activity and a cellular potency comparable to that of suberoylanilide hydroxamic acid (SAHA), recently approved for treatment of patients with advanced cutaneous T-cell lymphoma.

Voltage- and ATP-dependent structural rearrangements of the P2X2 receptor associated with the gating of the pore

PubMed Central

Keceli, Batu; Kubo, Yoshihiro

2014-01-01

P2X2 is an extracellular ATP-gated cation channel which has a voltage-dependent gating property even though it lacks a canonical voltage sensor. It is a trimer in which each subunit has two transmembrane helices and a large extracellular domain. The three inter-subunit ATP binding sites are linked to the pore forming transmembrane (TM) domains by β-strands. We analysed structural rearrangements of the linker strands between the ATP binding site and TM domains upon ligand binding and voltage change, electrophysiologically in Xenopus oocytes, using mutants carrying engineered thiol-modifiable cysteine residues. (1) We demonstrated that the double mutant D315C&I67C (at β-14 and β-1, respectively) shows a 2- to 4-fold increase in current amplitude after treatment with a reducing reagent, dithiothreitol (DTT). Application of the thiol-reactive metal Cd2+ induced current decline due to bond formation between D315C and I67C. This effect was not observed in wild type (WT) or in single point mutants. (2) Cd2+-induced current decline was analysed in hyperpolarized and depolarized conditions with different pulse protocols, and also in the presence and absence of ATP. (3) Current decline induced by Cd2+ could be clearly observed in the presence of ATP, but was not clear in the absence of ATP, showing a state-dependent modification. (4) In the presence of ATP, Cd2+ modification was significantly faster in hyperpolarized than in depolarized conditions, showing voltage-dependent structural rearrangements of the linker strands. (5) Experiments using tandem trimeric constructs (TTCs) with controlled number and position of mutations in the trimer showed that the bridging by Cd2+ between 315 and 67 was not intra- but inter-subunit. (6) Finally, we performed similar analyses of a pore mutant T339S, which makes the channel activation voltage insensitive. Cd2+ modification rates of T339S were similar in hyperpolarized and depolarized conditions. Taking these results together, we demonstrated that structural rearrangements of the linker region of the P2X2 receptor channel are induced not only by ligand binding but also by membrane potential change. PMID:25172943

Structure-based design of ligands for protein basic domains: Application to the HIV-1 Tat protein

NASA Astrophysics Data System (ADS)

Filikov, Anton V.; James, Thomas L.

1998-05-01

A methodology has been developed for designing ligands to bind a flexible basic protein domain where the structure of the domain is essentially known. It is based on an empirical binding free energy function developed for highly charged complexes and on Monte Carlo simulations in internal coordinates with both the ligand and the receptor being flexible. HIV-1 encodes a transactivating regulatory protein called Tat. Binding of the basic domain of Tat to TAR RNA is required for efficient transcription of the viral genome. The structure of a biologically active peptide containing the Tat basic RNA-binding domain is available from NMR studies. The goal of the current project is to design a ligand which will bind to that basic domain and potentially inhibit the TAR-Tat interaction. The basic domain contains six arginine and two lysine residues. Our strategy was to design a ligand for arginine first and then a superligand for the basic domain by joining arginine ligands with a linker. Several possible arginine ligands were obtained by searching the Available Chemicals Directory with DOCK 3.5 software. Phytic acid, which can potentially bind multiple arginines, was chosen as a building block for the superligand. Calorimetric binding studies of several compounds to methylguanidine and Arg-/Lys-containing peptides were performed. The data were used to develop an empirical binding free energy function for prediction of affinity of the ligands for the Tat basic domain. Modeling of the conformations of the complexes with both the superligand and the basic domain being flexible has been carried out via Biased Probability Monte Carlo (BPMC) simulations in internal coordinates (ICM 2.6 suite of programs). The simulations used parameters to ensure correct folding, i.e., consistent with the experimental NMR structure of a 25-residue Tat peptide, from a random starting conformation. Superligands for the basic domain were designed by joining together two molecules of phytic acid with peptidic and peptidomimetic linkers. The linkers were refined by varying the length and side chains of the linking residues, carrying out BPMC simulations, and evaluation of the binding free energy for the best energy conformation. The dissociation constant of the best ligand designed is estimated to be in the low- to mid-nanomolar range.

Galangin enhances TGF-β1-mediated growth inhibition by suppressing phosphorylation of threonine 179 residue in Smad3 linker region.

