Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly

PubMed Central

Cook, Jeremy D.; Kondapalli, Kalyan C.; Rawat, Swati; Childs, William C.; Murugesan, Yogapriya; Dancis, Andrew; Stemmler, Timothy L.

2010-01-01

Frataxin, a conserved nuclear encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich’s ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two: Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone n the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to better understand the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry (ITC). Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into a Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly. PMID:20815377

Molecular Details of the Yeast Frataxin-Isu1 Interaction during Mitochondrial Fe-S Cluster Assembly

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

Cook, J.; Kondapalli, K; Rawat, S

2010-01-01

Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural modulemore » to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.« less

Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.

PubMed

Cook, Jeremy D; Kondapalli, Kalyan C; Rawat, Swati; Childs, William C; Murugesan, Yogapriya; Dancis, Andrew; Stemmler, Timothy L

2010-10-12

Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.

Architectonics: Design of Molecular Architecture for Functional Applications.

PubMed

Avinash, M B; Govindaraju, Thimmaiah

2018-02-20

The term architectonics has its roots in the architectural and philosophical (as early as 1600s) literature that refers to "the theory of structure" and "the structure of theory", respectively. The concept of architectonics has been adapted to advance the field of molecular self-assembly and termed as molecular architectonics. In essence, the methodology of organizing molecular units in the required and controlled configurations to develop advanced functional systems for materials and biological applications comprises the field of molecular architectonics. This concept of designing noncovalent systems enables to focus on different functional aspects of designer molecules for biological and nonbiological applications and also strengthens our efforts toward the mastery over the art of controlled molecular self-assemblies. Programming complex molecular interactions and assemblies for specific functions has been one of the most challenging tasks in the modern era. Meticulously ordered molecular assemblies can impart remarkable developments in several areas spanning energy, health, and environment. For example, the well-defined nano-, micro-, and macroarchitectures of functional molecules with specific molecular ordering possess potential applications in flexible electronics, photovoltaics, photonic crystals, microreactors, sensors, drug delivery, biomedicine, and superhydrophobic coatings, among others. The functional molecular architectures having unparalleled properties are widely evident in various designs of Nature. By drawing inspirations from Nature, intended molecular architectures can be designed and developed to harvest various functions, as there is an inexhaustible resource and scope. In this Account, we present exquisite designer molecules developed by our group and others with an objective to master the art of molecular recognition and self-assembly for functional applications. We demonstrate the tailor-ability of molecular self-assemblies by employing biomolecules like amino acids and nucleobases as auxiliaries. Naphthalenediimide (NDI), perylenediimide (PDI), and few other molecular systems serve as functional modules. The effects of stereochemistry and minute structural modifications in the molecular designs on the supramolecular interactions, and construction of self-assembled zero-dimensional (OD), one-dimensional (1D), and two-dimensional (2D) nano- and microarchitectures like particles, spheres, cups, bowls, fibers, belts, helical belts, supercoiled helices, sheets, fractals, and honeycomb-like arrays are discussed in extensive detail. Additionally, we present molecular systems that showcase the elegant designs of coassembly, templated assembly, hierarchical assembly, transient self-assembly, chiral denaturation, retentive helical memory, self-replication, supramolecular regulation, supramolecular speciation, supernon linearity, dynamic pathway complexity, supramolecular heterojunction, living supramolecular polymerization, and molecular machines. Finally, we describe the molecular engineering principles learnt over the years that have led to several applications, namely, organic electronics, self-cleaning, high-mechanical strength, and tissue engineering.

Self-assembly of a double-helical complex of sodium.

PubMed

Bell, T W; Jousselin, H

1994-02-03

Spontaneous self-organization of helical and multiple-helical molecular structures occurs on several levels in living organisms. Key examples are alpha-helical polypeptides, double-helical nucleic acids and helical protein structures, including F-actin, microtubules and the protein sheath of the tobacco mosaic virus. Although the self-assembly of double-helical transition-metal complexes bears some resemblance to the molecular organization of double-stranded DNA, selection between monohelical, double-helical and triple-helical structures is determined largely by the size and geometrical preference of the tightly bound metal. Here we present an example of double-helical assembly induced by the weaker and non-directional interactions of an alkali-metal ion with an organic ligand that is pre-organized into a coil. We have characterized the resulting complex by two-dimensional NMR and fast-atom-bombardment mass spectrometry. These results provide a step toward the creation of molecular tubes or ion channels consisting of intertwined coils.

Molecular Assembly of Clostridium botulinum progenitor M complex of type E.

PubMed

Eswaramoorthy, Subramaniam; Sun, Jingchuan; Li, Huilin; Singh, Bal Ram; Swaminathan, Subramanyam

2015-12-07

Clostridium botulinum neurotoxin (BoNT) is released as a progenitor complex, in association with a non-toxic-non-hemagglutinin protein (NTNH) and other associated proteins. We have determined the crystal structure of M type Progenitor complex of botulinum neurotoxin E [PTC-E(M)], a heterodimer of BoNT and NTNH. The crystal structure reveals that the complex exists as a tight, interlocked heterodimer of BoNT and NTNH. The crystal structure explains the mechanism of molecular assembly of the complex and reveals several acidic clusters at the interface responsible for association at low acidic pH and disassociation at basic/neutral pH. The similarity of the general architecture between the PTC-E(M) and the previously determined PTC-A(M) strongly suggests that the progenitor M complexes of all botulinum serotypes may have similar molecular arrangement, although the neurotoxins apparently can take very different conformation when they are released from the M complex.

Proteins from Multiple Metabolic Pathways Associate with Starch Biosynthetic Enzymes in High Molecular Weight Complexes: A Model for Regulation of Carbon Allocation in Maize Amyloplasts1[C][W][OA

PubMed Central

Hennen-Bierwagen, Tracie A.; Lin, Qiaohui; Grimaud, Florent; Planchot, Véronique; Keeling, Peter L.; James, Martha G.; Myers, Alan M.

2009-01-01

Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds. PMID:19168640

An {Fe60} tetrahedral cage: building nanoscopic molecular assemblies through cyanometallate and alkoxo linkers.

PubMed

Jiménez, J-R; Mondal, A; Chamoreau, L-M; Fertey, P; Tuna, F; Julve, M; Bousseksou, A; Lescouëzec, R; Lisnard, L

2016-11-08

A nanoscopic {Fe 60 } coordination cage (approximately 3 nm) was prepared by the self assembly of a partially blocked tricyanidoferrate(iii) complex and tris(alkoxo)-based iron(iii) coordination motifs. This cage is a rare example of a mixed cyanido/alkoxo-bridged high nuclearity complex and it exemplifies the great potential of this new synthetic route to generate uncommon molecular architectures using cyanometallates as metalloligands versus alkoxo-based polynuclear entities.

BLNK: molecular scaffolding through ‘cis’-mediated organization of signaling proteins

PubMed Central

Chiu, Christopher W.; Dalton, Mark; Ishiai, Masamichi; Kurosaki, Tomohiro; Chan, Andrew C.

2002-01-01

Assembly of intracellular macromolecular complexes is thought to provide an important mechanism to coordinate the generation of second messengers upon receptor activation. We have previously identified a B cell linker protein, termed BLNK, which serves such a scaffolding function in B cells. We demonstrate here that phosphorylation of five tyrosine residues within human BLNK nucleates distinct signaling effectors following B cell antigen receptor activation. The phosphorylation of multiple tyrosine residues not only amplifies PLCγ-mediated signaling but also supports ‘cis’-mediated interaction between distinct signaling effectors within a large molecular complex. These data demonstrate the importance of coordinate phosphorylation of molecular scaffolds, and provide insights into how assembly of macromolecular complexes is required for normal receptor function. PMID:12456653

Molecular assembly of Clostridium botulinum progenitor M complex of type E

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

Eswaramoorthy, Subramaniam; Sun, Jingchuan; Li, Huilin

2015-12-07

Clostridium botulinum neurotoxin (BoNT) is released as a progenitor complex, in association with a non-toxic-non-hemagglutinin protein (NTNH) and other associated proteins. We have determined the crystal structure of M type Progenitor complex of botulinum neurotoxin E [PTC-E(M)], a heterodimer of BoNT and NTNH. The crystal structure reveals that the complex exists as a tight, interlocked heterodimer of BoNT and NTNH. The crystal structure explains the mechanism of molecular assembly of the complex and reveals several acidic clusters at the interface responsible for association at low acidic pH and disassociation at basic/neutral pH. Furthermore, the similarity of the general architecture betweenmore » the PTC-E(M) and the previously determined PTC-A(M) strongly suggests that the progenitor M complexes of all botulinum serotypes may have similar molecular arrangement, although the neurotoxins apparently can take very different conformation when they are released from the M complex.« less

Spintronic characteristics of self-assembled neurotransmitter acetylcholine molecular complexes enable quantum information processing in neural networks and brain

NASA Astrophysics Data System (ADS)

Tamulis, Arvydas; Majauskaite, Kristina; Kairys, Visvaldas; Zborowski, Krzysztof; Adhikari, Kapil; Krisciukaitis, Sarunas

2016-09-01

Implementation of liquid state quantum information processing based on spatially localized electronic spin in the neurotransmitter stable acetylcholine (ACh) neutral molecular radical is discussed. Using DFT quantum calculations we proved that this molecule possesses stable localized electron spin, which may represent a qubit in quantum information processing. The necessary operating conditions for ACh molecule are formulated in self-assembled dimer and more complex systems. The main quantum mechanical research result of this paper is that the neurotransmitter ACh systems, which were proposed, include the use of quantum molecular spintronics arrays to control the neurotransmission in neural networks.

Engineering On-Surface Spin Crossover: Spin-State Switching in a Self-Assembled Film of Vacuum-Sublimable Functional Molecule.

PubMed

Kumar, Kuppusamy Senthil; Studniarek, Michał; Heinrich, Benoît; Arabski, Jacek; Schmerber, Guy; Bowen, Martin; Boukari, Samy; Beaurepaire, Eric; Dreiser, Jan; Ruben, Mario

2018-03-01

The realization of spin-crossover (SCO)-based applications requires study of the spin-state switching characteristics of SCO complex molecules within nanostructured environments, especially on surfaces. Except for a very few cases, the SCO of a surface-bound thin molecular film is either quenched or heavily altered due to: (i) molecule-surface interactions and (ii) differing intermolecular interactions in films relative to the bulk. By fabricating SCO complexes on a weakly interacting surface, the interfacial quenching problem is tackled. However, engineering intermolecular interactions in thin SCO active films is rather difficult. Here, a molecular self-assembly strategy is proposed to fabricate thin spin-switchable surface-bound films with programmable intermolecular interactions. Molecular engineering of the parent complex system [Fe(H 2 B(pz) 2 ) 2 (bpy)] (pz = pyrazole, bpy = 2,2'-bipyridine) with a dodecyl (C 12 ) alkyl chain yields a classical amphiphile-like functional and vacuum-sublimable charge-neutral Fe II complex, [Fe(H 2 B(pz) 2 ) 2 (C 12 -bpy)] (C 12 -bpy = dodecyl[2,2'-bipyridine]-5-carboxylate). Both the bulk powder and 10 nm thin films sublimed onto either quartz glass or SiO x surfaces of the complex show comparable spin-state switching characteristics mediated by similar lamellar bilayer like self-assembly/molecular interactions. This unprecedented observation augurs well for the development of SCO-based applications, especially in molecular spintronics. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging.

PubMed

Peck, Evan M; Battles, Paul M; Rice, Douglas R; Roland, Felicia M; Norquest, Kathryn A; Smith, Bradley D

2016-05-18

A programmable pre-assembly method is described and shown to produce near-infrared fluorescent molecular probes with tunable multivalent binding properties. The modular assembly process threads one or two copies of a tetralactam macrocycle onto a fluorescent PEGylated squaraine scaffold containing a complementary number of docking stations. Appended to the macrocycle periphery are multiple copies of a ligand that is known to target a biomarker. The structure and high purity of each threaded complex was determined by independent spectrometric methods and also by gel electrophoresis. Especially helpful were diagnostic red-shift and energy transfer features in the absorption and fluorescence spectra. The threaded complexes were found to be effective multivalent molecular probes for fluorescence microscopy and in vivo fluorescence imaging of living subjects. Two multivalent probes were prepared and tested for targeting of bone in mice. A pre-assembled probe with 12 bone-targeting iminodiacetate ligands produced more bone accumulation than an analogous pre-assembled probe with six iminodiacetate ligands. Notably, there was no loss in probe fluorescence at the bone target site after 24 h in the living animal, indicating that the pre-assembled fluorescent probe maintained very high mechanical and chemical stability on the skeletal surface. The study shows how this versatile pre-assembly method can be used in a parallel combinatorial manner to produce libraries of near-infrared fluorescent multivalent molecular probes for different types of imaging and diagnostic applications, with incremental structural changes in the number of targeting groups, linker lengths, linker flexibility, and degree of PEGylation.

Assembling oppositely charged lock and key responsive colloids: A mesoscale analog of adaptive chemistry

PubMed Central

Mihut, Adriana M.; Stenqvist, Björn; Lund, Mikael; Schurtenberger, Peter; Crassous, Jérôme J.

2017-01-01

We have seen a considerable effort in colloid sciences to copy Nature’s successful strategies to fabricate complex functional structures through self-assembly. This includes attempts to design colloidal building blocks and their intermolecular interactions, such as creating the colloidal analogs of directional molecular interactions, molecular recognition, host-guest systems, and specific binding. We show that we can use oppositely charged thermoresponsive particles with complementary shapes, such as spherical and bowl-shaped particles, to implement an externally controllable lock-and-key self-assembly mechanism. The use of tunable electrostatic interactions combined with the temperature-dependent size and shape and van der Waals interactions of these building blocks provides an exquisite control over the selectivity and specificity of the interactions and self-assembly process. The dynamic nature of the mechanism allows for reversibly cycling through various structures that range from weakly structured dense liquids to well-defined molecule-shaped clusters with different configurations through variations in temperature and ionic strength. We link this complex and dynamic self-assembly behavior to the relevant molecular interactions, such as screened Coulomb and van der Waals forces and the geometrical complementarity of the two building blocks, and discuss our findings in the context of the concepts of adaptive chemistry recently introduced to molecular systems. PMID:28929133

Understanding self-assembly of charged-neutral block copolymer (BCP) and surfactant complexes using molecular dynamics (MD) simulation

NASA Astrophysics Data System (ADS)

Goswami, Monojoy; Sumpter, Bobby; Kilbey, Michael

Here we report the formation of phase separated BCP-surfactant complexes resulting from the electrostatic self-assembly of charge-neutral block copolymers with oppositely charged surfactants. Complexation behaviors of oppositely charged polyelectrolytes has gained considerable attention in the field of soft condensed matter physics due to their potential application as functional nanomaterials for batteries, wastewater treatment and drug delivery systems. Numerous experiments have examined the self-assembled structures resulting from complexation of charge-neutral BCP and surfactants, however, there is a lack of comprehensive understanding at the fundamental level. To help bridge this gap, we use, MD simulations to study self-assembly and dynamics of the BCP-surfactant complex at the molecular level. Our results show an overcharging effect in BCPs with hydrophobic neutral blocks and a formation of core-shell colloidal structure. Hydrophilic neutral blocks, on the other hand, show stable, hairy colloidal structures with neutral blocks forming a loosely-bound, fuzzy outer layer. Our results qualitatively agree with previous SANS and SAXS experiments. This work was supported by the U.S. Department of Energy (DOE), Office of Basic Energy Sciences, Materials Science and Engineering Division.

Modeling the assembly order of multimeric heteroprotein complexes

PubMed Central

Esquivel-Rodriguez, Juan; Terashi, Genki; Christoffer, Charles; Shin, Woong-Hee

2018-01-01

Protein-protein interactions are the cornerstone of numerous biological processes. Although an increasing number of protein complex structures have been determined using experimental methods, relatively fewer studies have been performed to determine the assembly order of complexes. In addition to the insights into the molecular mechanisms of biological function provided by the structure of a complex, knowing the assembly order is important for understanding the process of complex formation. Assembly order is also practically useful for constructing subcomplexes as a step toward solving the entire complex experimentally, designing artificial protein complexes, and developing drugs that interrupt a critical step in the complex assembly. There are several experimental methods for determining the assembly order of complexes; however, these techniques are resource-intensive. Here, we present a computational method that predicts the assembly order of protein complexes by building the complex structure. The method, named Path-LzerD, uses a multimeric protein docking algorithm that assembles a protein complex structure from individual subunit structures and predicts assembly order by observing the simulated assembly process of the complex. Benchmarked on a dataset of complexes with experimental evidence of assembly order, Path-LZerD was successful in predicting the assembly pathway for the majority of the cases. Moreover, when compared with a simple approach that infers the assembly path from the buried surface area of subunits in the native complex, Path-LZerD has the strong advantage that it can be used for cases where the complex structure is not known. The path prediction accuracy decreased when starting from unbound monomers, particularly for larger complexes of five or more subunits, for which only a part of the assembly path was correctly identified. As the first method of its kind, Path-LZerD opens a new area of computational protein structure modeling and will be an indispensable approach for studying protein complexes. PMID:29329283

Modeling the assembly order of multimeric heteroprotein complexes.

PubMed

Peterson, Lenna X; Togawa, Yoichiro; Esquivel-Rodriguez, Juan; Terashi, Genki; Christoffer, Charles; Roy, Amitava; Shin, Woong-Hee; Kihara, Daisuke

2018-01-01

Protein-protein interactions are the cornerstone of numerous biological processes. Although an increasing number of protein complex structures have been determined using experimental methods, relatively fewer studies have been performed to determine the assembly order of complexes. In addition to the insights into the molecular mechanisms of biological function provided by the structure of a complex, knowing the assembly order is important for understanding the process of complex formation. Assembly order is also practically useful for constructing subcomplexes as a step toward solving the entire complex experimentally, designing artificial protein complexes, and developing drugs that interrupt a critical step in the complex assembly. There are several experimental methods for determining the assembly order of complexes; however, these techniques are resource-intensive. Here, we present a computational method that predicts the assembly order of protein complexes by building the complex structure. The method, named Path-LzerD, uses a multimeric protein docking algorithm that assembles a protein complex structure from individual subunit structures and predicts assembly order by observing the simulated assembly process of the complex. Benchmarked on a dataset of complexes with experimental evidence of assembly order, Path-LZerD was successful in predicting the assembly pathway for the majority of the cases. Moreover, when compared with a simple approach that infers the assembly path from the buried surface area of subunits in the native complex, Path-LZerD has the strong advantage that it can be used for cases where the complex structure is not known. The path prediction accuracy decreased when starting from unbound monomers, particularly for larger complexes of five or more subunits, for which only a part of the assembly path was correctly identified. As the first method of its kind, Path-LZerD opens a new area of computational protein structure modeling and will be an indispensable approach for studying protein complexes.

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

PubMed Central

Skinner, Owen S.; Do Vale, Luis H. F.; Catherman, Adam D.; Havugimana, Pierre C.; Valle de Sousa, Marcelo; Domont, Gilberto B.; Kelleher, Neil L.; Compton, Philip D.

2016-01-01

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses. PMID:26967310

Stepwise Construction of Heterobimetallic Cages by an Extended Molecular Library Approach.

PubMed

Hardy, Matthias; Struch, Niklas; Topić, Filip; Schnakenburg, Gregor; Rissanen, Kari; Lützen, Arne

2018-04-02

Two novel heterobimetallic complexes, a trigonal-bipyramidal and a cubic one, have been synthesized and characterized using the same C 3 -symmetric metalloligand, prepared by a simple subcomponent self-assembly strategy. Adopting the molecular library approach, we chose a mononuclear, preorganized iron(II) complex as the metalloligand capable of self-assembly into a trigonal-bipyramidal or a cubic aggregate upon coordination to cis-protected C 2 -symmetric palladium(II) or unprotected tetravalent palladium(II) ions, respectively. The trigonal-bipyramidal complex was characterized by NMR and UV-vis spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and single-crystal X-ray diffraction. The cubic structure was characterized by NMR and UV-vis spectroscopy and ESI-MS.

Static Electricity-Responsive Supramolecular Assembly.