PubMed

Kwak, Mi-Kyung; Yang, Kyung-Min; Park, Jinah; Lee, Siyoung; Park, Yuna; Hong, Eunji; Sun, Eun Jin; An, Haein; Park, Sujin; Pang, Kyoungwha; Lee, Jihee; Kang, Jin Muk; Kim, Pyunggang; Ooshima, Akira; Kim, Seong-Jin

2017-12-16

Smad3 linker phosphorylation is a candidate target for several kinases that play important roles in cancer cell initiation, proliferation and progression. Also, Smad3 is an essential intracellular mediator of TGF-β1-induced transcriptional responses during carcinogenesis. Therefore, it is highly advantageous to identify and develop inhibitors targeting Smad3 linker phosphorylation for the treatment of cancers. Galangin (3,5,7-trihydroxyflavone) has been known to be an active flavonoid showing a cytotoxic effect on several cancer cells. However, the mechanism of action of galangin in various cancers remains unclear, and there has been no report concerning regulation of Smad3 phosphorylation by galangin. In the present study, we show that galangin significantly induced apoptosis and inhibited cell proliferation in the presence of TGF-β1 in both human prostate and pancreatic cancer cell lines. Particularly, galangin effectively inhibits phosphorylation of the Thr-179 site at Smad3 linker region through suppression of CDK4 phosphorylation. Thus, galangin can be a promising candidate as a selective inhibitor to suppress phosphorylation of Smad3 linker region. Copyright © 2017 Elsevier Inc. All rights reserved.

Transient inter-cellular polymeric linker.

PubMed

Ong, Siew-Min; He, Lijuan; Thuy Linh, Nguyen Thi; Tee, Yee-Han; Arooz, Talha; Tang, Guping; Tan, Choon-Hong; Yu, Hanry

2007-09-01

Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell-cell and cell-matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell-cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell-cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell-cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.

A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps.

PubMed

Bahira, Meriem; McCauley, Micah J; Almaqwashi, Ali A; Lincoln, Per; Westerlund, Fredrik; Rouzina, Ioulia; Williams, Mark C

2015-10-15

Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [μ-C4(cpdppz)2(phen)4Ru2](4+), which consists of two Ru(phen)2dppz(2+) moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Photoreactive, core-shell cross-linked/hollow microspheres prepared by delayed addition of cross-linker in dispersion polymerization for antifouling and immobilization of protein.

PubMed

Wang, Shengliu; Yue, Kai; Liu, Lianying; Yang, Wantai

2013-01-01

When dispersion polymerization of styrene (St) had run for 3h, after particle rapidly growing stage, 4,4'-dimethacryloyloxybenzophenone (DMABP) cross-linker was added to reaction system and photoreactive, core(PSt)-shell(Poly(St-co-DMABP)) particles with rich benzophenone (BP) groups on surface were prepared. Polymerization of DMABP could occurred mainly on the preformed core of PSt because its diffusion could be impeded by (1) compactness of particles formed at the moment of cross-linker addition (more than 80% of monomer had been consumed, particles were no longer fully swollen by monomer), (2) reduced polarity of continuous phase, and (3) immediate occurrence of cross-linking. Subsequently, photoreactive, cross-linked hollow particles were yielded by removal of uncross-linked core in THF. SEM and TEM observation demonstrated the formation of core-shell structure and improvement of shell thickness when DMABP content increased. UV-vis spectra analysis on polymer dissolved in THF indicated that there is no polymer of DMABP in core. FTIR spectra analysis and XPS measurement further revealed that BP component on particle surface was enriched when amount of DMABP increased. Finally, an anti-fouling polymer (poly (ethylene glycol), PEG) and protein of mouse IgG was immobilized on particle surface under UV irradiation, as confirmed by FTIR spectra analysis, SEM observation and TMB color reaction. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Preparation, structural analysis and bioactivity of ribonuclease A-albumin conjugate: tetra-conjugation or PEG as the linker.

PubMed

Li, Chunju; Lin, Qixun; Wang, Jun; Shen, Lijuan; Ma, Guanghui; Su, Zhiguo; Hu, Tao

2012-12-31

Ribonuclease A (RNase A) is a therapeutic enzyme with cytotoxic action against tumor cells. Its clinical application is limited by the short half-life and insufficient stability. Conjugation of albumin can overcome the limitation, whereas dramatically decrease the enzymatic activity of RNase A. Here, three strategies were proposed to prepare the RNase A-bovine serum albumin (BSA) conjugates. R-SMCC-B (a conjugate of four RNase A attached with one BSA) and R-PEG-B (a mono-conjugate) were prepared using Sulfo-SMCC (a short bifunctional linker) and mal-PEG-NHS (a bifunctional PEG), respectively. Mal-PEG-NHS and hexadecylamine (HDA) were used to prepare the mono-conjugate, R-HDA-B, where HDA was adopted to bind BSA. The PEG linker can elongate the proximity between RNase A and BSA. In contrast, four RNase A were closely located on BSA in R-SMCC-B. R-SMCC-B showed the lowest K(m) and the highest relative enzymatic activity and k(cat)/K(m) in the three conjugates. Presumably, the tetravalent interaction of RNase A in R-SMCC-B can increase the binding affinity to its substrate. In addition, the slow release of BSA from R-HDA-B may increase the enzymatic activity of R-HDA-B. Our study is expected to provide strategies to develop protein-albumin conjugate with high therapeutic potential. Copyright © 2012 Elsevier B.V. All rights reserved.

Lipophilic Polycation Vehicles Display High Plasmid DNA Delivery to Multiple Cell Types.

PubMed

Wu, Yaoying; Smith, Adam E; Reineke, Theresa M

2017-08-16

A class of cationic poly(alkylamidoamine)s (PAAAs) containing lipophilic methylene linkers were designed and examined as in vitro plasmid DNA (pDNA) delivery agents. The PAAAs were synthesized via step-growth polymerization between a diamine monomer and each of four different diacid chloride monomers with varying methylene linker lengths, including glutaryl chloride, adipoyl chloride, pimeloyl chloride, and suberoyl chloride, which served to systematically increase the lipophilicity of the polymers. The synthesized polymers successfully complexed with pDNA in reduced serum medium at N/P ratios of 5 and greater, resulting in polyplexes with hydrodynamic diameters of approximately 1 μm. These polyplexes were tested for in vitro transgene expression and cytotoxicity using HDFa (human dermal fibroblast), HeLa (human cervical carcinoma), HMEC (human mammary epithelial), and HUVEC (human umbilical vein endothelial) cells. Interestingly, select PAAA polyplex formulations were found to be more effective than Lipofectamine 2000 at promoting transgene expression (GFP) while maintaining comparable or higher cell viability. Transgene expression was highest in HeLa cells (∼90% for most formulations) and lowest in HDFa cells (up to ∼20%) as measured by GFP fluorescence. In addition, the cytotoxicity of PAAA polyplex formulations was significantly increased as the molecular weight, N/P ratio, and methylene linker length were increased. The PAAA vehicles developed herein provide a new delivery vehicle design strategy of displaying attributes of both polycations and lipids, which show promise as a tunable scaffold for refining the structure-activity-toxicity profiles for future genome editing studies.

Wall to membrane linkers, stretch activated channels, and the detection of tension, voltage, temperature, auxin, and pH

NASA Technical Reports Server (NTRS)

Pickard, B. G.

1992-01-01

Introduction. The higher plant is a heterogeneous, mechanically prestressed structure continually subject to shifting forces. When a cell grows in a plant at gravitropic equilibrium, it must create localized maxima of shear in walls of neighboring cells. Such mechanical stress and strain are likely detected in a variety of ways. However, tension-sensitive ion channels are of particular interest because it appears that they are elaborately evolved for sensory function. We hypothesize that 1) the patchy patterns of high shear are focused via wall-to-membrane linkers onto the plasma membrane, where 2) they are translated by mechanosensory cation channels into corresponding patterns of high cytosolic Ca2+, which 3) initiate local enhancement of wall expansion. Further, we hypothesize that the local promotion of enhancement is achieved at least in part by local intensification of auxin transport across the plasma membrane. By implication, when an organ is asymmetrically pressed, rubbed, or bent or when it is displaced in the gravitational field, the net asymmetry of shear stress occurring across the organ would lead to asymmetric redistribution of auxin and corrective asymmetric growth. We shall describe a representative mechanosensitive Ca(2+) -selective cation channel (MCaC) with susceptibilities to xenobiotics implicating it as a force transducer in thigmo- and gravitropism. Then, we shall consider whether a putative wall-to-membrane linker (WML) could be a key feature of the molecular architecture permitting the stress distributed in the wall system to be focused on the channels.