PubMed

Jintoku, Hirokuni; Ihara, Hirotaka; Matsuzawa, Yoko; Kihara, Hideyuki

2017-12-01

Stimuli-responsive materials can convert between molecular scale and macroscopic scale phenomena. Two macroscopic static electricity-responsive phenomena based on nanoscale supramolecular assemblies of a zinc porphyrin derivative are presented. One example involves the movement of supramolecular assemblies in response to static electricity. The assembly of a pyridine (Py) complex of the above-mentioned derivative in cyclohexane is drawn to a positively charged material, whereas the assembly of a 3,5-dimethylpyridine complex is drawn to a negatively charged material. The second phenomenon involves the movement of a non-polar solvent in response to static electrical stimulation. A cyclohexane solution containing a small quantity of the Py-complexed assembly exhibited a strong movement response towards negatively charged materials. Based on spectroscopic measurements and electron microscope observations, it was revealed that the assembled formation generates the observed response to static electricity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Complex molecular assemblies at hand via interactive simulations.

PubMed

Delalande, Olivier; Férey, Nicolas; Grasseau, Gilles; Baaden, Marc

2009-11-30

Studying complex molecular assemblies interactively is becoming an increasingly appealing approach to molecular modeling. Here we focus on interactive molecular dynamics (IMD) as a textbook example for interactive simulation methods. Such simulations can be useful in exploring and generating hypotheses about the structural and mechanical aspects of biomolecular interactions. For the first time, we carry out low-resolution coarse-grain IMD simulations. Such simplified modeling methods currently appear to be more suitable for interactive experiments and represent a well-balanced compromise between an important gain in computational speed versus a moderate loss in modeling accuracy compared to higher resolution all-atom simulations. This is particularly useful for initial exploration and hypothesis development for rare molecular interaction events. We evaluate which applications are currently feasible using molecular assemblies from 1900 to over 300,000 particles. Three biochemical systems are discussed: the guanylate kinase (GK) enzyme, the outer membrane protease T and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors complex involved in membrane fusion. We induce large conformational changes, carry out interactive docking experiments, probe lipid-protein interactions and are able to sense the mechanical properties of a molecular model. Furthermore, such interactive simulations facilitate exploration of modeling parameters for method improvement. For the purpose of these simulations, we have developed a freely available software library called MDDriver. It uses the IMD protocol from NAMD and facilitates the implementation and application of interactive simulations. With MDDriver it becomes very easy to render any particle-based molecular simulation engine interactive. Here we use its implementation in the Gromacs software as an example. Copyright 2009 Wiley Periodicals, Inc.

Complex between triple helix of collagen and double helix of DNA in aqueous solution.

PubMed

Mrevlishvili, George M; Svintradze, David V

2005-06-01

We demonstrate in this paper that one example of a biologically important and molecular self-assembling complex system is a collagen-DNA ordered aggregate which spontaneously forms in aqueous solutions. Interaction between the collagen and the DNA leads to destruction of the hydration shell of the triple helix and stabilization of the double helix structure. From a molecular biology point of view this nano-scale self-assembling superstructure could increase the stability of DNA against the nucleases during collagen diseases and the growth of collagen fibrills in the presence of DNA.

DNA assisted self-assembly of PAMAM dendrimers.

PubMed

Mandal, Taraknath; Kumar, Mattaparthi Venkata Satish; Maiti, Prabal K

2014-10-09

We report DNA assisted self-assembly of polyamidoamine (PAMAM) dendrimers using all atom Molecular Dynamics (MD) simulations and present a molecular level picture of a DNA-linked PAMAM dendrimer nanocluster, which was first experimentally reported by Choi et al. (Nano Lett., 2004, 4, 391-397). We have used single stranded DNA (ssDNA) to direct the self-assembly process. To explore the effect of pH on this mechanism, we have used both the protonated (low pH) and nonprotonated (high pH) dendrimers. In all cases studied here, we observe that the DNA strand on one dendrimer unit drives self-assembly as it binds to the complementary DNA strand present on the other dendrimer unit, leading to the formation of a DNA-linked dendrimer dimeric complex. However, this binding process strongly depends on the charge of the dendrimer and length of the ssDNA. We observe that the complex with a nonprotonated dendrimer can maintain a DNA length dependent inter-dendrimer distance. In contrast, for complexes with a protonated dendrimer, the inter-dendrimer distance is independent of the DNA length. We attribute this observation to the electrostatic complexation of a negatively charged DNA strand with the positively charged protonated dendrimer.

Assembly of the MHC I peptide-loading complex determined by a conserved ionic lock-switch

PubMed Central

Blees, Andreas; Reichel, Katrin; Trowitzsch, Simon; Fisette, Olivier; Bock, Christoph; Abele, Rupert; Hummer, Gerhard; Schäfer, Lars V.; Tampé, Robert

2015-01-01

Salt bridges in lipid bilayers play a decisive role in the dynamic assembly and downstream signaling of the natural killer and T-cell receptors. Here, we describe the identification of an inter-subunit salt bridge in the membrane within yet another key component of the immune system, the peptide-loading complex (PLC). The PLC regulates cell surface presentation of self-antigens and antigenic peptides via molecules of the major histocompatibility complex class I. We demonstrate that a single salt bridge in the membrane between the transporter associated with antigen processing TAP and the MHC I-specific chaperone tapasin is essential for the assembly of the PLC and for efficient MHC I antigen presentation. Molecular modeling and all-atom molecular dynamics simulations suggest an ionic lock-switch mechanism for the binding of TAP to tapasin, in which an unfavorable uncompensated charge in the ER-membrane is prevented through complex formation. Our findings not only deepen the understanding of the interaction network within the PLC, but also provide evidence for a general interaction principle of dynamic multiprotein membrane complexes in immunity. PMID:26611325

Coevolutionary constraints in the sequence-space of macromolecular complexes reflect their self-assembly pathways.

PubMed

Mallik, Saurav; Kundu, Sudip

2017-07-01

Is the order in which biomolecular subunits self-assemble into functional macromolecular complexes imprinted in their sequence-space? Here, we demonstrate that the temporal order of macromolecular complex self-assembly can be efficiently captured using the landscape of residue-level coevolutionary constraints. This predictive power of coevolutionary constraints is irrespective of the structural, functional, and phylogenetic classification of the complex and of the stoichiometry and quaternary arrangement of the constituent monomers. Combining this result with a number of structural attributes estimated from the crystal structure data, we find indications that stronger coevolutionary constraints at interfaces formed early in the assembly hierarchy probably promotes coordinated fixation of mutations that leads to high-affinity binding with higher surface area, increased surface complementarity and elevated number of molecular contacts, compared to those that form late in the assembly. Proteins 2017; 85:1183-1189. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Subtle spectral effects accompanying the assembly of bacteriochlorophylls into cyclic light harvesting complexes revealed by high-resolution fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Rätsep, Margus; Pajusalu, Mihkel; Linnanto, Juha Matti; Freiberg, Arvi

2014-10-01

We have observed that an assembly of the bacteriochloropyll a molecules into B850 and B875 groups of cyclic bacterial light-harvesting complexes LH2 and LH1, respectively, results an almost total loss of the intra-molecular vibronic structure in the fluorescence spectrum, and simultaneously, an essential enhancement of its phonon sideband due to electron-phonon coupling. While the suppression of the vibronic coupling in delocalized (excitonic) molecular systems is predictable, as also confirmed by our model calculations, a boost of the electron-phonon coupling is rather unexpected. The latter phenomenon is explained by exciton self-trapping, promoted by mixing the molecular exciton states with charge transfer states between the adjacent chromophores in the tightly packed B850 and B875 arrangements. Similar, although less dramatic trends were noted for the light-harvesting complexes containing chlorophyll pigments.

Structural determinants of APOBEC3B non-catalytic domain for molecular assembly and catalytic regulation

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

Xiao, Xiao; Yang, Hanjing; Arutiunian, Vagan

The catalytic activity of human cytidine deaminase APOBEC3B (A3B) has been correlated with kataegic mutational patterns within multiple cancer types. The molecular basis of how the N-terminal non-catalytic CD1 regulates the catalytic activity and consequently, biological function of A3B remains relatively unknown. Here, we report the crystal structure of a soluble human A3B-CD1 variant and delineate several structural elements of CD1 involved in molecular assembly, nucleic acid interactions and catalytic regulation of A3B. We show that (i) A3B expressed in human cells exists in hypoactive high-molecular-weight (HMW) complexes, which can be activated without apparent dissociation into low-molecular-weight (LMW) species aftermore » RNase A treatment. (ii) Multiple surface hydrophobic residues of CD1 mediate the HMW complex assembly and affect the catalytic activity, including one tryptophan residue W127 that likely acts through regulating nucleic acid binding. (iii) One of the highly positively charged surfaces on CD1 is involved in RNA-dependent attenuation of A3B catalysis. (iv) Surface hydrophobic residues of CD1 are involved in heterogeneous nuclear ribonucleoproteins (hnRNPs) binding to A3B. The structural and biochemical insights described here suggest that unique structural features on CD1 regulate the molecular assembly and catalytic activity of A3B through distinct mechanisms.« less

The Origin of Hierarchical Structure Formation in Highly Grafted Symmetric Supramolecular Double-Comb Diblock Copolymers.

PubMed

Hofman, Anton H; Reza, Mehedi; Ruokolainen, Janne; Ten Brinke, Gerrit; Loos, Katja

2017-09-01

Involving supramolecular chemistry in self-assembling block copolymer systems enables design of complex macromolecular architectures that, in turn, could lead to complex phase behavior. It is an elegant route, as complicated and sensitive synthesis techniques can be avoided. Highly grafted double-comb diblock copolymers based on symmetric double hydrogen bond accepting poly(4-vinylpyridine)-block-poly(N-acryloylpiperidine) diblock copolymers and donating 3-nonadecylphenol amphiphiles are realized and studied systematically by changing the molecular weight of the copolymer. Double perpendicular lamellae-in-lamellae are formed in all complexes, independent of the copolymer molecular weight. Temperature-resolved measurements demonstrate that the supramolecular nature and ability to crystallize are responsible for the formation of such multiblock-like structures. Because of these driving forces and severe plasticization of the complexes in the liquid crystalline state, this supramolecular approach can be useful for steering self-assembly of both low- and high-molecular-weight block copolymer systems. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mesoscale Graphene-like Honeycomb Mono- and Multilayers Constructed via Self-Assembly of Coclusters.

PubMed

Hou, Xue-Sen; Zhu, Guo-Long; Ren, Li-Jun; Huang, Zi-Han; Zhang, Rui-Bin; Ungar, Goran; Yan, Li-Tang; Wang, Wei

2018-02-07

Honeycomb structure endows graphene with extraordinary properties. But could a honeycomb monolayer superlattice also be generated via self-assembly of colloids or nanoparticles? Here we report the construction of mono- and multilayer molecular films with honeycomb structure that can be regarded as self-assembled artificial graphene (SAAG). We construct fan-shaped molecular building blocks by covalently connecting two kinds of clusters, one polyoxometalate and four polyhedral oligomeric silsesquioxanes. The precise shape control enables these complex molecules to self-assemble into a monolayer 2D honeycomb superlattice that mirrors that of graphene but on the mesoscale. The self-assembly of the SAAG was also reproduced via coarse-grained molecular simulations of a fan-shaped building block. It revealed a hierarchical process and the key role of intermediate states in determining the honeycomb structure. Experimental images also show a diversity of bi- and trilayer stacking modes. The successful creation of SAAG and its stacks opens up prospects for the preparation of novel self-assembled nanomaterials with unique properties.

Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes

PubMed Central

Torreira, Eva; Jha, Sudhakar; López-Blanco, José R.; Arias-Palomo, Ernesto; Chacón, Pablo; Cañas, Cristina; Ayora, Sylvia; Dutta, Anindya; Llorca, Oscar

2008-01-01

Summary Pontin and reptin belong to the AAA+ family and they are essential for the structural integrity and catalytic activity of several chromatin remodeling complexes. They are also indispensable for the assembly of several ribonucleoprotein complexes, including telomerase. Here, we propose a structural model of the yeast pontin/reptin complex based on a cryo-electron microscopy reconstruction at 13 Å. Pontin/reptin hetero-dodecamers were purified from in vivo assembled complexes forming a double ring. Two rings interact through flexible domains projecting from each hexamer, constituting an atypical asymmetric form of oligomerization. These flexible domains and the AAA+ cores reveal significant conformational changes when compared to the crystal structure of human pontin that generate enlarged channels. This structure of endogenously assembled pontin/reptin complexes is different to previously described structures, suggesting that pontin and reptin could acquire distinct structural states to regulate their broad functions as molecular motors and scaffolds for nucleic acids and proteins. PMID:18940606

Self-Organization in Coordination-Driven Self-Assembly

PubMed Central

Northrop, Brian H.; Zheng, Yao-Rong; Chi, Ki-Whan; Stang, Peter J.

2009-01-01

Conspectus Self-assembly allows for the preparation of highly complex molecular and supramolecular systems from relatively simple starting materials. Typically, self-assembled supramolecules are constructed by combining complementary pairs of two highly symmetric molecular components, thus limiting the chances of forming unwanted side products. Combining asymmetric molecular components or multiple complementary sets of molecules in one complex mixture can produce myriad different ordered and disordered supramolecular assemblies. Alternatively, spontaneous self-organization phenomena can promote the formation of specific product(s) out of a collection of multiple possibilities. Self-organization processes are common throughout much of nature and are especially common in biological systems. Recently, researchers have studied self-organized self-assembly in purely synthetic systems. This Account describes our investigations of self-organization in the coordination-driven self-assembly of platinum(II)-based metallosupramolecules. The modularity of the coordination-driven approach to self-assembly has allowed us to systematically study a wide variety of different factors that can control the extent of supramolecular self-organization. In particular, we have evaluated the effects of the symmetry and polarity of ambidentate donor subunits, differences in geometrical parameters (e.g. the size, angularity, and dimensionality) of Pt(II)-based acceptors and organic donors, the influence of temperature and solvent, and the effects of intermolecular steric interactions and hydrophobic interactions on self-organization. Our studies have shown that the extent of self-organization in the coordination-driven self-assembly of both 2D polygons and 3D polyhedra ranges from no organization (a statistical mixture of multiple products), to amplified organization (wherein a particular product or products are favored over others), and all the way to the absolute self-organization of discrete supramolecular assemblies. In many cases, inputs such as dipolar interactions, steric interactions, and differences in the geometric parameters of subunits—used either alone or as multiple factors simultaneously—can achieve absolute self-organization of discrete supramolecules. We have also observed instances where self-organization is not absolute and varies in its deviation from statistical results. Steric interactions are particularly useful control factors for driving such amplified self-organization because they can be subtly tuned through small structural variations. Having the ability to fully understand and control the self-organization of complex mixtures into specific synthetic supramolecules can provide a better understanding of analogous processes in biological systems. Furthermore, self-organization may allow for the facile synthesis of complex multifunctional, multicomponent systems from simply mixing a collection of much simpler, judiciously designed individual molecular components. PMID:19555073

The Self-Assembly of Particles with Multipolar Interactions

DTIC Science & Technology

2004-01-01

the LATEX template in which this thesis has been written. I also thank Kevin Van Workum and Jack Douglas for contributing simulation work and some...of the computational expense of simulating such complex self-assembly systems at the molecular level and a desire to understand the self-assembly at...Dissertation directed by: Professor Wolfgang Losert Department of Physics In this thesis , we describe results from investigations of the self-assembly of

Metallosupramolecular Architectures Obtained from Poly-N-heterocyclic Carbene Ligands.

PubMed

Sinha, Narayan; Hahn, F Ekkehardt

2017-09-19

Over the past two decades, self-assembly of supramolecular architectures has become a field of intensive research due to the wide range of applications for the resulting assemblies in various fields such as molecular encapsulation, supramolecular catalysis, drug delivery, metallopharmaceuticals, chemical and photochemical sensing, and light-emitting materials. For these purposes, a large number of coordination-driven metallacycles and metallacages featuring different sizes and shapes have been prepared and investigated. Almost all of these are Werner-type coordination compounds where metal centers are coordinated by nitrogen and/or oxygen donors of polydentate ligands. With the evolving interest in the coordination chemistry of N-heterocyclic carbenes (NHCs), discrete supramolecular complexes held together by M-C NHC bonds have recently become of interest. The construction of such metallosupramolecular assemblies requires the synthesis of suitable poly-NHC ligands where the NHC donors form labile bonds with metal centers thus enabling the formation of the thermodynamically most stable reaction product. In organometallic chemistry, these conditions are uniquely met by the combination of poly-NHCs and silver(I) ions where the resulting assemblies also offer the possibility to generate new structures by transmetalation of the poly-NHC ligands to additional metal centers forming more stable C NHC -M bonds. Stable metallosupramolecular assemblies obtained from poly-NHC ligands feature special properties such as good solubility in many less polar organic solvents and the presence of the often catalyticlly active {M(NHC) n } moiety as building block. In this Account, we review recent developments in organometallic supramolecular architectures derived from poly-NHC ligands. We describe dinuclear (M = Ag I , Au I , Cu I ) tetracarbene complexes obtained from bis-NHC ligands with an internal olefin or two external coumarin pendants and their postsynthetic modification via a photochemically induced single or double [2 + 2] cycloaddition to form dinuclear tetracarbene complexes featuring cyclobutane units. Even three-dimensional cage-like structures can be prepared by this postsynthetic strategy. Cylinder-like trinuclear, tetranuclear, and hexanuclear (M = Ag I , Au I , Cu I , Hg II , Pd II ) complexes have been obtained from benzene-bridged tris-, tetrakis-, or hexakis-NHC ligands. These complexes resemble polynuclear assemblies obtained from related polydentate Werner-type ligands. Contrary to the Werner-type complexes, cylinder-like assemblies with three, four, or six silver(I) ions sandwiched in between two tris-, tetrakis-, or hexakis-NHC ligands undergo a facile transmetalation reaction to give the complexes featuring more stable M-C NHC bonds, normally with retention of the metallosupramolecular structure. This unique behavior of NHC-Ag + complexes allows the prepration of assemblies containing various metals from the poly-NHC silver(I) assemblies. Narcissistic self-sorting phenomena have also been observed for mixtures of selected poly-NHC ligands and silver(I) ions. Even a very early type of metallosupramolecular assembly, the tetranuclear molecular square, can be prepared from four bridging dicarbene ligands and four transition metal ions either by a stepwise assembly or by a single-step protocol. At this point, it appears that procedures for the synthesis of metallosupramolecular assemblies using polydentate Werner-type ligands and metal ions can be transferred to organometallic chemistry by using suitable poly-NHC ligands. The resulting structures feature stable M-C NHC bonds (with the exception of the labile C NHC -Ag + bond) when compared to M-N/M-O bonds in classical Werner-type complexes. The generally good solubility of the compounds and the presence of the often catalytically active {M(NHC) n } moiety make organometallic supramolecular complexes a promising new class of molecular hosts for catalytic transformations and encapsulation of selected substrates.

Molecular engineering of polymersome surface topology

PubMed Central

Ruiz-Pérez, Lorena; Messager, Lea; Gaitzsch, Jens; Joseph, Adrian; Sutto, Ludovico; Gervasio, Francesco Luigi; Battaglia, Giuseppe

2016-01-01

Biological systems exploit self-assembly to create complex structures whose arrangements are finely controlled from the molecular to mesoscopic level. We report an example of using fully synthetic systems that mimic two levels of self-assembly. We show the formation of vesicles using amphiphilic copolymers whose chemical nature is chosen to control both membrane formation and membrane-confined interactions. We report polymersomes with patterns that emerge by engineering interfacial tension within the polymersome surface. This allows the formation of domains whose topology is tailored by chemical synthesis, paving the avenue to complex supramolecular designs functionally similar to those found in viruses and trafficking vesicles. PMID:27152331

Photoresponse of supramolecular self-assembled networks on graphene-diamond interfaces.