Equation Chapter 1 Section 1Sequence-To-Conformation Relationships of Disordered Regions Tethered to Folded Domains of Proteins.

PubMed

Mittal, Anuradha; Holehouse, Alex S; Cohan, Megan C; Pappu, Rohit V

2018-05-12

Intrinsically disordered proteins and regions (IDPs / IDRs) are characterized by well-defined sequence-to-conformation relationships (SCRs). These relationships refer to the sequence-specific preferences for average sizes, shapes, residue-specific secondary structure propensities, and amplitudes of multiscale conformational fluctuations. SCRs are discerned from the sequence-specific conformational ensembles of IDPs. A vast majority of IDPs are actually tethered to folded domains (FDs). This raises the question of whether or not SCRs inferred for IDPs are applicable to IDRs tethered to folded domains. Here, we use atomistic simulations based on a well-established forcefield paradigm and an enhanced sampling method to obtain comparative assessments of SCRs for thirteen archetypal IDRs modeled as autonomous units, as C-terminal tails connected to folded domains, and as linkers between pairs of folded domains. Our studies uncover a set of general observations regarding context-independent versus context-dependent SCRs of IDRs. SCRs are minimally perturbed upon tethering to folded domains if the IDRs are deficient in charged residues and for polyampholytic IDRs where the oppositely charged residues within the sequence of the IDR are separated into distinct blocks. In contrast, the interplay between IDRs and tethered folded domains has a significant modulatory effect on SCRs if the IDRs have intermediate fractions of charged residues or if they have sequence-intrinsic conformational preferences for canonical random coils. Our findings suggest that IDRs with context-independent SCRs might be independent evolutionary modules whereas IDRs with context-dependent intrinsic SCRs might co-evolve with the FDs to which they are tethered. Copyright © 2018. Published by Elsevier Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doan-Nguyen, Vicky V. T.; Subrahmanyam, Kota S.; Butala, Megan M.

Sulfur cathodes in conversion reaction batteries offer high gravimetric capacity but suffer from parasitic polysulfide shuttling. We demonstrate here that transition metal chalcogels of approximate formula MoS 3.4 achieve a high gravimetric capacity close to 600 mAh g –1 (close to 1000 mAh g –1 on a sulfur basis) as electrode materials for lithium-ion batteries. Transition metal chalcogels are amorphous and comprise polysulfide chains connected by inorganic linkers. The linkers appear to act as a “glue” in the electrode to prevent polysulfide shuttling. The Mo chalcogels function as electrodes in carbonate- and ether-based electrolytes, which further provides evidence of polysulfidemore » solubility not being a limiting issue. We employ X-ray spectroscopy and operando pair distribution function techniques to elucidate the structural evolution of the electrode. Raman and X-ray photoelectron spectroscopy track the chemical moieties that arise during the anion-redox-driven processes. As a result, we find the redox state of Mo remains unchanged across the electrochemical cycling and, correspondingly, the redox is anion-driven.« less

Modeling light-induced charge transfer dynamics across a metal-molecule-metal junction: Bridging classical electrodynamics and quantum dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Zixuan; Ratner, Mark A.; Seideman, Tamar, E-mail: t-seideman@northwestern.edu

2014-12-14

We develop a numerical approach for simulating light-induced charge transport dynamics across a metal-molecule-metal conductance junction. The finite-difference time-domain method is used to simulate the plasmonic response of the metal structures. The Huygens subgridding technique, as adapted to Lorentz media, is used to bridge the vastly disparate length scales of the plasmonic metal electrodes and the molecular system, maintaining accuracy. The charge and current densities calculated with classical electrodynamics are transformed to an electronic wavefunction, which is then propagated through the molecular linker via the Heisenberg equations of motion. We focus mainly on development of the theory and exemplify ourmore » approach by a numerical illustration of a simple system consisting of two silver cylinders bridged by a three-site molecular linker. The electronic subsystem exhibits fascinating light driven dynamics, wherein the charge density oscillates at the driving optical frequency, exhibiting also the natural system timescales, and a resonance phenomenon leads to strong conductance enhancement.« less

Recognition of the bacterial alarmone ZMP through long-distance association of two RNA subdomains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, Christopher P.; Ferré-D'Amaré, Adrian R.