PubMed

Wieghold, Sarah; Li, Juan; Simon, Patrick; Krause, Maximilian; Avlasevich, Yuri; Li, Chen; Garrido, Jose A; Heiz, Ueli; Samorì, Paolo; Müllen, Klaus; Esch, Friedrich; Barth, Johannes V; Palma, Carlos-Andres

2016-02-25

Nature employs self-assembly to fabricate the most complex molecularly precise machinery known to man. Heteromolecular, two-dimensional self-assembled networks provide a route to spatially organize different building blocks relative to each other, enabling synthetic molecularly precise fabrication. Here we demonstrate optoelectronic function in a near-to-monolayer molecular architecture approaching atomically defined spatial disposition of all components. The active layer consists of a self-assembled terrylene-based dye, forming a bicomponent supramolecular network with melamine. The assembly at the graphene-diamond interface shows an absorption maximum at 740 nm whereby the photoresponse can be measured with a gallium counter electrode. We find photocurrents of 0.5 nA and open-circuit voltages of 270 mV employing 19 mW cm(-2) irradiation intensities at 710 nm. With an ex situ calculated contact area of 9.9 × 10(2) μm(2), an incident photon to current efficiency of 0.6% at 710 nm is estimated, opening up intriguing possibilities in bottom-up optoelectronic device fabrication with molecular resolution.

Photoresponse of supramolecular self-assembled networks on graphene–diamond interfaces

PubMed Central

Wieghold, Sarah; Li, Juan; Simon, Patrick; Krause, Maximilian; Avlasevich, Yuri; Li, Chen; Garrido, Jose A.; Heiz, Ueli; Samorì, Paolo; Müllen, Klaus; Esch, Friedrich; Barth, Johannes V.; Palma, Carlos-Andres

2016-01-01

Nature employs self-assembly to fabricate the most complex molecularly precise machinery known to man. Heteromolecular, two-dimensional self-assembled networks provide a route to spatially organize different building blocks relative to each other, enabling synthetic molecularly precise fabrication. Here we demonstrate optoelectronic function in a near-to-monolayer molecular architecture approaching atomically defined spatial disposition of all components. The active layer consists of a self-assembled terrylene-based dye, forming a bicomponent supramolecular network with melamine. The assembly at the graphene-diamond interface shows an absorption maximum at 740 nm whereby the photoresponse can be measured with a gallium counter electrode. We find photocurrents of 0.5 nA and open-circuit voltages of 270 mV employing 19 mW cm−2 irradiation intensities at 710 nm. With an ex situ calculated contact area of 9.9 × 102 μm2, an incident photon to current efficiency of 0.6% at 710 nm is estimated, opening up intriguing possibilities in bottom-up optoelectronic device fabrication with molecular resolution. PMID:26911248

Integrating DNA strand-displacement circuitry with DNA tile self-assembly

PubMed Central

Zhang, David Yu; Hariadi, Rizal F.; Choi, Harry M.T.; Winfree, Erik

2013-01-01

DNA nanotechnology has emerged as a reliable and programmable way of controlling matter at the nanoscale through the specificity of Watson–Crick base pairing, allowing both complex self-assembled structures with nanometer precision and complex reaction networks implementing digital and analog behaviors. Here we show how two well-developed frameworks, DNA tile self-assembly and DNA strand-displacement circuits, can be systematically integrated to provide programmable kinetic control of self-assembly. We demonstrate the triggered and catalytic isothermal self-assembly of DNA nanotubes over 10 μm long from precursor DNA double-crossover tiles activated by an upstream DNA catalyst network. Integrating more sophisticated control circuits and tile systems could enable precise spatial and temporal organization of dynamic molecular structures. PMID:23756381

Toward a molecular programming language for algorithmic self-assembly

NASA Astrophysics Data System (ADS)

Patitz, Matthew John

Self-assembly is the process whereby relatively simple components autonomously combine to form more complex objects. Nature exhibits self-assembly to form everything from microscopic crystals to living cells to galaxies. With a desire to both form increasingly sophisticated products and to understand the basic components of living systems, scientists have developed and studied artificial self-assembling systems. One such framework is the Tile Assembly Model introduced by Erik Winfree in 1998. In this model, simple two-dimensional square 'tiles' are designed so that they self-assemble into desired shapes. The work in this thesis consists of a series of results which build toward the future goal of designing an abstracted, high-level programming language for designing the molecular components of self-assembling systems which can perform powerful computations and form into intricate structures. The first two sets of results demonstrate self-assembling systems which perform infinite series of computations that characterize computably enumerable and decidable languages, and exhibit tools for algorithmically generating the necessary sets of tiles. In the next chapter, methods for generating tile sets which self-assemble into complicated shapes, namely a class of discrete self-similar fractal structures, are presented. Next, a software package for graphically designing tile sets, simulating their self-assembly, and debugging designed systems is discussed. Finally, a high-level programming language which abstracts much of the complexity and tedium of designing such systems, while preventing many of the common errors, is presented. The summation of this body of work presents a broad coverage of the spectrum of desired outputs from artificial self-assembling systems and a progression in the sophistication of tools used to design them. By creating a broader and deeper set of modular tools for designing self-assembling systems, we hope to increase the complexity which is attainable. These tools provide a solid foundation for future work in both the Tile Assembly Model and explorations into more advanced models.

Interdependency of formation and localisation of the Min complex controls symmetric plastid division.

PubMed

Maple, Jodi; Møller, Simon G

2007-10-01

Plastid division represents a fundamental biological process essential for plant development; however, the molecular basis of symmetric plastid division is unclear. AtMinE1 plays a pivotal role in selection of the plastid division site in concert with AtMinD1. AtMinE1 localises to discrete foci in chloroplasts and interacts with AtMinD1, which shows a similar localisation pattern. Here, we investigate the importance of Min protein complex formation during the chloroplast division process. Dissection of the assembly of the Min protein complex and determination of the interdependency of complex assembly and localisation in planta allow us to present a model of the molecular basis of selection of the division site in plastids. Moreover, functional analysis of AtMinE1 in bacteria demonstrates the level of functional conservation and divergence of the plastidic MinE proteins.

TIMMDC1/C3orf1 functions as a membrane-embedded mitochondrial complex I assembly factor through association with the MCIA complex.

PubMed

Guarani, Virginia; Paulo, Joao; Zhai, Bo; Huttlin, Edward L; Gygi, Steven P; Harper, J Wade

2014-03-01

Complex I (CI) of the electron transport chain, a large membrane-embedded NADH dehydrogenase, couples electron transfer to the release of protons into the mitochondrial inner membrane space to promote ATP production through ATP synthase. In addition to being a central conduit for ATP production, CI activity has been linked to neurodegenerative disorders, including Parkinson's disease. CI is built in a stepwise fashion through the actions of several assembly factors. We employed interaction proteomics to interrogate the molecular associations of 15 core subunits and assembly factors previously linked to human CI deficiency, resulting in a network of 101 proteins and 335 interactions (edges). TIMMDC1, a predicted 4-pass membrane protein, reciprocally associated with multiple members of the MCIA CI assembly factor complex and core CI subunits and was localized in the mitochondrial inner membrane, and its depletion resulted in reduced CI activity and cellular respiration. Quantitative proteomics demonstrated a role for TIMMDC1 in assembly of membrane-embedded and soluble arms of the complex. This study defines a new membrane-embedded CI assembly factor and provides a resource for further analysis of CI biology.

Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

PubMed

Thomas, Jemima C; Matak-Vinkovic, Dijana; Van Molle, Inge; Ciulli, Alessio

2013-08-06

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

Multimeric Complexes among Ankyrin-Repeat and SOCS-box Protein 9 (ASB9), ElonginBC, and Cullin 5: Insights into the Structure and Assembly of ECS-type Cullin-RING E3 Ubiquitin Ligases

PubMed Central

2013-01-01

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC–Cullin–SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM–MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9–EloBC–Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM–MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems. PMID:23837592

Toward understanding of the mechanisms of Mediator function in vivo: Focus on the preinitiation complex assembly.

PubMed

Eychenne, Thomas; Werner, Michel; Soutourina, Julie

2017-01-01

Mediator is a multisubunit complex conserved in eukaryotes that plays an essential coregulator role in RNA polymerase (Pol) II transcription. Despite intensive studies of the Mediator complex, the molecular mechanisms of its function in vivo remain to be fully defined. In this review, we will discuss the different aspects of Mediator function starting with its interactions with specific transcription factors, its recruitment to chromatin and how, as a coregulator, it contributes to the assembly of transcription machinery components within the preinitiation complex (PIC) in vivo and beyond the PIC formation.

Nanoscale Structure and Interaction of Compact Assemblies of Carbon Nano-Materials

NASA Astrophysics Data System (ADS)

Timsina, Raju; Qiu, Xiangyun

Carbon-based nano-materials (CNM) are a diverse family of multi-functional materials under research and development world wide. Our work is further motivated by the predictive power of the physical understanding of the underlying structure-interaction-function relationships. Here we present results form recent studies of the condensed phases of several model CNMs in complexation with biologically derived molecules. Specifically, we employ X-ray diffraction (XRD) to determine nanoscale structures and use the osmotic stress method to quantify their interactions. The systems under investigation are dsDNA-dispersed carbon nanotubes (dsDNA-CNT), bile-salt-dispersed carbon nanotubes, and surfactant-assisted assemblies of graphene oxides. We found that salt and molecular crowding are both effective in condensing CNMs but the resultant structures show disparate phase behaviors. The molecular interactions driving the condensation/assembly sensitively depend on the nature of CNM complex surface chemistry and range from hydrophobic to electrostatic to entropic forces.

3D Printing of Molecular Models

ERIC Educational Resources Information Center

Gardner, Adam; Olson, Arthur

2016-01-01

Physical molecular models have played a valuable role in our understanding of the invisible nano-scale world. We discuss 3D printing and its use in producing models of the molecules of life. Complex biomolecular models, produced from 3D printed parts, can demonstrate characteristics of molecular structure and function, such as viral self-assembly,…

Quantitative self-assembly prediction yields targeted nanomedicines

NASA Astrophysics Data System (ADS)

Shamay, Yosi; Shah, Janki; Işık, Mehtap; Mizrachi, Aviram; Leibold, Josef; Tschaharganeh, Darjus F.; Roxbury, Daniel; Budhathoki-Uprety, Januka; Nawaly, Karla; Sugarman, James L.; Baut, Emily; Neiman, Michelle R.; Dacek, Megan; Ganesh, Kripa S.; Johnson, Darren C.; Sridharan, Ramya; Chu, Karen L.; Rajasekhar, Vinagolu K.; Lowe, Scott W.; Chodera, John D.; Heller, Daniel A.

2018-02-01

Development of targeted nanoparticle drug carriers often requires complex synthetic schemes involving both supramolecular self-assembly and chemical modification. These processes are generally difficult to predict, execute, and control. We describe herein a targeted drug delivery system that is accurately and quantitatively predicted to self-assemble into nanoparticles based on the molecular structures of precursor molecules, which are the drugs themselves. The drugs assemble with the aid of sulfated indocyanines into particles with ultrahigh drug loadings of up to 90%. We devised quantitative structure-nanoparticle assembly prediction (QSNAP) models to identify and validate electrotopological molecular descriptors as highly predictive indicators of nano-assembly and nanoparticle size. The resulting nanoparticles selectively targeted kinase inhibitors to caveolin-1-expressing human colon cancer and autochthonous liver cancer models to yield striking therapeutic effects while avoiding pERK inhibition in healthy skin. This finding enables the computational design of nanomedicines based on quantitative models for drug payload selection.

Constructing a molecular theory of self-assembly: Interplay of ideas from surfactants and block copolymers.

PubMed

Nagarajan, Ramanathan

2017-06-01

Low molecular weight surfactants and high molecular weight block copolymers display analogous self-assembly behavior in solutions and at interfaces, generating nanoscale structures of different shapes. Understanding the link between the molecular structure of these amphiphiles and their self-assembly behavior has been the goal of theoretical studies. Despite the analogies between surfactants and block copolymers, models predicting their self-assembly behavior have evolved independent of one another, each overlooking the molecular feature considered critical to the other. In this review, we focus on the interplay of ideas pertaining to surfactants and block copolymers in three areas of self-assembly. First, we show how improved free energy models have evolved by applying ideas from surfactants to block copolymers and vice versa, giving rise to a unitary theoretical framework and better predictive capabilities for both classes of amphiphiles. Second we show that even though molecular packing arguments are often used to explain aggregate shape transitions resulting from self-assembly, the molecular packing considerations are more relevant in the case of surfactants whereas free energy criteria are relevant for block copolymers. Third, we show that even though the surfactant and block copolymer aggregates are small nanostructures, the size differences between them is significant enough to make the interfacial effects control the solubilization of molecules in surfactant micelles while the bulk interactions control the solubilization in block copolymer micelles. Finally, we conclude by identifying recent theoretical progress in adapting the micelle model to a wide variety of self-assembly phenomena and the challenges to modeling posed by emerging novel classes of amphiphiles with complex biological, inorganic or nanoparticle moieties. Published by Elsevier B.V.

Creation of Functional Micro/Nano Systems through Top-down and Bottom-up Approaches

PubMed Central

Wong, Tak-Sing; Brough, Branden; Ho, Chih-Ming

2009-01-01

Mimicking nature’s approach in creating devices with similar functional complexity is one of the ultimate goals of scientists and engineers. The remarkable elegance of these naturally evolved structures originates from bottom-up self-assembly processes. The seamless integration of top-down fabrication and bottom-up synthesis is the challenge for achieving intricate artificial systems. In this paper, technologies necessary for guided bottom-up assembly such as molecular manipulation, molecular binding, and the self assembling of molecules will be reviewed. In addition, the current progress of synthesizing mechanical devices through top-down and bottom-up approaches will be discussed. PMID:19382535

Meshing complex macro-scale objects into self-assembling bricks

PubMed Central

Hacohen, Adar; Hanniel, Iddo; Nikulshin, Yasha; Wolfus, Shuki; Abu-Horowitz, Almogit; Bachelet, Ido

2015-01-01

Self-assembly provides an information-economical route to the fabrication of objects at virtually all scales. However, there is no known algorithm to program self-assembly in macro-scale, solid, complex 3D objects. Here such an algorithm is described, which is inspired by the molecular assembly of DNA, and based on bricks designed by tetrahedral meshing of arbitrary objects. Assembly rules are encoded by topographic cues imprinted on brick faces while attraction between bricks is provided by embedded magnets. The bricks can then be mixed in a container and agitated, leading to properly assembled objects at high yields and zero errors. The system and its assembly dynamics were characterized by video and audio analysis, enabling the precise time- and space-resolved characterization of its performance and accuracy. Improved designs inspired by our system could lead to successful implementation of self-assembly at the macro-scale, allowing rapid, on-demand fabrication of objects without the need for assembly lines. PMID:26226488

A Cobalt Supramolecular Triple-Stranded Helicate-based Discrete Molecular Cage

PubMed Central

Mai, Hien Duy; Kang, Philjae; Kim, Jin Kyung; Yoo, Hyojong

2017-01-01

We report a strategy to achieve a discrete cage molecule featuring a high level of structural hierarchy through a multiple-assembly process. A cobalt (Co) supramolecular triple-stranded helicate (Co-TSH)-based discrete molecular cage (1) is successfully synthesized and fully characterized. The solid-state structure of 1 shows that it is composed of six triple-stranded helicates interconnected by four linking cobalt species. This is an unusual example of a highly symmetric cage architecture resulting from the coordination-driven assembly of metallosupramolecular modules. The molecular cage 1 shows much higher CO2 uptake properties and selectivity compared with the separate supramolecular modules (Co-TSH, complex 2) and other molecular platforms. PMID:28262690

Respiratory chain supercomplexes associate with the cysteine desulfurase complex of the iron–sulfur cluster assembly machinery

PubMed Central

Böttinger, Lena; Mårtensson, Christoph U.; Song, Jiyao; Zufall, Nicole; Wiedemann, Nils; Becker, Thomas

2018-01-01

Mitochondria are the powerhouses of eukaryotic cells. The activity of the respiratory chain complexes generates a proton gradient across the inner membrane, which is used by the F1FO-ATP synthase to produce ATP for cellular metabolism. In baker’s yeast, Saccharomyces cerevisiae, the cytochrome bc1 complex (complex III) and cytochrome c oxidase (complex IV) associate in respiratory chain supercomplexes. Iron–sulfur clusters (ISC) form reactive centers of respiratory chain complexes. The assembly of ISC occurs in the mitochondrial matrix and is essential for cell viability. The cysteine desulfurase Nfs1 provides sulfur for ISC assembly and forms with partner proteins the ISC-biogenesis desulfurase complex (ISD complex). Here, we report an unexpected interaction of the active ISD complex with the cytochrome bc1 complex and cytochrome c oxidase. The individual deletion of complex III or complex IV blocks the association of the ISD complex with respiratory chain components. We conclude that the ISD complex binds selectively to respiratory chain supercomplexes. We propose that this molecular link contributes to coordination of iron–sulfur cluster formation with respiratory activity. PMID:29386296

Molecular Effects on Coacervate-Driven Block Copolymer Self Assembly

NASA Astrophysics Data System (ADS)

Lytle, Tyer; Radhakrishna, Mithun; Sing, Charles

Two oppositely charged polymers can undergo associative phase separation in a salt solution in a process known as \\x98complex coacervation. Recent work has used this as a motif to control the self-assembly behavior of a mixture of oppositely-charged block copolymers which form nanoscale structures. The materials formed from these complex coacervate-block copolymers (BCPs) have potential use as drug delivery systems, gels, and sensors. We have developed a hybrid Monte Carlo-Single Chain in a Mean Field (MC-SCMF) simulation method that is able to determine morphological phase diagrams for BCPs. This technique is an efficient way to calculate morphological phase diagrams and provides a clear link between molecular level features and self-assembly behaviors. Morphological phase diagrams showing the effects of polymer concentration, salt concentration, chain length, and charge-block fraction at large charge densities on self-assembly behavior have been determined. An unexpected phase transition from disorder to hexagonal packing at large salt concentrations has been observed for charge-block fractions equal to and larger than 0.5. This is attributed to the salt filling space stabilizing the morphology of the BCP.

Geometry induced sequence of nanoscale Frank–Kasper and quasicrystal mesophases in giant surfactants

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

Yue, Kan; Huang, Mingjun; Marson, Ryan L.

Frank–Kasper (F-K) and quasicrystal phases were originally identified in metal alloys and only sporadically reported in soft materials. These unconventional sphere-packing schemes open up possibilities to design materials with different properties. The challenge in soft materials is how to correlate complex phases built from spheres with the tunable parameters of chemical composition and molecular architecture. Here, we report a complete sequence of various highly ordered mesophases by the self-assembly of specifically designed and synthesized giant surfactants, which are conjugates of hydrophilic polyhedral oligomeric silsesquioxane cages tethered with hydrophobic polystyrene tails. We show that the occurrence of these mesophases results frommore » nanophase separation between the heads and tails and thus is critically dependent on molecular geometry. Variations in molecular geometry achieved by changing the number of tails from one to four not only shift compositional phase boundaries but also stabilize F-K and quasicrystal phases in regions where simple phases of spheroidal micelles are typically observed. These complex self-assembled nanostructures have been identified by combining X-ray scattering techniques and real-space electron microscopy images. Brownian dynamics simulations based on a simplified molecular model confirm the architecture-induced sequence of phases. Our results demonstrate the critical role of molecular architecture in dictating the formation of supramolecular crystals with “soft” spheroidal motifs and provide guidelines to the design of unconventional self-assembled nanostructures.« less

Geometry induced sequence of nanoscale Frank–Kasper and quasicrystal mesophases in giant surfactants

PubMed Central

Yue, Kan; Huang, Mingjun; Marson, Ryan L.; He, Jinlin; Huang, Jiahao; Zhou, Zhe; Wang, Jing; Liu, Chang; Yan, Xuesheng; Wu, Kan; Guo, Zaihong; Liu, Hao; Ni, Peihong; Wesdemiotis, Chrys; Zhang, Wen-Bin; Glotzer, Sharon C.; Cheng, Stephen Z. D.