The bacterial alarmone 5-aminoimidazole-4-carboxamide riboside 5'-triphosphate (AICAR triphosphate or ZTP), derived from the monophosphorylated purine precursor ZMP, accumulates during folate starvation. ZTP regulates genes involved in purine and folate metabolism through a cognate riboswitch. The linker connecting this riboswitch's two subdomains varies in length by over 100 nucleotides. In this paper, we report the cocrystal structure of the Fusobacterium ulcerans riboswitch bound to ZMP, which spans the two subdomains whose interface also comprises a pseudoknot and ribose zipper. The riboswitch recognizes the carboxamide oxygen of ZMP through an unprecedented inner-sphere coordination with a Mg 2+ ion. We show that themore » affinity of the riboswitch for ZMP is modulated by the linker length. Notably, ZMP can simultaneously bind to the two subdomains even when they are synthesized as separate RNAs. Finally, the ZTP riboswitch demonstrates how specific small-molecule binding can drive association of distant noncoding-RNA domains to regulate gene expression.« less

Design and synthesis of polyoxometalate-framework materials from cluster precursors

NASA Astrophysics Data System (ADS)

Vilà-Nadal, Laia; Cronin, Leroy

2017-10-01

Inorganic oxide materials are used in semiconductor electronics, ion exchange, catalysis, coatings, gas sensors and as separation materials. Although their synthesis is well understood, the scope for new materials is reduced because of the stability limits imposed by high-temperature processing and top-down synthetic approaches. In this Review, we describe the derivatization of polyoxometalate (POM) clusters, which enables their assembly into a range of frameworks by use of organic or inorganic linkers. Additionally, bottom-up synthetic approaches can be used to make metal oxide framework materials, and the features of the molecular POM precursors are retained in these structures. Highly robust all-inorganic frameworks can be made using metal-ion linkers, which combine molecular synthetic control without the need for organic components. The resulting frameworks have high stability, and high catalytic, photochemical and electrochemical activity. Conceptually, these inorganic oxide materials bridge the gap between zeolites and metal-organic frameworks (MOFs) and establish a new class of all-inorganic POM frameworks that can be designed using topological and reactivity principles similar to MOFs.

Chirality- and sequence-selective successive self-sorting via specific homo- and complementary-duplex formations

PubMed Central

Makiguchi, Wataru; Tanabe, Junki; Yamada, Hidekazu; Iida, Hiroki; Taura, Daisuke; Ousaka, Naoki; Yashima, Eiji

2015-01-01

Self-recognition and self-discrimination within complex mixtures are of fundamental importance in biological systems, which entirely rely on the preprogrammed monomer sequences and homochirality of biological macromolecules. Here we report artificial chirality- and sequence-selective successive self-sorting of chiral dimeric strands bearing carboxylic acid or amidine groups joined by chiral amide linkers with different sequences through homo- and complementary-duplex formations. A mixture of carboxylic acid dimers linked by racemic-1,2-cyclohexane bis-amides with different amide sequences (NHCO or CONH) self-associate to form homoduplexes in a completely sequence-selective way, the structures of which are different from each other depending on the linker amide sequences. The further addition of an enantiopure amide-linked amidine dimer to a mixture of the racemic carboxylic acid dimers resulted in the formation of a single optically pure complementary duplex with a 100% diastereoselectivity and complete sequence specificity stabilized by the amidinium–carboxylate salt bridges, leading to the perfect chirality- and sequence-selective duplex formation. PMID:26051291

Meso-oblate spheroids of thermal-stabile linker-free aggregates with size-tunable subunits for reversible lithium storage.

PubMed

Deng, Da; Lee, Jim Yang

2014-01-22

The organization of nanoscale materials as building units into extended structures with specific geometry and functional properties is a challenging endeavor. Hereby, an environmentally benign, simple, and scalable method for preparation of stable, linker-free, self-supported, high-order 3D meso-oblate spheroids of CuO nanoparticle aggregates with size-tunable building nanounits for reversible lithium-ion storage is reported. In contrast to traditional spherical nanoparticle aggregation, a unique oblate spheroid morphology is achieved. The formation mechanism of the unusual oblate spheroid of aggregated nanoparticles is proposed. When tested for reversible lithium ion storage, the unique 3D meso-oblate spheroids of CuO nanoparticle aggregate demonstrated highly improved electrochemical performance (around ∼600 mAh/g over 20 cycles), which could be ascribed to the nanoporous aggregated mesostructure with abundant crystalline imperfection. Furthermore, the size of building units can be controlled (12 and 21 nm were tested) to further improve their electrochemical performance.