2016-01-01

Frank–Kasper (F-K) and quasicrystal phases were originally identified in metal alloys and only sporadically reported in soft materials. These unconventional sphere-packing schemes open up possibilities to design materials with different properties. The challenge in soft materials is how to correlate complex phases built from spheres with the tunable parameters of chemical composition and molecular architecture. Here, we report a complete sequence of various highly ordered mesophases by the self-assembly of specifically designed and synthesized giant surfactants, which are conjugates of hydrophilic polyhedral oligomeric silsesquioxane cages tethered with hydrophobic polystyrene tails. We show that the occurrence of these mesophases results from nanophase separation between the heads and tails and thus is critically dependent on molecular geometry. Variations in molecular geometry achieved by changing the number of tails from one to four not only shift compositional phase boundaries but also stabilize F-K and quasicrystal phases in regions where simple phases of spheroidal micelles are typically observed. These complex self-assembled nanostructures have been identified by combining X-ray scattering techniques and real-space electron microscopy images. Brownian dynamics simulations based on a simplified molecular model confirm the architecture-induced sequence of phases. Our results demonstrate the critical role of molecular architecture in dictating the formation of supramolecular crystals with “soft” spheroidal motifs and provide guidelines to the design of unconventional self-assembled nanostructures. PMID:27911786

Single-Molecule Analysis for RISC Assembly and Target Cleavage.

PubMed

Sasaki, Hiroshi M; Tadakuma, Hisashi; Tomari, Yukihide

2018-01-01

RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

Diastereoselective chain-elongation reactions using microreactors for applications in complex molecule assembly.

PubMed

Carter, Catherine F; Lange, Heiko; Sakai, Daiki; Baxendale, Ian R; Ley, Steven V

2011-03-14

Diastereoselective chain-elongation reactions are important transformations for the assembly of complex molecular structures, such as those present in polyketide natural products. Here we report new methods for performing crotylation reactions and homopropargylation reactions by using newly developed low-temperature flow-chemistry technology. In-line purification protocols are described, as well as the application of the crotylation protocol in an automated multi-step sequence. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Self-assembly of polyelectrolyte surfactant complexes using large scale MD simulation

NASA Astrophysics Data System (ADS)

Goswami, Monojoy; Sumpter, Bobby

2014-03-01

Polyelectrolytes (PE) and surfactants are known to form interesting structures with varied properties in aqueous solutions. The morphological details of the PE-surfactant complexes depend on a combination of polymer backbone, electrostatic interactions and hydrophobic interactions. We study the self-assembly of cationic PE and anionic surfactants complexes in dilute condition. The importance of such complexes of PE with oppositely charged surfactants can be found in biological systems, such as immobilization of enzymes in polyelectrolyte complexes or nonspecific association of DNA with protein. Many useful properties of PE surfactant complexes come from the highly ordered structures of surfactant self-assembly inside the PE aggregate which has applications in industry. We do large scale molecular dynamics simulation using LAMMPS to understand the structure and dynamics of PE-surfactant systems. Our investigation shows highly ordered pearl-necklace structures that have been observed experimentally in biological systems. We investigate many different properties of PE-surfactant complexation for different parameter ranges that are useful for pharmaceutical, engineering and biological applications.

Synthesis of single-molecule nanocars.

PubMed

Vives, Guillaume; Tour, James M

2009-03-17

The drive to miniaturize devices has led to a variety of molecular machines inspired by macroscopic counterparts such as molecular motors, switches, shuttles, turnstiles, barrows, elevators, and nanovehicles. Such nanomachines are designed for controlled mechanical motion and the transport of nanocargo. As researchers miniaturize devices, they can consider two complementary approaches: (1) the "top-down" approach, which reduces the size of macroscopic objects to reach an equivalent microscopic entity using photolithography and related techniques and (2) the "bottom-up" approach, which builds functional microscopic or nanoscopic entities from molecular building blocks. The top-down approach, extensively used by the semiconductor industry, is nearing its scaling limits. On the other hand, the bottom-up approach takes advantage of the self-assembly of smaller molecules into larger networks by exploiting typically weak molecular interactions. But self-assembly alone will not permit complex assembly. Using nanomachines, we hope to eventually consider complex, enzyme-like directed assembly. With that ultimate goal, we are currently exploring the control of nanomachines that would provide a basis for the future bottom-up construction of complex systems. This Account describes the synthesis of a class of molecular machines that resemble macroscopic vehicles. We designed these so-called nanocars for study at the single-molecule level by scanning probe microscopy (SPM). The vehicles have a chassis connected to wheel-terminated axles and convert energy inputs such as heat, electric fields, or light into controlled motion on a surface, ultimately leading to transport of nanocargo. At first, we used C(60) fullerenes as wheels, which allowed the demonstration of a directional rolling mechanism of a nanocar on a gold surface by STM. However, because of the low solubility of the fullerene nanocars and the incompatibility of fullerenes with photochemical processes, we developed new p-carborane- and ruthenium-based wheels with greater solubility in organic solvents. Although fullerene wheels must be attached in the final synthetic step, p-carborane- and ruthenium-based wheels do not inhibit organometallic coupling reactions, which allows a more convergent synthesis of molecular machines. We also prepared functional nanotrucks for the transport of atoms and molecules, as well as self-assembling nanocars and nanotrains. Although engineering challenges such as movement over long distance and non-atomically flat surfaces remain, the greatest current research challenge is imaging. The detailed study of nanocars requires complementary single molecule imaging techniques such as STM, AFM, TEM, or single-molecule fluorescence microscopy. Further developments in engineering and synthesis could lead to enzyme-like manipulation and assembly of atoms and small molecules in nonbiological environments.

Learning surface molecular structures via machine vision

NASA Astrophysics Data System (ADS)

Ziatdinov, Maxim; Maksov, Artem; Kalinin, Sergei V.

2017-08-01

Recent advances in high resolution scanning transmission electron and scanning probe microscopies have allowed researchers to perform measurements of materials structural parameters and functional properties in real space with a picometre precision. In many technologically relevant atomic and/or molecular systems, however, the information of interest is distributed spatially in a non-uniform manner and may have a complex multi-dimensional nature. One of the critical issues, therefore, lies in being able to accurately identify (`read out') all the individual building blocks in different atomic/molecular architectures, as well as more complex patterns that these blocks may form, on a scale of hundreds and thousands of individual atomic/molecular units. Here we employ machine vision to read and recognize complex molecular assemblies on surfaces. Specifically, we combine Markov random field model and convolutional neural networks to classify structural and rotational states of all individual building blocks in molecular assembly on the metallic surface visualized in high-resolution scanning tunneling microscopy measurements. We show how the obtained full decoding of the system allows us to directly construct a pair density function—a centerpiece in analysis of disorder-property relationship paradigm—as well as to analyze spatial correlations between multiple order parameters at the nanoscale, and elucidate reaction pathway involving molecular conformation changes. The method represents a significant shift in our way of analyzing atomic and/or molecular resolved microscopic images and can be applied to variety of other microscopic measurements of structural, electronic, and magnetic orders in different condensed matter systems.

Supramolecular domains in mixed peptide self-assembled monolayers on gold nanoparticles.

PubMed

Duchesne, Laurence; Wells, Geoff; Fernig, David G; Harris, Sarah A; Lévy, Raphaël

2008-09-01

Self-organization in mixed self-assembled monolayers of small molecules provides a route towards nanoparticles with complex molecular structures. Inspired by structural biology, a strategy based on chemical cross-linking is introduced to probe proximity between functional peptides embedded in a mixed self-assembled monolayer at the surface of a nanoparticle. The physical basis of the proximity measurement is a transition from intramolecular to intermolecular cross-linking as the functional peptides get closer. Experimental investigations of a binary peptide self-assembled monolayer show that this transition happens at an extremely low molar ratio of the functional versus matrix peptide. Molecular dynamics simulations of the peptide self-assembled monolayer are used to calculate the volume explored by the reactive groups. Comparison of the experimental results with a probabilistic model demonstrates that the peptides are not randomly distributed at the surface of the nanoparticle, but rather self-organize into supramolecular domains.

Robust hydrogen-bonded self-assemblies from biimidazole complexes. Synthesis and structural characterization of [M(biimidazole)2(OH2)2]2+ (M = Co2+, Ni2+) complexes and carboxylate modules.

PubMed

Atencio, Reinaldo; Chacón, Mirbel; González, Teresa; Briceño, Alexander; Agrifoglio, Giuseppe; Sierraalta, Anibal

2004-02-21

A robust heteromeric hydrogen-bonded synthon [R2(2) (9)-Id] is exploited to drive the modular self-assembly of four coordination complexes [M(H2biim)2(OH2)2]2+ (M = Co2+, Ni2+) and carboxylate counterions. This strategy allowed us to build molecular architectures of 0-, 1-, and 2-dimensions. A hydrogen-bonded 2D-network with cavities has been designed, which maintains its striking integrity after reversible water desorption-resorption processes.

Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway.

PubMed

Pham, John W; Sontheimer, Erik J

2005-11-25

Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). Here we show that R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer alone can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute 2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex, which also requires Argonaute 2 for assembly into RISC. We have compared R2 to the RISC-loading complex and show that the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. We have examined the requirements for RISC formation and show that the siRNA 5'-termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2'-position of the 5'-terminal nucleotide also affects RISC assembly, because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference pathway.

Self-Assembling Molecular Logic Gates Based on DNA Crossover Tiles.

PubMed

Campbell, Eleanor A; Peterson, Evan; Kolpashchikov, Dmitry M

2017-07-05

DNA-based computational hardware has attracted ever-growing attention due to its potential to be useful in the analysis of complex mixtures of biological markers. Here we report the design of self-assembling logic gates that recognize DNA inputs and assemble into crossover tiles when the output signal is high; the crossover structures disassemble to form separate DNA stands when the output is low. The output signal can be conveniently detected by fluorescence using a molecular beacon probe as a reporter. AND, NOT, and OR logic gates were designed. We demonstrate that the gates can connect to each other to produce other logic functions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

γ-Tubulin complex in Trypanosoma brucei: molecular composition, subunit interdependence and requirement for axonemal central pair protein assembly.

PubMed

Zhou, Qing; Li, Ziyin

2015-11-01

γ-Tubulin complex constitutes a key component of the microtubule-organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ-tubulin small complex (γTuSC) composed of γ-tubulin, gamma-tubulin complex protein (GCP)2 and GCP3, whereas animals contain the γ-tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ-tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ-tubulin complex in T. brucei is composed of γ-tubulin and three GCP proteins, GCP2-GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ-tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex. © 2015 John Wiley & Sons Ltd.

TAF11 assembles RISC loading complex to enhance RNAi efficiency

PubMed Central

Liang, Chunyang; Wang, Yibing; Murota, Yukiko; Liu, Xiang; Smith, Dean; Siomi, Mikiko C.; Liu, Qinghua

2015-01-01

SUMMARY Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains Dicer-2(Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila melanogaster. However, the precise composition and action mechanism of Drosophila RLC remain unclear. Here, we identified the missing factor of RLC as TATA-binding protein associated factor 11 (TAF11) by genetic screen. Although an annotated nuclear transcription factor, we found that TAF11 also associated with Dcr-2/R2D2 and localized to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11−/− ovary extract, we reconstituted the RLC in vitro using recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, we showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, our genetic and biochemical studies define the molecular nature of Drosophila RLC and elucidate a novel cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency. PMID:26257286

WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

PubMed Central

Havrylenko, Svitlana; Noguera, Philippe; Abou-Ghali, Majdouline; Manzi, John; Faqir, Fahima; Lamora, Audrey; Guérin, Christophe; Blanchoin, Laurent; Plastino, Julie

2015-01-01

The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP's ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly. PMID:25355952

Complex supramolecular interfacial tessellation through convergent multi-step reaction of a dissymmetric simple organic precursor

NASA Astrophysics Data System (ADS)

Zhang, Yi-Qi; Paszkiewicz, Mateusz; Du, Ping; Zhang, Liding; Lin, Tao; Chen, Zhi; Klyatskaya, Svetlana; Ruben, Mario; Seitsonen, Ari P.; Barth, Johannes V.; Klappenberger, Florian

2018-03-01

Interfacial supramolecular self-assembly represents a powerful tool for constructing regular and quasicrystalline materials. In particular, complex two-dimensional molecular tessellations, such as semi-regular Archimedean tilings with regular polygons, promise unique properties related to their nontrivial structures. However, their formation is challenging, because current methods are largely limited to the direct assembly of precursors, that is, where structure formation relies on molecular interactions without using chemical transformations. Here, we have chosen ethynyl-iodophenanthrene (which features dissymmetry in both geometry and reactivity) as a single starting precursor to generate the rare semi-regular (3.4.6.4) Archimedean tiling with long-range order on an atomically flat substrate through a multi-step reaction. Intriguingly, the individual chemical transformations converge to form a symmetric alkynyl-Ag-alkynyl complex as the new tecton in high yields. Using a combination of microscopy and X-ray spectroscopy tools, as well as computational modelling, we show that in situ generated catalytic Ag complexes mediate the tecton conversion.

Molecular architecture of the Spire-actin nucleus and its implication for actin filament assembly.

PubMed

Sitar, Tomasz; Gallinger, Julia; Ducka, Anna M; Ikonen, Teemu P; Wohlhoefler, Michael; Schmoller, Kurt M; Bausch, Andreas R; Joel, Peteranne; Trybus, Kathleen M; Noegel, Angelika A; Schleicher, Michael; Huber, Robert; Holak, Tad A

2011-12-06

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs--even a single WH2 repeat--sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.

The Atg1-kinase complex tethers Atg9-vesicles to initiate autophagy

NASA Astrophysics Data System (ADS)

Rao, Yijian; Perna, Marco G.; Hofmann, Benjamin; Beier, Viola; Wollert, Thomas

2016-01-01

Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation. Their biogenesis is initiated by recruitment of Atg9-vesicles to the phagophore assembly site. This process depends on the regulated activation of the Atg1-kinase complex. However, the underlying molecular mechanism remains unclear. Here we reconstitute this early step in autophagy from purified components in vitro. We find that on assembly from its cytoplasmic subcomplexes, the Atg1-kinase complex becomes activated, enabling it to recruit and tether Atg9-vesicles. The scaffolding protein Atg17 targets the Atg1-kinase complex to autophagic membranes by specifically recognizing the membrane protein Atg9. This interaction is inhibited by the two regulatory subunits Atg31 and Atg29. Engagement of the Atg1-Atg13 subcomplex restores the Atg9-binding and membrane-tethering activity of Atg17. Our data help to unravel the mechanism that controls Atg17-mediated tethering of Atg9-vesicles, providing the molecular basis to understand initiation of autophagosome-biogenesis.

Mass spectrometric characterization of membrane integral low molecular weight proteins from photosystem II in barley etioplasts.

PubMed

Plöscher, Matthias; Granvogl, Bernhard; Zoryan, Mikael; Reisinger, Veronika; Eichacker, Lutz Andreas

2009-02-01

In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN-PAGE separation of membrane protein complexes and selective MS that the accumulation of one-helix proteins from PSII is light independent and occurs in etioplasts. In contrast, in chloroplasts isolated from light-grown plants, low molecular weight proteins were found to specifically accumulate in PSI and II complexes. Our results demonstrate how plants grown in darkness prepare for the induction of chlorophyll dependent photosystem assembly upon light perception. We anticipate that our investigation will provide the essential means for the analysis of protein assembly in any membrane utilizing low molecular weight protein subunits.

Directing folding pathways for multi-component DNA origami nanostructures with complex topology

NASA Astrophysics Data System (ADS)

Marras, A. E.; Zhou, L.; Kolliopoulos, V.; Su, H.-J.; Castro, C. E.

2016-05-01

Molecular self-assembly has become a well-established technique to design complex nanostructures and hierarchical mesoscale assemblies. The typical approach is to design binding complementarity into nucleotide or amino acid sequences to achieve the desired final geometry. However, with an increasing interest in dynamic nanodevices, the need to design structures with motion has necessitated the development of multi-component structures. While this has been achieved through hierarchical assembly of similar structural units, here we focus on the assembly of topologically complex structures, specifically with concentric components, where post-folding assembly is not feasible. We exploit the ability to direct folding pathways to program the sequence of assembly and present a novel approach of designing the strand topology of intermediate folding states to program the topology of the final structure, in this case a DNA origami slider structure that functions much like a piston-cylinder assembly in an engine. The ability to program the sequence and control orientation and topology of multi-component DNA origami nanostructures provides a foundation for a new class of structures with internal and external moving parts and complex scaffold topology. Furthermore, this work provides critical insight to guide the design of intermediate states along a DNA origami folding pathway and to further understand the details of DNA origami self-assembly to more broadly control folding states and landscapes.

Pydna: a simulation and documentation tool for DNA assembly strategies using python.

PubMed

Pereira, Filipa; Azevedo, Flávio; Carvalho, Ângela; Ribeiro, Gabriela F; Budde, Mark W; Johansson, Björn

2015-05-02

Recent advances in synthetic biology have provided tools to efficiently construct complex DNA molecules which are an important part of many molecular biology and biotechnology projects. The planning of such constructs has traditionally been done manually using a DNA sequence editor which becomes error-prone as scale and complexity of the construction increase. A human-readable formal description of cloning and assembly strategies, which also allows for automatic computer simulation and verification, would therefore be a valuable tool. We have developed pydna, an extensible, free and open source Python library for simulating basic molecular biology DNA unit operations such as restriction digestion, ligation, PCR, primer design, Gibson assembly and homologous recombination. A cloning strategy expressed as a pydna script provides a description that is complete, unambiguous and stable. Execution of the script automatically yields the sequence of the final molecule(s) and that of any intermediate constructs. Pydna has been designed to be understandable for biologists with limited programming skills by providing interfaces that are semantically similar to the description of molecular biology unit operations found in literature. Pydna simplifies both the planning and sharing of cloning strategies and is especially useful for complex or combinatorial DNA molecule construction. An important difference compared to existing tools with similar goals is the use of Python instead of a specifically constructed language, providing a simulation environment that is more flexible and extensible by the user.

The γ-tubulin complex in Trypanosoma brucei: molecular composition, subunit interdependence and requirement for axonemal central pair protein assembly

PubMed Central

Zhou, Qing; Li, Ziyin

2015-01-01

The γ-tubulin complex constitutes a key component of the microtubule-organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ-tubulin small complex (γTuSC) composed of γ-tubulin, GCP2 and GCP3, whereas animals contain the γ-tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ-tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ-tubulin complex in T. brucei is composed of γ-tubulin and three GCP proteins, GCP2-GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ-tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex. PMID:26224545

The optimization of peptide cargo bound to MHC class I molecules by the peptide-loading complex.

PubMed

Elliott, Tim; Williams, Anthony

2005-10-01

Major histocompatibility complex (MHC) class I complexes present peptides from both self and foreign intracellular proteins on the surface of most nucleated cells. The assembled heterotrimeric complexes consist of a polymorphic glycosylated heavy chain, non-polymorphic beta(2) microglobulin, and a peptide of typically nine amino acids in length. Assembly of the class I complexes occurs in the endoplasmic reticulum and is assisted by a number of chaperone molecules. A multimolecular unit termed the peptide-loading complex (PLC) is integral to this process. The PLC contains a peptide transporter (transporter associated with antigen processing), a thiooxido-reductase (ERp57), a glycoprotein chaperone (calreticulin), and tapasin, a class I-specific chaperone. We suggest that class I assembly involves a process of optimization where the peptide cargo of the complex is edited by the PLC. Furthermore, this selective peptide loading is biased toward peptides that have a longer off-rate from the assembled complex. We suggest that tapasin is the key chaperone that directs this action of the PLC with secondary contributions from calreticulin and possibly ERp57. We provide a framework model for how this may operate at the molecular level and draw parallels with the proposed mechanism of action of human leukocyte antigen-DM for MHC class II complex optimization.

3D DNA Crystals and Nanotechnology

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

Paukstelis, Paul; Seeman, Nadrian

DNA's molecular recognition properties have made it one of the most widely used biomacromolecular construction materials. The programmed assembly of DNA oligonucleotides has been used to create complex 2D and 3D self-assembled architectures and to guide the assembly of other molecules. The origins of DNA nanotechnology are rooted in the goal of assembling DNA molecules into designed periodic arrays, i.e., crystals. Here, we highlight several DNA crystal structures, the progress made in designing DNA crystals, and look at the current prospects and future directions of DNA crystals in nanotechnology.

3D DNA Crystals and Nanotechnology

DOE PAGES

Paukstelis, Paul; Seeman, Nadrian

2016-08-18

DNA's molecular recognition properties have made it one of the most widely used biomacromolecular construction materials. The programmed assembly of DNA oligonucleotides has been used to create complex 2D and 3D self-assembled architectures and to guide the assembly of other molecules. The origins of DNA nanotechnology are rooted in the goal of assembling DNA molecules into designed periodic arrays, i.e., crystals. Here, we highlight several DNA crystal structures, the progress made in designing DNA crystals, and look at the current prospects and future directions of DNA crystals in nanotechnology.

Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin-biotin recognition.

PubMed

Borovok, Natalia; Iram, Natalie; Zikich, Dragoslav; Ghabboun, Jamal; Livshits, Gideon I; Porath, Danny; Kotlyar, Alexander B

2008-09-01

We describe a method for the preparation of novel long (hundreds of nanometers), uniform, inter-molecular G4-DNA molecules composed of four parallel G-strands. The only long continuous G4-DNA reported so far are intra-molecular structures made of a single G-strand. To enable a tetra-molecular assembly of the G-strands we developed a novel approach based on avidin-biotin biological recognition. The steps of the G4-DNA production include: (i) Enzymatic synthesis of long poly(dG)-poly(dC) molecules with biotinylated poly(dG)-strand; (ii) Formation of a complex between avidin-tetramer and four biotinylated poly(dG)-poly(dC) molecules; (iii) Separation of the poly(dC) strands from the poly(dG)-strands, which are connected to the avidin; (iv) Assembly of the four G-strands attached to the avidin into tetra-molecular G4-DNA. The average contour length of the formed structures, as measured by AFM, is equal to that of the initial poly(dG)-poly(dC) molecules, suggesting a tetra-molecular mechanism of the G-strands assembly. The height of tetra-molecular G4-nanostructures is larger than that of mono-molecular G4-DNA molecules having similar contour length. The CD spectra of the tetra- and mono-molecular G4-DNA are markedly different, suggesting different structural organization of these two types of molecules. The tetra-molecular G4-DNA nanostructures showed clear electrical polarizability. This suggests that they may be useful for molecular electronics.

Advanced Cloning Tools for Construction of Designer Cellulosomes.

PubMed

Kahn, Amaranta; Bayer, Edward A; Moraïs, Sarah

2018-01-01

Cellulose deconstruction is achieved in nature through two main enzymatic paradigms, i.e., free enzymes and enzymatic complexes (called cellulosomes). Gaining insights into the mechanism of action and synergy among the different cellulases is of high interest, notably in the field of renewable energy, and specifically, for the conversion of cellulosic biomass to soluble sugars, en route to biofuels. In this context, designer cellulosomes are artificially assembled, chimaeric protein complexes that are used as a tool to comparatively study cellulose degradation by different enzymatic paradigms, and could also serve to improve cellulose deconstruction. Various molecular biology techniques are employed in order to design and engineer the various components of designer cellulosomes. In this chapter, we describe the cloning processes through which the appropriate modules are selected and assembled at the molecular level.

Self-assembly of discrete metal complexes in aqueous solution via block copolypeptide amphiphiles.

PubMed

Kuroiwa, Keita; Masaki, Yoshitaka; Koga, Yuko; Deming, Timothy J

2013-01-21

The integration of discrete metal complexes has been attracting significant interest due to the potential of these materials for soft metal-metal interactions and supramolecular assembly. Additionally, block copolypeptide amphiphiles have been investigated concerning their capacity for self-assembly into structures such as nanoparticles, nanosheets and nanofibers. In this study, we combined these two concepts by investigating the self-assembly of discrete metal complexes in aqueous solution using block copolypeptides. Normally, discrete metal complexes such as [Au(CN)(2)]-, when molecularly dispersed in water, cannot interact with one another. Our results demonstrated, however, that the addition of block copolypeptide amphiphiles such as K(183)L(19) to [Au(CN)(2)]- solutions induced one-dimensional integration of the discrete metal complex, resulting in photoluminescence originating from multinuclear complexes with metal-metal interactions. Transmission electron microscopy (TEM) showed a fibrous nanostructure with lengths and widths of approximately 100 and 20 nm, respectively, which grew to form advanced nanoarchitectures, including those resembling the weave patterns of Waraji (traditional Japanese straw sandals). This concept of combining block copolypeptide amphiphiles with discrete coordination compounds allows the design of flexible and functional supramolecular coordination systems in water.

Getting a Grip on Complexes

PubMed Central

Nie, Yan; Viola, Cristina; Bieniossek, Christoph; Trowitzsch, Simon; Vijay-achandran, Lakshmi Sumitra; Chaillet, Maxime; Garzoni, Frederic; Berger, Imre

2009-01-01

We are witnessing tremendous advances in our understanding of the organization of life. Complete genomes are being deciphered with ever increasing speed and accuracy, thereby setting the stage for addressing the entire gene product repertoire of cells, towards understanding whole biological systems. Advances in bioinformatics and mass spectrometric techniques have revealed the multitude of interactions present in the proteome. Multiprotein complexes are emerging as a paramount cornerstone of biological activity, as many proteins appear to participate, stably or transiently, in large multisubunit assemblies. Analysis of the architecture of these assemblies and their manifold interactions is imperative for understanding their function at the molecular level. Structural genomics efforts have fostered the development of many technologies towards achieving the throughput required for studying system-wide single proteins and small interaction motifs at high resolution. The present shift in focus towards large multiprotein complexes, in particular in eukaryotes, now calls for a likewise concerted effort to develop and provide new technologies that are urgently required to produce in quality and quantity the plethora of multiprotein assemblies that form the complexome, and to routinely study their structure and function at the molecular level. Current efforts towards this objective are summarized and reviewed in this contribution. PMID:20514218

Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly.

PubMed

O'Neill, Sharon; Mathis, Magalie; Kovačič, Lidija; Zhang, Suisheng; Reinhardt, Jürgen; Scholz, Dimitri; Schopfer, Ulrich; Bouhelal, Rochdi; Knaus, Ulla G

2018-06-08

Protein-protein interactions critically regulate many biological systems, but quantifying functional assembly of multipass membrane complexes in their native context is still challenging. Here, we combined modeling-assisted protein modification and information from human disease variants with a minimal-size fusion tag, split-luciferase-based approach to probe assembly of the NADPH oxidase 4 (NOX4)-p22 phox enzyme, an integral membrane complex with unresolved structure, which is required for electron transfer and generation of reactive oxygen species (ROS). Integrated analyses of heterodimerization, trafficking, and catalytic activity identified determinants for the NOX4-p22 phox interaction, such as heme incorporation into NOX4 and hot spot residues in transmembrane domains 1 and 4 in p22 phox Moreover, their effect on NOX4 maturation and ROS generation was analyzed. We propose that this reversible and quantitative protein-protein interaction technique with its small split-fragment approach will provide a protein engineering and discovery tool not only for NOX research, but also for other intricate membrane protein complexes, and may thereby facilitate new drug discovery strategies for managing NOX-associated diseases. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Self-assembling DNA nanotubes to connect molecular landmarks

NASA Astrophysics Data System (ADS)

Mohammed, Abdul M.; Šulc, Petr; Zenk, John; Schulman, Rebecca

2017-05-01

Within cells, nanostructures are often organized using local assembly rules that produce long-range order. Because these rules can take into account the cell's current structure and state, they can enable complexes, organelles or cytoskeletal structures to assemble around existing cellular components to form architectures. Although many methods for self-assembling biomolecular nanostructures have been developed, few can be programmed to assemble structures whose form depends on the identity and organization of structures already present in the environment. Here, we demonstrate that DNA nanotubes can grow to connect pairs of molecular landmarks with different separation distances and relative orientations. DNA tile nanotubes nucleate at these landmarks and grow while their free ends diffuse. The nanotubes can then join end to end to form stable connections, with unconnected nanotubes selectively melted away. Connections form between landmark pairs separated by 1-10 µm in more than 75% of cases and can span a surface or three dimensions. This point-to-point assembly process illustrates how self-assembly kinetics can be designed to produce structures with a desired physical property rather than a specific shape.

Dissecting the molecular assembly of the Toxoplasma gondii MyoA motility complex.

PubMed

Powell, Cameron J; Jenkins, Meredith L; Parker, Michelle L; Ramaswamy, Raghavendran; Kelsen, Anne; Warshaw, David M; Ward, Gary E; Burke, John E; Boulanger, Martin J

2017-11-24

Apicomplexan parasites such as Toxoplasma gondii rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA-light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775-818) using a combination of hydrogen-deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm K d measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a K d of 12 nm). Using the full-length MyoA motor (residues 1-831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Learning surface molecular structures via machine vision

DOE PAGES

Ziatdinov, Maxim; Maksov, Artem; Kalinin, Sergei V.

2017-08-10

Recent advances in high resolution scanning transmission electron and scanning probe microscopies have allowed researchers to perform measurements of materials structural parameters and functional properties in real space with a picometre precision. In many technologically relevant atomic and/or molecular systems, however, the information of interest is distributed spatially in a non-uniform manner and may have a complex multi-dimensional nature. One of the critical issues, therefore, lies in being able to accurately identify (‘read out’) all the individual building blocks in different atomic/molecular architectures, as well as more complex patterns that these blocks may form, on a scale of hundreds andmore » thousands of individual atomic/molecular units. Here we employ machine vision to read and recognize complex molecular assemblies on surfaces. Specifically, we combine Markov random field model and convolutional neural networks to classify structural and rotational states of all individual building blocks in molecular assembly on the metallic surface visualized in high-resolution scanning tunneling microscopy measurements. We show how the obtained full decoding of the system allows us to directly construct a pair density function—a centerpiece in analysis of disorder-property relationship paradigm—as well as to analyze spatial correlations between multiple order parameters at the nanoscale, and elucidate reaction pathway involving molecular conformation changes. Here, the method represents a significant shift in our way of analyzing atomic and/or molecular resolved microscopic images and can be applied to variety of other microscopic measurements of structural, electronic, and magnetic orders in different condensed matter systems.« less

Learning surface molecular structures via machine vision

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

Ziatdinov, Maxim; Maksov, Artem; Kalinin, Sergei V.

Recent advances in high resolution scanning transmission electron and scanning probe microscopies have allowed researchers to perform measurements of materials structural parameters and functional properties in real space with a picometre precision. In many technologically relevant atomic and/or molecular systems, however, the information of interest is distributed spatially in a non-uniform manner and may have a complex multi-dimensional nature. One of the critical issues, therefore, lies in being able to accurately identify (‘read out’) all the individual building blocks in different atomic/molecular architectures, as well as more complex patterns that these blocks may form, on a scale of hundreds andmore » thousands of individual atomic/molecular units. Here we employ machine vision to read and recognize complex molecular assemblies on surfaces. Specifically, we combine Markov random field model and convolutional neural networks to classify structural and rotational states of all individual building blocks in molecular assembly on the metallic surface visualized in high-resolution scanning tunneling microscopy measurements. We show how the obtained full decoding of the system allows us to directly construct a pair density function—a centerpiece in analysis of disorder-property relationship paradigm—as well as to analyze spatial correlations between multiple order parameters at the nanoscale, and elucidate reaction pathway involving molecular conformation changes. Here, the method represents a significant shift in our way of analyzing atomic and/or molecular resolved microscopic images and can be applied to variety of other microscopic measurements of structural, electronic, and magnetic orders in different condensed matter systems.« less

Evidence that the assembly of the yeast cytochrome bc1 complex involves the formation of a large core structure in the inner mitochondrial membrane.

PubMed

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-04-01

The assembly status of the cytochrome bc(1) complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc(1) subunits were deleted. In all the yeast strains tested, a bc(1) sub-complex of approximately 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS/PAGE and immunodecoration, revealed the presence of the two catalytic subunits, cytochrome b and cytochrome c(1), associated with the noncatalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Together, these bc(1) subunits build up the core structure of the cytochrome bc(1) complex, which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc(1) core structure may represent a true assembly intermediate during the maturation of the bc(1) complex; first, because of its wide distribution in distinct yeast deletion strains and, second, for its characteristics of stability, which resemble those of the intact homodimeric bc(1) complex. By contrast, the bc(1) core structure is unable to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc(1) complex provides a number of new elements clarifying the molecular events leading to the maturation of the yeast cytochrome bc(1) complex in the inner mitochondrial membrane.

Evidence that assembly of the yeast cytochrome bc1 complex involves formation of a large core structure in the inner mitochondrial membrane

PubMed Central

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L.

2009-01-01

The assembly status of the cytochrome bc1 complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc1 subunits had been deleted. In all the yeast strains tested a bc1 sub-complex of about 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS-PAGE and immunodecoration, revealed the presence of the two catalytic subunits cytochrome b and cytochrome c1, associated with the non catalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Altogether these bc1 subunits build up the core structure of the cytochrome bc1 complex which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc1 core structure may represent a true assembly intermediate during the maturation of the bc1 complex, first because of its wide distribution in distinct yeast deletion strains and second for its characteristics of stability which resemble those of the intact homodimeric bc1 complex. Differently from this latter, however, the bc1 core structure is not able to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc1 complex provides a number of new elements for clarification of the molecular events leading to the maturation of the yeast cytochrome bc1 complex in the inner mitochondrial membrane. PMID:19236481

Structural Study of the RIPoptosome Core Reveals a Helical Assembly for Kinase Recruitment

PubMed Central

2015-01-01

Receptor interaction protein kinase 1 (RIP1) is a molecular cell-fate switch. RIP1, together with Fas-associated protein with death domain (FADD) and caspase-8, forms the RIPoptosome that activates apoptosis. RIP1 also associates with RIP3 to form the necrosome that triggers necroptosis. The RIPoptosome assembles through interactions between the death domains (DDs) of RIP1 and FADD and between death effector domains (DEDs) of FADD and caspase-8. In this study, we analyzed the overall structure of the RIP1 DD/FADD DD complex, the core of the RIPoptosome, by negative-stain electron microscopy and modeling. The results show that RIP1 DD and FADD DD form a stable complex in vitro similar to the previously described Fas DD/FADD DD complex, suggesting that the RIPoptosome and the Fas death-inducing signaling complex share a common assembly mechanism. Both complexes adopt a helical conformation that requires type I, II, and III interactions between the death domains. PMID:25119434

Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery.

PubMed

Gakh, Oleksandr; Ranatunga, Wasantha; Smith, Douglas Y; Ahlgren, Eva-Christina; Al-Karadaghi, Salam; Thompson, James R; Isaya, Grazia

2016-09-30

Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN 42-210 (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with ∼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN 42-210 ] 24 ·[NFS1] 24 ·[ISD11] 24 ·[ISCU] 24 complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN 42-210 ] 24 ·[ISCU] 24 sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN 42-210 trimer at each of its eight vertices. Binding of 12 [NFS1] 2 ·[ISD11] 2 sub-complexes to the surface results in a globular macromolecule with a diameter of ∼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN 42-210 to ISCU. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Lock and Key Colloids through Polymerization-Induced Buckling of Monodispersed Silicon Oil Droplets

NASA Astrophysics Data System (ADS)

Sacanna, Stefano; Irvine, William T. M.; Chaikin, Paul M.; Pine, David J.

2010-03-01

Colloidal particles can spontaneously associate into larger structured aggregates when driven by selective and directional interactions. Colloidal organization can be programmed by engineering shapes and interactions of basic building blocks in a manner similar to molecular self-assembly. Examples of successful strategies that allow non-trivial assembly of particles include template-directed patterning, capillary forces and, most commonly, the functionalization of the particle surfaces with ``sticky patches'' of biological or synthetic molecules. The level of complexity of the realizable assemblies, increases when particles with well defined shape anisotropies are used. In particular depletion forces and specific surface treatments in combination with non spherical particles have proven to be powerful tools to self-assembly complex microstructures. We describe a simple, high yield, synthetic pathway to fabricate monodisperse hybrid silica spheres with well defined cavities. Because the particle morphologies are reproducible and tunable with precision, the resulting particles can be used as basic building blocks in the assembly of larger monodisperse clusters. This is demonstrated using depletion to drive the self-assembly.

Role of Bassoon and Piccolo in Assembly and Molecular Organization of the Active Zone

PubMed Central

Gundelfinger, Eckart D.; Reissner, Carsten; Garner, Craig C.

2016-01-01

Bassoon and Piccolo are two very large scaffolding proteins of the cytomatrix assembled at the active zone (CAZ) where neurotransmitter is released. They share regions of high sequence similarity distributed along their entire length and seem to share both overlapping and distinct functions in organizing the CAZ. Here, we survey our present knowledge on protein-protein interactions and recent progress in understanding of molecular functions of these two giant proteins. These include roles in the assembly of active zones (AZ), the localization of voltage-gated Ca2+ channels (VGCCs) in the vicinity of release sites, synaptic vesicle (SV) priming and in the case of Piccolo, a role in the dynamic assembly of the actin cytoskeleton. Piccolo and Bassoon are also important for the maintenance of presynaptic structure and function, as well as for the assembly of CAZ specializations such as synaptic ribbons. Recent findings suggest that they are also involved in the regulation activity-dependent communication between presynaptic boutons and the neuronal nucleus. Together these observations suggest that Bassoon and Piccolo use their modular structure to organize super-molecular complexes essential for various aspects of presynaptic function. PMID:26793095

α-SNAP interferes with the zippering of the SNARE protein membrane fusion machinery.

PubMed

Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

2014-06-06

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural basis of the pH-dependent assembly of a botulinum neurotoxin complex.

PubMed

Matsui, Tsutomu; Gu, Shenyan; Lam, Kwok-Ho; Carter, Lester G; Rummel, Andreas; Mathews, Irimpan I; Jin, Rongsheng

2014-11-11

Botulinum neurotoxins (BoNTs) are among the most poisonous biological substances known. They assemble with non-toxic non-hemagglutinin (NTNHA) protein to form the minimally functional progenitor toxin complexes (M-PTC), which protects BoNT in the gastrointestinal tract and releases it upon entry into the circulation. Here we provide molecular insight into the assembly between BoNT/A and NTNHA-A using small-angle X-ray scattering. We found that the free form BoNT/A maintains a pH-independent conformation with limited domain flexibility. Intriguingly, the free form NTNHA-A adopts pH-dependent conformational changes due to a torsional motion of its C-terminal domain. Once forming a complex at acidic pH, they each adopt a stable conformation that is similar to that observed in the crystal structure of the M-PTC. Our results suggest that assembly of the M-PTC depends on the environmental pH and that the complex form of BoNT/A is induced by interacting with NTNHA-A at acidic pH. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication.

PubMed

Mattiroli, Francesca; Gu, Yajie; Yadav, Tejas; Balsbaugh, Jeremy L; Harris, Michael R; Findlay, Eileen S; Liu, Yang; Radebaugh, Catherine A; Stargell, Laurie A; Ahn, Natalie G; Whitehouse, Iestyn; Luger, Karolin

2017-03-18

Nucleosome assembly in the wake of DNA replication is a key process that regulates cell identity and survival. Chromatin assembly factor 1 (CAF-1) is a H3-H4 histone chaperone that associates with the replisome and orchestrates chromatin assembly following DNA synthesis. Little is known about the mechanism and structure of this key complex. Here we investigate the CAF-1•H3-H4 binding mode and the mechanism of nucleosome assembly. We show that yeast CAF-1 binding to a H3-H4 dimer activates the Cac1 winged helix domain interaction with DNA. This drives the formation of a transient CAF-1•histone•DNA intermediate containing two CAF-1 complexes, each associated with one H3-H4 dimer. Here, the (H3-H4) 2 tetramer is formed and deposited onto DNA. Our work elucidates the molecular mechanism for histone deposition by CAF-1, a reaction that has remained elusive for other histone chaperones, and it advances our understanding of how nucleosomes and their epigenetic information are maintained through DNA replication.

Acetylcholine molecular arrays enable quantum information processing

NASA Astrophysics Data System (ADS)

Tamulis, Arvydas; Majauskaite, Kristina; Talaikis, Martynas; Zborowski, Krzysztof; Kairys, Visvaldas

2017-09-01

We have found self-assembly of four neurotransmitter acetylcholine (ACh) molecular complexes in a water molecules environment by using geometry optimization with DFT B97d method. These complexes organizes to regular arrays of ACh molecules possessing electronic spins, i.e. quantum information bits. These spin arrays could potentially be controlled by the application of a non-uniform external magnetic field. The proper sequence of resonant electromagnetic pulses would then drive all the spin groups into the 3-spin entangled state and proceed large scale quantum information bits.