Recognition of the bacterial alarmone ZMP through long-distance association of two RNA subdomains

DOE PAGES

Jones, Christopher P.; Ferré-D'Amaré, Adrian R.

2015-08-17

The bacterial alarmone 5-aminoimidazole-4-carboxamide riboside 5'-triphosphate (AICAR triphosphate or ZTP), derived from the monophosphorylated purine precursor ZMP, accumulates during folate starvation. ZTP regulates genes involved in purine and folate metabolism through a cognate riboswitch. The linker connecting this riboswitch's two subdomains varies in length by over 100 nucleotides. In this paper, we report the cocrystal structure of the Fusobacterium ulcerans riboswitch bound to ZMP, which spans the two subdomains whose interface also comprises a pseudoknot and ribose zipper. The riboswitch recognizes the carboxamide oxygen of ZMP through an unprecedented inner-sphere coordination with a Mg 2+ ion. We show that themore » affinity of the riboswitch for ZMP is modulated by the linker length. Notably, ZMP can simultaneously bind to the two subdomains even when they are synthesized as separate RNAs. Finally, the ZTP riboswitch demonstrates how specific small-molecule binding can drive association of distant noncoding-RNA domains to regulate gene expression.« less

Atomically dispersed metal sites in MOF-based materials for electrocatalytic and photocatalytic energy conversion.

PubMed

Liang, Zibin; Qu, Chong; Xia, Dingguo; Zou, Ruqiang; Xu, Qiang

2018-02-19

Metal sites play an essential role for both electrocatalytic and photocatalytic energy conversion applications. The highly ordered arrangements of the organic linkers and metal nodes and the well-defined pore structures of metal-organic frameworks (MOFs) make them ideal substrates to support atomically dispersed metal sites (ADMSs) located in their metal nodes, linkers, and pores. Besides, porous carbon materials doped with ADMSs can be derived from these ADMS-incorporated MOF precursors through controlled treatments. These ADMSs incorporated in pristine MOFs and MOF-derived carbon materials possess unique merits over the molecular or the bulk metal-based catalysts, bridging the gap between homogeneous and heterogeneous catalysts for energy conversion applications. In this review, recent progress and perspective of design and incorporation of ADMSs in pristine MOFs and MOF-derived materials for energy conversion applications are highlighted, which will hopefully promote further developments of advanced MOF-based catalysts in foreseeable future. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Long-range allosteric signaling in red light–regulated diguanylyl cyclases

PubMed Central

Gourinchas, Geoffrey; Etzl, Stefan; Göbl, Christoph; Vide, Uršula; Madl, Tobias; Winkler, Andreas

2017-01-01

Nature has evolved an astonishingly modular architecture of covalently linked protein domains with diverse functionalities to enable complex cellular networks that are critical for cell survival. The coupling of sensory modules with enzymatic effectors allows direct allosteric regulation of cellular signaling molecules in response to diverse stimuli. We present molecular details of red light–sensing bacteriophytochromes linked to cyclic dimeric guanosine monophosphate–producing diguanylyl cyclases. Elucidation of the first crystal structure of a full-length phytochrome with its enzymatic effector, in combination with the characterization of light-induced changes in conformational dynamics, reveals how allosteric light regulation is fine-tuned by the architecture and composition of the coiled-coil sensor-effector linker and also the central helical spine. We anticipate that consideration of molecular principles of sensor-effector coupling, going beyond the length of the characteristic linker, and the appreciation of dynamically driven allostery will open up new directions for the design of novel red light–regulated optogenetic tools. PMID:28275738

Combined Biology and Bioinformatics Approaches to Breast Cancer

DTIC Science & Technology

2006-04-01

In these experiments, LMO4 interacted with the MH1 and linker domains of Smad3 ; no interaction was found with the MH2 domain (Figure 4b). Figure 2...transcriptional response to TGFb by interacting with Smad proteins, and that both the MH1 and linker domains of Smad3 participate in the interaction. LMO4 can...LMO4 interacts with the MH1 and linker regions of Smad proteins. (a) Full-length, 35S-labeled Smad2, Smad3 , Smad4, Smad5, and Smad8 were incubated with

Chemotactic Signaling by Single-Chain Chemoreceptors

PubMed Central

Mowery, Patricia; Ames, Peter; Reiser, Rebecca H.; Parkinson, John S.

2015-01-01

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors. PMID:26709829