Reversible Self-Assembly of Supramolecular Vesicles and Nanofibers Driven by Chalcogen-Bonding Interactions.

PubMed

Chen, Liang; Xiang, Jun; Zhao, Yue; Yan, Qiang

2018-05-29

Chalcogen-bonding interactions have been viewed as new noncovalent forces in supramolecular chemistry. However, harnessing chalcogen bonds to drive molecular self-assembly processes is still unexplored. Here we report for the first time a novel class of supra-amphiphiles formed by Te···O or Se···O chalcogen-bonding interactions, and their self-assembly into supramolecular vesicles and nanofibers. A quasi-calix[4]chalcogenadiazole (C4Ch) as macrocyclic donor and a tailed pyridine N-oxide surfactant as molecular acceptor are designed to construct the donor-acceptor complex via chalcogen-chalcogen connection between the chalcogenadiazole moieties and oxide anion. The affinity of such chalcogen-bonding can dictate the geometry of supra-amphiphiles, driving diverse self-assembled morphologies. Furthermore, the reversible disassembly of these nanostructures can be promoted by introducing competing anions, such as halide ions, or by decreasing the systemic pH value.

Electrostatic Interactions and Self-Assembly in Polymeric Systems

NASA Astrophysics Data System (ADS)

Dobrynin, Andrey

Electrostatic interactions between macroions play an important role in different areas ranging from materials science to biophysics. They are main driving forces behind layer-by-layer assembly technique that allows self-assembly of multilayer films from synthetic polyelectrolytes, DNA, proteins and nanoparticles. They are responsible for complexation and reversible gelation between polyelectrolytes and proteins. In this talk, using results of the molecular dynamics simulations and analytical calculations, I will demonstrate what effect electrostatic interactions, counterion condensation and polymer solvent affinity have on a collapse of polyelectrolyte chain in a poor solvent conditions for the polymer backbone, on complexations and reversible gelation between polyelectrolytes and polyamholytes (unstructured proteins), on microphase separation transitions in spherical and planar charged brushes, and on a layer-by-layer assembly of charged nanoparticles and linear polyelectrolytes on charged surfaces. NSF DMR-1004576 DMR-1409710.

Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo

PubMed Central

Schaefer, Kristina N.; Williams, Clara E.; Roberts, David M.; McKay, Daniel J.

2018-01-01

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity. PMID:29641560

Superresolution Imaging Captures Carbohydrate Utilization Dynamics in Human Gut Symbionts

PubMed Central

Karunatilaka, Krishanthi S.; Cameron, Elizabeth A.; Martens, Eric C.; Koropatkin, Nicole M.

2014-01-01

ABSTRACT Gut microbes play a key role in human health and nutrition by catabolizing a wide variety of glycans via enzymatic activities that are not encoded in the human genome. The ability to recognize and process carbohydrates strongly influences the structure of the gut microbial community. While the effects of diet on the microbiota are well documented, little is known about the molecular processes driving metabolism. To provide mechanistic insight into carbohydrate catabolism in gut symbionts, we studied starch processing in real time in the model Bacteroides thetaiotaomicron starch utilization system (Sus) by single-molecule fluorescence. Although previous studies have explored Sus protein structure and function, the transient interactions, assembly, and collaboration of these outer membrane proteins have not yet been elucidated in live cells. Our live-cell superresolution imaging reveals that the polymeric starch substrate dynamically recruits Sus proteins, serving as an external scaffold for bacterial membrane assembly of the Sus complex, which may promote efficient capturing and degradation of starch. Furthermore, by simultaneously localizing multiple Sus outer membrane proteins on the B. thetaiotaomicron cell surface, we have characterized the dynamics and stoichiometry of starch-induced Sus complex assembly on the molecular scale. Finally, based on Sus protein knockout strains, we have discerned the mechanism of starch-induced Sus complex assembly in live anaerobic cells with nanometer-scale resolution. Our insights into the starch-induced outer membrane protein assembly central to this conserved nutrient uptake mechanism pave the way for the development of dietary or pharmaceutical therapies to control Bacteroidetes in the intestinal tract to enhance human health and treat disease. PMID:25389179

TAF11 Assembles the RISC Loading Complex to Enhance RNAi Efficiency.

PubMed

Liang, Chunyang; Wang, Yibing; Murota, Yukiko; Liu, Xiang; Smith, Dean; Siomi, Mikiko C; Liu, Qinghua

2015-09-03

Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains the Dicer-2 (Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila melanogaster. However, the precise composition and action mechanism of Drosophila RLC remain unclear. Here we identified the missing factor of RLC as TATA-binding protein-associated factor 11 (TAF11) by genetic screen. Although it is an annotated nuclear transcription factor, we found that TAF11 also associated with Dcr-2/R2D2 and localized to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11(-/-) ovary extract, we reconstituted the RLC in vitro using the recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, we showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, our genetic and biochemical studies define the molecular nature of the Drosophila RLC and elucidate a cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency. Copyright © 2015 Elsevier Inc. All rights reserved.

"Chemical transformers" from nanoparticle ensembles operated with logic.

PubMed

Motornov, Mikhail; Zhou, Jian; Pita, Marcos; Gopishetty, Venkateshwarlu; Tokarev, Ihor; Katz, Evgeny; Minko, Sergiy

2008-09-01

The pH-responsive nanoparticles were coupled with information-processing enzyme-based systems to yield "smart" signal-responsive hybrid systems with built-in Boolean logic. The enzyme systems performed AND/OR logic operations, transducing biochemical input signals into reversible structural changes (signal-directed self-assembly) of the nanoparticle assemblies, thus resulting in the processing and amplification of the biochemical signals. The hybrid system mimics biological systems in effective processing of complex biochemical information, resulting in reversible changes of the self-assembled structures of the nanoparticles. The bioinspired approach to the nanostructured morphing materials could be used in future self-assembled molecular robotic systems.

Stimulus-responsive light-harvesting complexes based on the pillararene-induced co-assembly of β-carotene and chlorophyll

NASA Astrophysics Data System (ADS)

Sun, Yan; Guo, Fang; Zuo, Tongfei; Hua, Jingjing; Diao, Guowang

2016-06-01

The locations and arrangements of carotenoids at the subcellular level are responsible for their designated functions, which reinforces the necessity of developing methods for constructing carotenoid-based suprastructures beyond the molecular level. Because carotenoids lack the binding sites necessary for controlled interactions, functional structures based on carotenoids are not easily obtained. Here, we show that carotene-based suprastructures were formed via the induction of pillararene through a phase-transfer-mediated host-guest interaction. More importantly, similar to the main component in natural photosynthesis, complexes could be synthesized after chlorophyll was introduced into the carotene-based suprastructure assembly process. Remarkably, compared with molecular carotene or chlorophyll, this synthesized suprastructure exhibits some photocatalytic activity when exposed to light, which can be exploited for photocatalytic reaction studies of energy capture and solar conversion in living organisms.

Kinetic control over pathway complexity in supramolecular polymerization through modulating the energy landscape by rational molecular design.

PubMed

Ogi, Soichiro; Fukui, Tomoya; Jue, Melinda L; Takeuchi, Masayuki; Sugiyasu, Kazunori

2014-12-22

Far-from-equilibrium thermodynamic systems that are established as a consequence of coupled equilibria are the origin of the complex behavior of biological systems. Therefore, research in supramolecular chemistry has recently been shifting emphasis from a thermodynamic standpoint to a kinetic one; however, control over the complex kinetic processes is still in its infancy. Herein, we report our attempt to control the time evolution of supramolecular assembly in a process in which the supramolecular assembly transforms from a J-aggregate to an H-aggregate over time. The transformation proceeds through a delicate interplay of these two aggregation pathways. We have succeeded in modulating the energy landscape of the respective aggregates by a rational molecular design. On the basis of this understanding of the energy landscape, programming of the time evolution was achieved through adjusting the balance between the coupled equilibria. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular architecture of the Spire–actin nucleus and its implication for actin filament assembly

PubMed Central

Sitar, Tomasz; Gallinger, Julia; Ducka, Anna M.; Ikonen, Teemu P.; Wohlhoefler, Michael; Schmoller, Kurt M.; Bausch, Andreas R.; Joel, Peteranne; Trybus, Kathleen M.; Noegel, Angelika A.; Schleicher, Michael; Huber, Robert; Holak, Tad A.

2011-01-01

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire–actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire–actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott–Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire–actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire–actin module. In addition, we find that preformed, isolated Spire–actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs—even a single WH2 repeat—sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem. PMID:22106272

Minimalistic peptide supramolecular co-assembly: expanding the conformational space for nanotechnology.

PubMed

Makam, Pandeeswar; Gazit, Ehud

2018-05-21

Molecular self-assembly is a ubiquitous process in nature and central to bottom-up nanotechnology. In particular, the organization of peptide building blocks into ordered supramolecular structures has gained much interest due to the unique properties of the products, including biocompatibility, chemical and structural diversity, robustness and ease of large-scale synthesis. In addition, peptides, as short as dipeptides, contain all the molecular information needed to spontaneously form well-ordered structures at both the nano- and the micro-scale. Therefore, peptide supramolecular assembly has been effectively utilized to produce novel materials with tailored properties for various applications in the fields of material science, engineering, medicine, and biology. To further expand the conformational space of peptide assemblies in terms of structural and functional complexity, multicomponent (two or more) peptide supramolecular co-assembly has recently evolved as a promising extended approach, similar to the structural diversity of natural sequence-defined biopolymers (proteins) as well as of synthetic covalent co-polymers. The use of this methodology was recently demonstrated in various applications, such as nanostructure physical dimension control, the creation of non-canonical complex topologies, mechanical strength modulation, the design of light harvesting soft materials, fabrication of electrically conducting devices, induced fluorescence, enzymatic catalysis and tissue engineering. In light of these significant advancements in the field of peptide supramolecular co-assembly in the last few years, in this tutorial review, we provide an updated overview and future prospects of this emerging subject.

Harnessing Thin-Film Continuous-Flow Assembly Lines.

PubMed

Britton, Joshua; Castle, Jared W; Weiss, Gregory A; Raston, Colin L

2016-07-25

Inspired by nature's ability to construct complex molecules through sequential synthetic transformations, an assembly line synthesis of α-aminophosphonates has been developed. In this approach, simple starting materials are continuously fed through a thin-film reactor where the intermediates accrue molecular complexity as they progress through the flow system. Flow chemistry allows rapid multistep transformations to occur via reaction compartmentalization, an approach not amenable to using conventional flasks. Thin film processing can also access facile in situ solvent exchange to drive reaction efficiency, and through this method, α-aminophosphonate synthesis requires only 443 s residence time to produce 3.22 g h(-1) . Assembly-line synthesis allows unprecedented reaction flexibility and processing efficiency. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Correlating the magic numbers of inorganic nanomolecular assemblies with a {Pd84} molecular-ring Rosetta Stone

PubMed Central

Xu, Feng; Miras, Haralampos N.; Scullion, Rachel A.; Long, De-Liang; Thiel, Johannes; Cronin, Leroy

2012-01-01

Molecular self-assembly has often been suggested as the ultimate route for the bottom-up construction of building blocks atom-by-atom for functional nanotechnology, yet structural design or prediction of nanomolecular assemblies is still far from reach. Whereas nature uses complex machinery such as the ribosome, chemists use painstakingly engineered step-by-step approaches to build complex molecules but the size and complexity of such molecules, not to mention the accessible yields, can be limited. Herein we present the discovery of a palladium oxometalate {Pd84}-ring cluster 3.3 nm in diameter; [Pd84O42(OAc)28(PO4)42]70- ({Pd84} ≡ {Pd12}7) that is formed in water just by mixing two reagents at room temperature, giving crystals of the compound in just a few days. The structure of the {Pd84}-ring has sevenfold symmetry, comprises 196 building blocks, and we also show, using mass spectrometry, that a large library of other related nanostructures is present in solution. Finally, by analysis of the symmetry and the building block library that construct the {Pd84} we show that the correlation of the symmetry, subunit number, and overall cluster nuclearity can be used as a “Rosetta Stone” to rationalize the “magic numbers” defining a number of other systems. This is because the discovery of {Pd84} allows the relationship between seemingly unrelated families of molecular inorganic nanosystems to be decoded from the overall cluster magic-number nuclearity, to the symmetry and building blocks that define such structures allowing the prediction of other members of these nanocluster families. PMID:22753516

Bcs1p can rescue a large and productive cytochrome bc(1) complex assembly intermediate in the inner membrane of yeast mitochondria.

PubMed

Conte, Laura; Trumpower, Bernard L; Zara, Vincenzo

2011-01-01

The yeast cytochrome bc(1) complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc(1) complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc(1) assembly and the formation of a functionally inactive bc(1) core structure of about 500-kDa. This immature bc(1) core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc(1) core structure leading to the formation of the functional homodimeric bc(1) complex. Following Bcs1p expression, the mature bc(1) complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc(1) complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc(1) complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc(1) core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc(1) complex and gives new insights into the molecular mechanisms involved in the last steps of bc(1) assembly. Copyright © 2010 Elsevier B.V. All rights reserved.

Self-assembly of Janus dendrimers into uniform dendrimersomes and other complex architectures.

PubMed

Percec, Virgil; Wilson, Daniela A; Leowanawat, Pawaret; Wilson, Christopher J; Hughes, Andrew D; Kaucher, Mark S; Hammer, Daniel A; Levine, Dalia H; Kim, Anthony J; Bates, Frank S; Davis, Kevin P; Lodge, Timothy P; Klein, Michael L; DeVane, Russell H; Aqad, Emad; Rosen, Brad M; Argintaru, Andreea O; Sienkowska, Monika J; Rissanen, Kari; Nummelin, Sami; Ropponen, Jarmo

2010-05-21

Self-assembled nanostructures obtained from natural and synthetic amphiphiles serve as mimics of biological membranes and enable the delivery of drugs, proteins, genes, and imaging agents. Yet the precise molecular arrangements demanded by these functions are difficult to achieve. Libraries of amphiphilic Janus dendrimers, prepared by facile coupling of tailored hydrophilic and hydrophobic branched segments, have been screened by cryogenic transmission electron microscopy, revealing a rich palette of morphologies in water, including vesicles, denoted dendrimersomes, cubosomes, disks, tubular vesicles, and helical ribbons. Dendrimersomes marry the stability and mechanical strength obtainable from polymersomes with the biological function of stabilized phospholipid liposomes, plus superior uniformity of size, ease of formation, and chemical functionalization. This modular synthesis strategy provides access to systematic tuning of molecular structure and of self-assembled architecture.

Off-pathway assembly of fimbria subunits is prevented by chaperone CfaA of CFA/I fimbriae from enterotoxigenic E. coli.

PubMed

Bao, Rui; Liu, Yang; Savarino, Stephen J; Xia, Di

2016-12-01

The assembly of the class 5 colonization factor antigen I (CFA/I) fimbriae of enterotoxigenic E. coli was proposed to proceed via the alternate chaperone-usher pathway. Here, we show that in the absence of the chaperone CfaA, CfaB, the major pilin subunit of CFA/I fimbriae, is able to spontaneously refold and polymerize into cyclic trimers. CfaA kinetically traps CfaB to form a metastable complex that can be stabilized by mutations. Crystal structure of the stabilized complex reveals distinctive interactions provided by CfaA to trap CfaB in an assembly competent state through donor-strand complementation (DSC) and cleft-mediated anchorage. Mutagenesis indicated that DSC controls the stability of the chaperone-subunit complex and the cleft-mediated anchorage of the subunit C-terminus additionally assist in subunit refolding. Surprisingly, over-stabilization of the chaperone-subunit complex led to delayed fimbria assembly, whereas destabilizing the complex resulted in no fimbriation. Thus, CfaA acts predominantly as a kinetic trap by stabilizing subunit to avoid its off-pathway self-polymerization that results in energetically favorable trimers and could serve as a driving force for CFA/I pilus assembly, representing an energetic landscape unique to class 5 fimbria assembly. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Molecular Microbiology published by John Wiley & Sons Ltd.

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

Llave, Ezequiel de la; Herrera, Santiago E.; Adam, Catherine

The molecular and electronic structure of Os(II) complexes covalently bonded to self-assembled monolayers (SAMs) on Au(111) surfaces was studied by means of polarization modulation infrared reflection absorption spectroscopy, photoelectron spectroscopies, scanning tunneling microscopy, scanning tunneling spectroscopy, and density functional theory calculations. Attachment of the Os complex to the SAM proceeds via an amide covalent bond with the SAM alkyl chain 40° tilted with respect to the surface normal and a total thickness of 26 Å. The highest occupied molecular orbital of the Os complex is mainly based on the Os(II) center located 2.2 eV below the Fermi edge and themore » LUMO molecular orbital is mainly based on the bipyridine ligands located 1.5 eV above the Fermi edge.« less

DNA-programmed dynamic assembly of quantum dots for molecular computation.

PubMed

He, Xuewen; Li, Zhi; Chen, Muzi; Ma, Nan

2014-12-22

Despite the widespread use of quantum dots (QDs) for biosensing and bioimaging, QD-based bio-interfaceable and reconfigurable molecular computing systems have not yet been realized. DNA-programmed dynamic assembly of multi-color QDs is presented for the construction of a new class of fluorescence resonance energy transfer (FRET)-based QD computing systems. A complete set of seven elementary logic gates (OR, AND, NOR, NAND, INH, XOR, XNOR) are realized using a series of binary and ternary QD complexes operated by strand displacement reactions. The integration of different logic gates into a half-adder circuit for molecular computation is also demonstrated. This strategy is quite versatile and straightforward for logical operations and would pave the way for QD-biocomputing-based intelligent molecular diagnostics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Assembling the Tat protein translocase

PubMed Central

Alcock, Felicity; Stansfeld, Phillip J; Basit, Hajra; Habersetzer, Johann; Baker, Matthew AB; Palmer, Tracy; Wallace, Mark I; Berks, Ben C

2016-01-01

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes. DOI: http://dx.doi.org/10.7554/eLife.20718.001 PMID:27914200

Structural and Functional Analyses of the Proteins Involved in the Iron-Sulfur Cluster Biosynthesis

NASA Astrophysics Data System (ADS)

Wada, Kei

The iron-sulfur (Fe-S) clusters are ubiquitous prosthetic groups that are required to maintain such fundamental life processes as respiratory chain, photosynthesis and the regulation of gene expression. Assembly of intracellular Fe-S cluster requires the sophisticated biosynthetic systems called ISC and SUF machineries. To shed light on the molecular mechanism of Fe-S cluster assembly mediated by SUF machinery, several structures of the SUF components and their sub-complex were determined. The structural findings together with biochemical characterization of the core-complex (SufB-SufC-SufD complex) have led me to propose a working model for the cluster biosynthesis in the SUF machinery.

Molecular architecture of the human Mediator-RNA polymerase II-TFIIF assembly.

PubMed

Bernecky, Carrie; Grob, Patricia; Ebmeier, Christopher C; Nogales, Eva; Taatjes, Dylan J

2011-03-01

The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator-pol II interface is not well-characterized, whereas attempts to structurally define the Mediator-pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator-pol II-TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator-pol II-TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator-pol II-TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator-CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator-pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing pol II orientation within the assembly.

Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly

PubMed Central

Bernecky, Carrie; Grob, Patricia; Ebmeier, Christopher C.; Nogales, Eva; Taatjes, Dylan J.

2011-01-01

The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator–pol II interface is not well-characterized, whereas attempts to structurally define the Mediator–pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator–pol II–TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator–pol II–TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator–pol II–TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator–CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator–pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing pol II orientation within the assembly. PMID:21468301

Fabrication of sophisticated two-dimensional organic nanoarchitectures thought hydrogen bond mediated molecular self assembly

NASA Astrophysics Data System (ADS)

Silly, Fabien

2012-02-01

Complex supramolecular two-dimensional (2D) networks are attracting considerable interest as highly ordered functional materials for applications in nanotechnology. The challenge consists in tailoring the ordering of one or more molecular species into specific architectures over an extended length scale with molecular precision. Highly organized supramolecular arrays can be obtained through self-assembly of complementary molecules which can interlock via intermolecular interactions. Molecules forming hydrogen bonds (H-bonds) are especially interesting building blocks for creating sophisticated organic architectures due to high selectivity and directionality of these bindings. We used scanning tunnelling microscopy to investigate at the atomic scale the formation of H-bonded 2D organic nanoarchitectures on surfaces. We mixed perylene derivatives having rectangular shape with melamine and DNA base having triangular and non symmetric shape respectively. We observe that molecule substituents play a key role in formation of the multicomponent H-bonded architectures. We show that the 2D self-assembly of these molecules can be tailored by adjusting the temperature and molecular ratio. We used these stimuli to successfully create numerous close-packed and porous 2D multicomponent structures.

Modular assembly of chimeric phi29 packaging RNAs that support DNA packaging.

PubMed

Fang, Yun; Shu, Dan; Xiao, Feng; Guo, Peixuan; Qin, Peter Z

2008-08-08

The bacteriophage phi29 DNA packaging motor is a protein/RNA complex that can produce strong force to condense the linear-double-stranded DNA genome into a pre-formed protein capsid. The RNA component, called the packaging RNA (pRNA), utilizes magnesium-dependent inter-molecular base-pairing interactions to form ring-shaped complexes. The pRNA is a class of non-coding RNA, interacting with phi29 motor proteins to enable DNA packaging. Here, we report a two-piece chimeric pRNA construct that is fully competent in interacting with partner pRNA to form ring-shaped complexes, in packaging DNA via the motor, and in assembling infectious phi29 virions in vitro. This is the first example of a fully functional pRNA assembled using two non-covalently interacting fragments. The results support the notion of modular pRNA architecture in the phi29 packaging motor.

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery: THE SUB-COMPLEX FORMED BY THE IRON DONOR, Yfh1 PROTEIN, AND THE SCAFFOLD, Isu1 PROTEIN.

PubMed

Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K; Smith, Douglas Y; Söderberg, Christopher A G; Al-Karadaghi, Salam; Thompson, James R; Isaya, Grazia

2016-05-06

The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24 Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Pwp2 mediates UTP-B assembly via two structurally independent domains.

PubMed

Boissier, Fanny; Schmidt, Christina Maria; Linnemann, Jan; Fribourg, Sébastien; Perez-Fernandez, Jorge

2017-06-09

The SSU processome constitutes a large ribonucleoprotein complex involved in the early steps of ribosome biogenesis. UTP-B is one of the first multi-subunit protein complexes that associates with the pre-ribosomal RNA to form the SSU processome. To understand the molecular basis of the hierarchical assembly of the SSU-processome, we have undergone a structural and functional analysis of the UTP-B subunit Pwp2p. We show that Pwp2p is required for the proper assembly of UTP-B and for a productive association of UTP-B with pre-rRNA. These two functions are mediated by two distinct structural domains. The N-terminal domain of Pwp2p folds into a tandem WD-repeat (tWD) that associates with Utp21p, Utp18p, and Utp6p to form a core complex. The CTDs of Pwp2p and Utp21p mediate the assembly of the heterodimer Utp12p:Utp13p that is required for the stable incorporation of the UTP-B complex in the SSU processome. Finally, we provide evidence suggesting a role of UTP-B as a platform for the binding of assembly factors during the maturation of 20S rRNA precursors.

Phosphatidylinositol 4,5-bisphosphate clusters the cell adhesion molecule CD44 and assembles a specific CD44-Ezrin heterocomplex, as revealed by small angle neutron scattering.

PubMed

Chen, Xiaodong; Khajeh, Jahan Ali; Ju, Jeong Ho; Gupta, Yogesh K; Stanley, Christopher B; Do, Changwoo; Heller, William T; Aggarwal, Aneel K; Callaway, David J E; Bu, Zimei

2015-03-06

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular insights into early stage aggregation of di-Fmoc-L-lysine in binary mixture of organic solvent and water

NASA Astrophysics Data System (ADS)

Huda, Md Masrul; Rai, Neeraj

Molecular gels are relatively new class of soft materials, which are formed by the supramolecular aggregation of low molecular weight gelators (LMWGs) in organic solvents and/or water. Hierarchical self-assembly of small gelator molecules lead to three-dimensional complex fibrillar networks, which restricts the flow of solvents and results in viscous solid like materials or gels. These gels have drawn significant attentions for their potential applications for drug delivery, tissue engineering, materials for sensors etc. As of now, self-assembly of gelator molecules into one-dimensional fibers is not well understood, although that is very important to design new gelators for desired applications. Here, we present molecular dynamics study that provides molecular level insight into early stage aggregation of selected gelator, di-Fmoc-L-lysine in binary mixture of organic solvent and water. We will present the role of different functional groups of gelator molecule such as aromatic ring, amide, and carboxylic group on aggregation. We will also present the effect of concentrations of gelator and solvent on self-assembly of gelators. This study has captured helical fiber growth and branching of fiber, which is in good agreement with experimental observations.

Templated electrokinetic directed chemical assembly for the fabrication of close-packed plasmonic metamolecules

NASA Astrophysics Data System (ADS)

Thrift, W. J.; Darvishzadeh-Varcheie, M.; Capolino, F.; Ragan, R.

2017-08-01

Colloidal self-assembly combined with templated surfaces holds the promise of fabricating large area devices in a low cost facile manner. This directed assembly approach improves the complexity of assemblies that can be achieved with self-assembly while maintaining advantages of molecular scale control. In this work, electrokinetic driving forces, i.e., electrohydrodynamic flow, are paired with chemical crosslinking between colloidal particles to form close-packed plasmonic metamolecules. This method addresses challenges of obtaining uniformity in nanostructure geometry and nanometer scale gap spacings in structures. Electrohydrodynamic flows yield robust driving forces between the template and nanoparticles as well as between nanoparticles on the surface promoting the assembly of close-packed metamolecules. Here, electron beam lithography defined Au pillars are used as seed structures that generate electrohydrodynamic flows. Chemical crosslinking between Au surfaces enables molecular control over gap spacings between nanoparticles and Au pillars. An as-fabricated structure is analyzed via full wave electromagnetic simulations and shown to produce large magnetic field enhancements on the order of 3.5 at optical frequencies. This novel method for directed self-assembly demonstrates the synergy between colloidal driving forces and chemical crosslinking for the fabrication of plasmonic metamolecules with unique electromagnetic properties.

Meeting Report: Structural Determination of Environmentally Responsive Proteins

PubMed Central

Reinlib, Leslie

2005-01-01

The three-dimensional structure of gene products continues to be a missing lynchpin between linear genome sequences and our understanding of the normal and abnormal function of proteins and pathways. Enhanced activity in this area is likely to lead to better understanding of how discrete changes in molecular patterns and conformation underlie functional changes in protein complexes and, with it, sensitivity of an individual to an exposure. The National Institute of Environmental Health Sciences convened a workshop of experts in structural determination and environmental health to solicit advice for future research in structural resolution relative to environmentally responsive proteins and pathways. The highest priorities recommended by the workshop were to support studies of structure, analysis, control, and design of conformational and functional states at molecular resolution for environmentally responsive molecules and complexes; promote understanding of dynamics, kinetics, and ligand responses; investigate the mechanisms and steps in posttranslational modifications, protein partnering, impact of genetic polymorphisms on structure/function, and ligand interactions; and encourage integrated experimental and computational approaches. The workshop participants also saw value in improving the throughput and purity of protein samples and macromolecular assemblies; developing optimal processes for design, production, and assembly of macromolecular complexes; encouraging studies on protein–protein and macromolecular interactions; and examining assemblies of individual proteins and their functions in pathways of interest for environmental health. PMID:16263521

Self-assembly of chlorophenols in water

PubMed Central

Rogalska, Ewa; Rogalski, Marek; Gulik-Krzywicki, Tadeusz; Gulik, Annette; Chipot, Christophe

1999-01-01

In saturated solutions of some di- and trichlorophenols, structures with complex morphologies, consisting of thin, transparent sheets often coiling into helices and ultimately twisting into filaments, were observed under the optical microscope. Freeze-fracture electron microscopy, x-ray diffraction, phase diagrams, and molecular modeling were performed to elucidate the observed phenomena. Here, we present evidence that the chlorophenols studied, when interacting with water, self-assemble into bilayers. The fact that some chlorophenols form the same supramolecular structures as those described previously for structurally nonrelated surfactants sheds light on the mechanisms of self-assembly. PMID:10359753

An auto-biotinylated bifunctional protein nanowire for ultra-sensitive molecular biosensing.

PubMed

Men, Dong; Zhang, Zhi-Ping; Guo, Yong-Chao; Zhu, Duan-Hao; Bi, Li-Jun; Deng, Jiao-Yu; Cui, Zong-Qiang; Wei, Hong-Ping; Zhang, Xian-En

2010-12-15

In order to obtain an ultra-sensitive molecular biosensor, we designed an auto-biotinylated bifunctional protein nanowire (bFPNw) based on the self-assembly of a yeast amyloid protein, Sup35, to which protein G and a biotin acceptor peptide (BAP) were genetically fused. These auto-biotinylated bFPNws can transfer hundreds of commercially available diagnostic enzymes to an antigen-antibody complex via the biotin-avidin system, greatly enhancing the sensitivity of immune-biosensing. Compared to our previously reported seeding-induced bFPNws (Men et al., 2009), these auto-biotinylated bFPNws gave greater signal amplification, reduced non-specific binding and improved stability. The auto-biotinylated self-assembled bFPNw molecular biosensors were applied to detect Yersinia pestis (Y. pestis) F1 antigen and showed a 2000- to 4000-fold increase in sensitivity compared to traditional immunoassays, demonstrating the potential use of these self-assembling protein nanowires in biosensing. Copyright © 2010 Elsevier B.V. All rights reserved.

Direct Nanoscale Characterization of Submolecular Mobility in Complex Organic Non-linear Optical Systems

NASA Astrophysics Data System (ADS)

Knorr, Daniel; Gray, Tomoko; Kim, Tae-Dong; Luo, Jingdong; Jen, Alex; Overney, Rene

2008-03-01

For organic non-linear optical (NLO) materials composed of intricate molecular building blocks, the challenge is to deduce meaningful molecular scale mobility information to understand complex relaxation and phase behavior. This is crucial, as the process of achieving a robust acentric alignment strongly depends on the availability of inter- and intra-molecular mobilities outside the temperature range of the device operation window. Here, we introduce a nanoscale methodology based on scanning probe microscopy that provides direct insight into structural relaxations and shows great potential to direct material design of sophisticated macromolecules. It also offers a means by which mesoscale dynamics and cooperativity involved in relaxation processes can be quantified in terms of dynamic entropy and enthalpy. This study demonstrates this methodology to describe the mesocale dynamics of two systems (1) organic networking dendronized NLO molecular glasses that self-assemble into physically linked polymers due to quadrupolar phenyl-perfluorophenyl interactions and (2) dendronized side-chain electro-optic (EO) polymers. For the self assembling glasses, the degree of intermolecular cooperativity can be deduced using this methodology, while for the dendronized side-chain polymers, specific side chain mobilities are exploited to improve EO properties.

Stimulus-responsive light-harvesting complexes based on the pillararene-induced co-assembly of β-carotene and chlorophyll

PubMed Central

Sun, Yan; Guo, Fang; Zuo, Tongfei; Hua, Jingjing; Diao, Guowang

2016-01-01

The locations and arrangements of carotenoids at the subcellular level are responsible for their designated functions, which reinforces the necessity of developing methods for constructing carotenoid-based suprastructures beyond the molecular level. Because carotenoids lack the binding sites necessary for controlled interactions, functional structures based on carotenoids are not easily obtained. Here, we show that carotene-based suprastructures were formed via the induction of pillararene through a phase-transfer-mediated host–guest interaction. More importantly, similar to the main component in natural photosynthesis, complexes could be synthesized after chlorophyll was introduced into the carotene-based suprastructure assembly process. Remarkably, compared with molecular carotene or chlorophyll, this synthesized suprastructure exhibits some photocatalytic activity when exposed to light, which can be exploited for photocatalytic reaction studies of energy capture and solar conversion in living organisms. PMID:27345928

Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling

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

Lin, Su-Chang; Lo, Yu-Chih; Wu, Hao

2010-08-23

MyD88, IRAK4 and IRAK2 are critical signalling mediators of the TLR/IL1-R superfamily. Here we report the crystal structure of the MyD88-IRAK4-IRAK2 death domain (DD) complex, which surprisingly reveals a left-handed helical oligomer that consists of 6 MyD88, 4 IRAK4 and 4 IRAK2 DDs. Assembly of this helical signalling tower is hierarchical, in which MyD88 recruits IRAK4 and the MyD88-IRAK4 complex recruits the IRAK4 substrates IRAK2 or the related IRAK1. Formation of these Myddosome complexes brings the kinase domains of IRAKs into proximity for phosphorylation and activation. Composite binding sites are required for recruitment of the individual DDs in the complex,more » which are confirmed by mutagenesis and previously identified signalling mutations. Specificities in Myddosome formation are dictated by both molecular complementarity and correspondence of surface electrostatics. The MyD88-IRAK4-IRAK2 complex provides a template for Toll signalling in Drosophila and an elegant mechanism for versatile assembly and regulation of DD complexes in signal transduction.« less

Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.

PubMed

Iki, Taichiro; Yoshikawa, Manabu; Meshi, Tetsuo; Ishikawa, Masayuki

2012-01-18

Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90-AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants.

KDM2B Recruitment of the Polycomb Group Complex, PRC1.1, Requires Cooperation between PCGF1 and BCORL1.

PubMed

Wong, Sarah J; Gearhart, Micah D; Taylor, Alexander B; Nanyes, David R; Ha, Daniel J; Robinson, Angela K; Artigas, Jason A; Lee, Oliver J; Demeler, Borries; Hart, P John; Bardwell, Vivian J; Kim, Chongwoo A

2016-10-04

KDM2B recruits H2A-ubiquitinating activity of a non-canonical Polycomb Repression Complex 1 (PRC1.1) to CpG islands, facilitating gene repression. We investigated the molecular basis of recruitment using in vitro assembly assays to identify minimal components, subcomplexes, and domains required for recruitment. A minimal four-component PRC1.1 complex can be assembled by combining two separately isolated subcomplexes: the DNA-binding KDM2B/SKP1 heterodimer and the heterodimer of BCORL1 and PCGF1, a core component of PRC1.1. The crystal structure of the KDM2B/SKP1/BCORL1/PCGF1 complex illustrates the crucial role played by the PCGF1/BCORL1 heterodimer. The BCORL1 PUFD domain positions residues preceding the RAWUL domain of PCGF1 to create an extended interface for interaction with KDM2B, which is unique to the PCGF1-containing PRC1.1 complex. The structure also suggests how KDM2B might simultaneously function in PRC1.1 and an SCF ubiquitin ligase complex and the possible molecular consequences of BCOR PUFD internal tandem duplications found in pediatric kidney and brain tumors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modeling of the U1 snRNP assembly pathway in alternative splicing in human cells using Petri nets.

PubMed

Kielbassa, J; Bortfeldt, R; Schuster, S; Koch, I

2009-02-01

The investigation of spliceosomal processes is currently a topic of intense research in molecular biology. In the molecular mechanism of alternative splicing, a multi-protein-RNA complex - the spliceosome - plays a crucial role. To understand the biological processes of alternative splicing, it is essential to comprehend the biogenesis of the spliceosome. In this paper, we propose the first abstract model of the regulatory assembly pathway of the human spliceosomal subunit U1. Using Petri nets, we describe its highly ordered assembly that takes place in a stepwise manner. Petri net theory represents a mathematical formalism to model and analyze systems with concurrent processes at different abstraction levels with the possibility to combine them into a uniform description language. There exist many approaches to determine static and dynamic properties of Petri nets, which can be applied to analyze biochemical systems. In addition, Petri net tools usually provide intuitively understandable graphical network representations, which facilitate the dialog between experimentalists and theoreticians. Our Petri net model covers binding, transport, signaling, and covalent modification processes. Through the computation of structural and behavioral Petri net properties and their interpretation in biological terms, we validate our model and use it to get a better understanding of the complex processes of the assembly pathway. We can explain the basic network behavior, using minimal T-invariants which represent special pathways through the network. We find linear as well as cyclic pathways. We determine the P-invariants that represent conserved moieties in a network. The simulation of the net demonstrates the importance of the stability of complexes during the maturation pathway. We can show that complexes that dissociate too fast, hinder the formation of the complete U1 snRNP.

Self-Assembly Strategies for Integrating Light Harvesting and Charge Separation in Artificial Photosynthetic Systems

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

Wasielewski, Michael R.

In natural photosynthesis, organisms optimize solar energy conversion through organized assemblies of photofunctional chromophores and catalysts within proteins that provide specifically tailored environments for chemical reactions. As with their natural counterparts, artificial photosynthetic systems for practical solar fuels production must collect light energy, separate charge, and transport charge to catalytic sites where multielectron redox processes will occur. While encouraging progress has been made on each aspect of this complex problem, researchers have not yet developed self-ordering and self-assembling components and the tailored environments necessary to realize a fully-functional artificial system. Previously researchers have used complex, covalent molecular systems comprised ofmore » chromophores, electron donors, and electron acceptors to mimic both the light-harvesting and the charge separation functions of photosynthetic proteins. These systems allow for study of the dependencies of electron transfer rate constants on donor?acceptor distance and orientation, electronic interaction, and the free energy of the reaction. The most useful and informative systems are those in which structural constraints control both the distance and the orientation between the electron donors and acceptors. Self-assembly provides a facile means for organizing large numbers of molecules into supramolecular structures that can bridge length scales from nanometers to macroscopic dimensions. The resulting structures must provide pathways for migration of light excitation energy among antenna chromophores, and from antennas to reaction centers. They also must incorporate charge conduits, that is, molecular 'wires' that can efficiently move electrons and holes between reaction centers and catalytic sites. The central scientific challenge is to develop small, functional building blocks with a minimum number of covalent linkages, which also have the appropriate molecular recognition properties to facilitate self-assembly of complete, functional artificial photosynthetic systems. In this Account, we explore how self-assembly strategies involving ?-stacking can be used to integrate light harvesting with charge separation and transport.« less

Distinctive Roles for Periplasmic Proteases in the Maintenance of Essential Outer Membrane Protein Assembly.

PubMed

Soltes, Garner R; Martin, Nicholas R; Park, Eunhae; Sutterlin, Holly A; Silhavy, Thomas J

2017-10-15

Outer membrane protein (OMP) biogenesis in Escherichia coli is a robust process essential to the life of the organism. It is catalyzed by the β-barrel assembly machine (Bam) complex, and a number of quality control factors, including periplasmic chaperones and proteases, maintain the integrity of this trafficking pathway. Little is known, however, about how periplasmic proteases recognize and degrade OMP substrates when assembly is compromised or whether different proteases recognize the same substrate at distinct points in the assembly pathway. In this work, we use well-defined assembly-defective mutants of LptD, the essential lipopolysaccharide assembly translocon, to show that the periplasmic protease DegP degrades substrates with assembly defects that prevent or impair initial contact with Bam, causing the mutant protein to accumulate in the periplasm. In contrast, another periplasmic protease, BepA, degrades a LptD mutant substrate that has engaged the Bam complex and formed a nearly complete barrel. Furthermore, we describe the role of the outer membrane lipoprotein YcaL, a protease of heretofore unknown function, in the degradation of a LptD substrate that has engaged the Bam complex but is stalled at an earlier step in the assembly process that is not accessible to BepA. Our results demonstrate that multiple periplasmic proteases monitor OMPs at distinct points in the assembly process. IMPORTANCE OMP assembly is catalyzed by the essential Bam complex and occurs in a cellular environment devoid of energy sources. Assembly intermediates that misfold can compromise this essential molecular machine. Here we demonstrate distinctive roles for three different periplasmic proteases that can clear OMP substrates with folding defects that compromise assembly at three different stages. These quality control factors help ensure the integrity of the permeability barrier that contributes to the intrinsic resistance of Gram-negative organisms to many antibiotics. Copyright © 2017 American Society for Microbiology.

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

PubMed

Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon; Forbes, Douglass J

2010-12-01

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

Soil Organic Matter (SOM): Molecular Simulations

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

Andersen, Amity

Molecular simulation is a powerful tool used to gain an atomistic, molecular, and nanoscale level understanding of the structure, dynamics, and interactions from adsorption on minerals and assembly in aggregates of soil organic matter (SOM). Given the importance of SOM fate and persistence in soils and the current knowledge gaps, applications of atomistic scale simulations to study the complex compounds in SOM and their interactions in self-assembled aggregates composed of different organic matter compounds and with mineral surfaces of different types common in soils are few and far between. Here, we describe various molecular simulation methods that are currently inmore » use in various areas and applicable to SOM research, followed by a brief survey of specific applications to SOM research and an illustration with our own recent efforts in this area. We conclude with an outlook and the challenges for future research in this area.« less

Using Synchrotron Radiation and Electron Microscopy to Map the Huge Structural Changes that Occur in Viruses During Their Life Cycle

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

Rossman, Michael

2011-09-07

The crystallographic techniques for structure determination of proteins and neucleic acids at near atomic resolution using synchrotron X-radiation has become almost automatic. However the limits of this procedure are determined by the availability of crystals. As the size and complexity of the molecular assemblies being studied increases, the likelihood of growing useful crystals diminishes. Cryo electron microscopy and tomography have extended the range of biological objects that can be determined at near atomic resolution. Furthermore it is now becoming apparent that the function of the molecular assemblies most often requires very large conformational changes that could never be contained withinmore » a crystal, Examples will be presented of the structural changes that occur in viruses as they assembly and prepare to infect new cells.« less

Sample preparation for SFM imaging of DNA, proteins, and DNA-protein complexes.

PubMed

Ristic, Dejan; Sanchez, Humberto; Wyman, Claire

2011-01-01

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate, and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nanometer resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA-bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA, and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

A high quantum yield molecule-protein complex fluorophore for near-infrared II imaging

PubMed Central

Antaris, Alexander L.; Chen, Hao; Diao, Shuo; Ma, Zhuoran; Zhang, Zhe; Zhu, Shoujun; Wang, Joy; Lozano, Alexander X.; Fan, Quli; Chew, Leila; Zhu, Mark; Cheng, Kai; Hong, Xuechuan; Dai, Hongjie; Cheng, Zhen

2017-01-01

Fluorescence imaging in the second near-infrared window (NIR-II) allows visualization of deep anatomical features with an unprecedented degree of clarity. NIR-II fluorophores draw from a broad spectrum of materials spanning semiconducting nanomaterials to organic molecular dyes, yet unfortunately all water-soluble organic molecules with >1,000 nm emission suffer from low quantum yields that have limited temporal resolution and penetration depth. Here, we report tailoring the supramolecular assemblies of protein complexes with a sulfonated NIR-II organic dye (CH-4T) to produce a brilliant 110-fold increase in fluorescence, resulting in the highest quantum yield molecular fluorophore thus far. The bright molecular complex allowed for the fastest video-rate imaging in the second NIR window with ∼50-fold reduced exposure times at a fast 50 frames-per-second (FPS) capable of resolving mouse cardiac cycles. In addition, we demonstrate that the NIR-II molecular complexes are superior to clinically approved ICG for lymph node imaging deep within the mouse body. PMID:28524850

A high quantum yield molecule-protein complex fluorophore for near-infrared II imaging

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

Antaris, Alexander L.; Chen, Hao; Diao, Shuo

Fluorescence imaging in the second near-infrared window (NIR-II) allows visualization of deep anatomical features with an unprecedented degree of clarity. NIR-II fluorophores draw from a broad spectrum of materials spanning semiconducting nanomaterials to organic molecular dyes, yet unfortunately all water-soluble organic molecules with 41,000 nm emission suffer from low quantum yields that have limited temporal resolution and penetration depth. We report tailoring the supramolecular assemblies of protein complexes with a sulfonated NIR-II organic dye (CH-4T) to produce a brilliant 110-fold increase in fluorescence, resulting in the highest quantum yield molecular fluorophore thus far. The bright molecular complex allowed for themore » fastest video-rate imaging in the second NIR window with B50-fold reduced exposure times at a fast 50 frames-per-second (FPS) capable of resolving mouse cardiac cycles. Additionally, we demonstrate that the NIR-II molecular complexes are superior to clinically approved ICG for lymph node imaging deep within the mouse body.« less

A high quantum yield molecule-protein complex fluorophore for near-infrared II imaging

DOE PAGES

Antaris, Alexander L.; Chen, Hao; Diao, Shuo; ...

2017-05-19

Fluorescence imaging in the second near-infrared window (NIR-II) allows visualization of deep anatomical features with an unprecedented degree of clarity. NIR-II fluorophores draw from a broad spectrum of materials spanning semiconducting nanomaterials to organic molecular dyes, yet unfortunately all water-soluble organic molecules with 41,000 nm emission suffer from low quantum yields that have limited temporal resolution and penetration depth. We report tailoring the supramolecular assemblies of protein complexes with a sulfonated NIR-II organic dye (CH-4T) to produce a brilliant 110-fold increase in fluorescence, resulting in the highest quantum yield molecular fluorophore thus far. The bright molecular complex allowed for themore » fastest video-rate imaging in the second NIR window with B50-fold reduced exposure times at a fast 50 frames-per-second (FPS) capable of resolving mouse cardiac cycles. Additionally, we demonstrate that the NIR-II molecular complexes are superior to clinically approved ICG for lymph node imaging deep within the mouse body.« less

Understanding the role of dynamics in the iron sulfur cluster molecular machine.

PubMed

di Maio, Danilo; Chandramouli, Balasubramanian; Yan, Robert; Brancato, Giuseppe; Pastore, Annalisa

2017-01-01

The bacterial proteins IscS, IscU and CyaY, the bacterial orthologue of frataxin, play an essential role in the biological machine that assembles the prosthetic FeS cluster groups on proteins. They form functionally binary and ternary complexes both in vivo and in vitro. Yet, the mechanism by which they work remains unclear. We carried out extensive molecular dynamics simulations to understand the nature of their interactions and the role of dynamics starting from the crystal structure of a IscS-IscU complex and the experimentally-based model of a ternary IscS-IscU-CyaY complex and used nuclear magnetic resonance to experimentally test the interface. We show that, while being firmly anchored to IscS, IscU has a pivotal motion around the interface. Our results also describe how the catalytic loop of IscS can flip conformation to allow FeS cluster assembly. This motion is hampered in the ternary complex explaining its inhibitory properties in cluster formation. We conclude that the observed 'fluid' IscS-IscU interface provides the binary complex with a functional adaptability exploited in partner recognition and unravels the molecular determinants of the reported inhibitory action of CyaY in the IscS-IscU-CyaY complex explained in terms of the hampering effect on specific IscU-IscS movements. Our study provides the first mechanistic basis to explain how the IscS-IscU complex selects its binding partners and supports the inhibitory role of CyaY in the ternary complex. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A triaxial supramolecular weave

NASA Astrophysics Data System (ADS)

Lewandowska, Urszula; Zajaczkowski, Wojciech; Corra, Stefano; Tanabe, Junki; Borrmann, Ruediger; Benetti, Edmondo M.; Stappert, Sebastian; Watanabe, Kohei; Ochs, Nellie A. K.; Schaeublin, Robin; Li, Chen; Yashima, Eiji; Pisula, Wojciech; Müllen, Klaus; Wennemers, Helma

2017-11-01

Despite recent advances in the synthesis of increasingly complex topologies at the molecular level, nano- and microscopic weaves have remained difficult to achieve. Only a few diaxial molecular weaves exist—these were achieved by templation with metals. Here, we present an extended triaxial supramolecular weave that consists of self-assembled organic threads. Each thread is formed by the self-assembly of a building block comprising a rigid oligoproline segment with two perylene-monoimide chromophores spaced at 18 Å. Upon π stacking of the chromophores, threads form that feature alternating up- and down-facing voids at regular distances. These voids accommodate incoming building blocks and establish crossing points through CH-π interactions on further assembly of the threads into a triaxial woven superstructure. The resulting micrometre-scale supramolecular weave proved to be more robust than non-woven self-assemblies of the same building block. The uniform hexagonal pores of the interwoven network were able to host iridium nanoparticles, which may be of interest for practical applications.

Self-Assembly of Diblock Molecular Polymer Brushes in the Spherical Confinement of Nanoemulsion Droplets.

PubMed

Steinhaus, Andrea; Pelras, Théophile; Chakroun, Ramzi; Gröschel, André H; Müllner, Markus

2018-05-02

Understanding the self-assembly behavior of polymers of various topologies is key to a reliable design of functional polymer materials. Self-assembly under confinement conditions emerges as a versatile avenue to design polymer particles with complex internal morphologies while simultaneously facilitating scale-up. However, only linear block copolymers have been studied to date, despite the increasing control over macromolecule composition and architecture available. This study extends the investigation of polymer self-assembly in confinement from regular diblock copolymers to diblock molecular polymer brushes (MPBs). Block-type MPBs with polystyrene (PS) and polylactide (PLA) compartments of different sizes are incorporated into surfactant-stabilized oil-in-water (chloroform/water) emulsions. The increasing confinement in the nanoemulsion droplets during solvent evaporation directs the MPBs to form solid nano/microparticles. Microscopy studies reveal an intricate internal particle structure, including interpenetrating networks and axially stacked lamellae of PS and PLA, depending on the PS/PLA ratio of the brushes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metal-directed design of supramolecular protein assemblies

PubMed Central

Bailey, Jake B.; Subramanian, Rohit H.; Churchfield, Lewis A.

2016-01-01

Owing to their central roles in cellular signaling, construction, and biochemistry, protein-protein interactions (PPIs) and protein self-assembly have become a major focus of molecular design and synthetic biology. In order to circumvent the complexity of constructing extensive non-covalent interfaces, which are typically involved in natural PPIs and protein self-assembly, we have developed two design strategies, Metal-Directed Protein Self-Assembly (MDPSA) and Metal-Templated Interface Redesign (MeTIR). These strategies, inspired by both the proposed evolutionary roles of metals and their prevalence in natural PPIs, take advantage of the favorable properties of metal coordination (bonding strength, directionality, and reversibility) to guide protein self-assembly with minimal design and engineering. Using a small, monomeric protein (cytochrome cb562) as a model building block, we employed MDPSA and MeTIR to create a diverse array of functional supramolecular architectures which range from structurally tunable oligomers to metalloprotein complexes that can properly self-assemble in living cells into novel metalloenzymes. The design principles and strategies outlined herein should be readily applicable to other protein systems with the goal of creating new PPIs and protein assemblies with structures and functions not yet produced by natural evolution. PMID:27586336

Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly.

PubMed

Rebane, Aleksander A; Wang, Bigeng; Ma, Lu; Qu, Hong; Coleman, Jeff; Krishnakumar, Shyam; Rothman, James E; Zhang, Yongli

2018-02-16

Synaptic exocytosis relies on assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by stepwise zippering of the vesicle-associated SNARE (v-SNARE) onto a binary SNARE complex on the target plasma membrane (t-SNARE). Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering, partly due to difficulties to quantify the energetics and kinetics of SNARE assembly. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE synaptosomal-associated protein of 25kDa (SNAP-25B), which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~10 k B T, but did not affect N-terminal assembly. In addition, we observed that both mutations lead to unfolding of the C-terminal region in the t-SNARE complex. Our findings suggest that both SNAP-25B mutations impair synaptic exocytosis by destabilizing SNARE assembly, rather than stabilizing SNARE assembly as previously proposed. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations. Copyright © 2017 Elsevier Ltd. All rights reserved.

pH-Controlled Assembly of DNA Tiles

DOE PAGES

Amodio, Alessia; Adedeji, Abimbola Feyisara; Castronovo, Matteo; ...

2016-09-15

We demonstrate a strategy to trigger and finely control the assembly of supramolecular DNA nanostructures with pH. Control is achieved via a rationally designed strand displacement circuit that responds to pH and activates a downstream DNA tile self-assembly process. We observe that the DNA structures form under neutral/basic conditions, while the self-assembly process is suppressed under acidic conditions. The strategy presented here demonstrates a modular approach toward building systems capable of processing biochemical inputs and finely controlling the assembly of DNA-based nanostructures under isothermal conditions. In particular, the presented architecture is relevant for the development of complex DNA devices ablemore » to sense and respond to molecular markers associated with abnormal metabolism.« less

pH-Controlled Assembly of DNA Tiles

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

Amodio, Alessia; Adedeji, Abimbola Feyisara; Castronovo, Matteo

We demonstrate a strategy to trigger and finely control the assembly of supramolecular DNA nanostructures with pH. Control is achieved via a rationally designed strand displacement circuit that responds to pH and activates a downstream DNA tile self-assembly process. We observe that the DNA structures form under neutral/basic conditions, while the self-assembly process is suppressed under acidic conditions. The strategy presented here demonstrates a modular approach toward building systems capable of processing biochemical inputs and finely controlling the assembly of DNA-based nanostructures under isothermal conditions. In particular, the presented architecture is relevant for the development of complex DNA devices ablemore » to sense and respond to molecular markers associated with abnormal metabolism.« less

Drosophila Chitinase 2 is expressed in chitin producing organs for cuticle formation.

PubMed

Pesch, Yanina-Yasmin; Riedel, Dietmar; Behr, Matthias

2017-01-01

The architecture of the outer body wall cuticle is fundamental to protect arthropods against invading pathogens and numerous other harmful stresses. Such robust cuticles are formed by parallel running chitin microfibrils. Molting and also local wounding leads to dynamic assembly and disassembly of the chitin-matrix throughout development. However, the underlying molecular mechanisms that organize proper chitin-matrix formation are poorly known. Recently we identified a key region for cuticle thickening at the apical cell surface, the cuticle assembly zone, where Obstructor-A (Obst-A) coordinates the formation of the chitin-matrix. Obst-A binds chitin and the deacetylase Serpentine (Serp) in a core complex, which is required for chitin-matrix maturation and preservation. Here we present evidence that Chitinase 2 (Cht2) could be essential for this molecular machinery. We show that Cht2 is expressed in the chitin-matrix of epidermis, trachea, and the digestive system. There, Cht2 is enriched at the apical cell surface and the dense chitin-matrix. We further show that in Cht2 knockdown larvae the assembly zone is rudimentary, preventing normal cuticle formation and pore canal organization. As sequence similarities of Cht2 and the core complex proteins indicate evolutionarily conserved molecular mechanisms, our findings suggest that Cht2 is involved in chitin formation also in other insects. Copyright © 2016 Elsevier Ltd. All rights reserved.

Guest Controlled Nonmonotonic Deep Cavity Cavitand Assembly State Switching.

PubMed

Tang, Du; Barnett, J Wesley; Gibb, Bruce C; Ashbaugh, Henry S

2017-11-30

Octa-acid (OA) and tetra-endo-methyl octa-acid (TEMOA) are water-soluble, deep-cavity cavitands with nanometer-sized nonpolar pockets that readily bind complementary guests, such as n-alkanes. Experimentally, OA exhibits a progression of 1:1 to 2:2 to 2:1 host/guest complexes (X:Y where X is the number of hosts and Y is the number of guests) with increasing alkane chain length from methane to tetradecane. Differing from OA only by the addition of four methyl groups ringing the portal of the pocket, TEMOA exhibits a nonmonotonic progression of assembly states from 1:1 to 2:2 to 1:1 to 2:1 with increasing guest length. Here we present a systematic molecular simulation study to parse the molecular and thermodynamic determinants that distinguish the succession of assembly stoichiometries observed for these similar hosts. Potentials of mean force between hosts and guests, determined via umbrella sampling, are used to characterize association free energies. These free energies are subsequently used in a reaction network model to predict the equilibrium distributions of assemblies. Our models accurately reproduce the experimentally observed trends, showing that TEMOA's endo-methyl units constrict the opening of the binding pocket, limiting the conformations available to bound guests and disrupting the balance between monomeric complexes and dimeric capsules. The success of our simulations demonstrate their utility at interpreting the impact of even simple chemical modifications on supramolecular assembly and highlight their potential to aid bottom-up design.

Evolution of an ancient protein function involved in organized multicellularity in animals.

PubMed

Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

2016-01-07

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which - the evolution of GKPID's capacity to bind the cortical marker protein - can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals.

Molecular Precision at Micrometer Length Scales: Hierarchical Assembly of DNA-Protein Nanostructures.

PubMed

Schiffels, Daniel; Szalai, Veronika A; Liddle, J Alexander

2017-07-25

Robust self-assembly across length scales is a ubiquitous feature of biological systems but remains challenging for synthetic structures. Taking a cue from biology-where disparate molecules work together to produce large, functional assemblies-we demonstrate how to engineer microscale structures with nanoscale features: Our self-assembly approach begins by using DNA polymerase to controllably create double-stranded DNA (dsDNA) sections on a single-stranded template. The single-stranded DNA (ssDNA) sections are then folded into a mechanically flexible skeleton by the origami method. This process simultaneously shapes the structure at the nanoscale and directs the large-scale geometry. The DNA skeleton guides the assembly of RecA protein filaments, which provides rigidity at the micrometer scale. We use our modular design strategy to assemble tetrahedral, rectangular, and linear shapes of defined dimensions. This method enables the robust construction of complex assemblies, greatly extending the range of DNA-based self-assembly methods.

Functional Assembly of Soluble and Membrane Recombinant Proteins of Mammalian NADPH Oxidase Complex.

PubMed

Souabni, Hajer; Ezzine, Aymen; Bizouarn, Tania; Baciou, Laura

2017-01-01

Activation of phagocyte cells from an innate immune system is associated with a massive consumption of molecular oxygen to generate highly reactive oxygen species (ROS) as microbial weapons. This is achieved by a multiprotein complex, the so-called NADPH oxidase. The activity of phagocyte NADPH oxidase relies on an assembly of more than five proteins, among them the membrane heterodimer named flavocytochrome b 558 (Cytb 558 ), constituted by the tight association of the gp91 phox (also named Nox2) and p22 phox proteins. The Cytb 558 is the membrane catalytic core of the NADPH oxidase complex, through which the reducing equivalent provided by NADPH is transferred via the associated prosthetic groups (one flavin and two hemes) to reduce dioxygen into superoxide anion. The other major proteins (p47 phox , p67 phox , p40 phox , Rac) requisite for the complex activity are cytosolic proteins. Thus, the NADPH oxidase functioning relies on a synergic multi-partner assembly that in vivo can be hardly studied at the molecular level due to the cell complexity. Thus, a cell-free assay method has been developed to study the NADPH oxidase activity that allows measuring and eventually quantifying the ROS generation based on optical techniques following reduction of cytochrome c. This setup is a valuable tool for the identification of protein interactions, of crucial components and additives for a functional enzyme. Recently, this method was improved by the engineering and the production of a complete recombinant NADPH oxidase complex using the combination of purified proteins expressed in bacterial and yeast host cells. The reconstitution into artificial membrane leads to a fully controllable system that permits fine functional studies.

Imaging of DNA and Protein by SFM and Combined SFM-TIRF Microscopy.

PubMed

Grosbart, Małgorzata; Ristić, Dejan; Sánchez, Humberto; Wyman, Claire

2018-01-01

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nm resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

Creating Prebiotic Sanctuary: Self-Assembling Supramolecular Peptide Structures Bind and Stabilize RNA

NASA Astrophysics Data System (ADS)

Carny, Ohad; Gazit, Ehud

2011-04-01

Any attempt to uncover the origins of life must tackle the known `blind watchmaker problem'. That is to demonstrate the likelihood of the emergence of a prebiotic system simple enough to be formed spontaneously and yet complex enough to allow natural selection that will lead to Darwinistic evolution. Studies of short aromatic peptides revealed their ability to self-assemble into ordered and stable structures. The unique physical and chemical characteristics of these peptide assemblies point out to their possible role in the origins of life. We have explored mechanisms by which self-assembling short peptides and RNA fragments could interact together and go through a molecular co-evolution, using diphenylalanine supramolecular assemblies as a model system. The spontaneous formation of these self-assembling peptides under prebiotic conditions, through the salt-induced peptide formation (SIPF) pathway was demonstrated. These peptide assemblies possess the ability to bind and stabilize ribonucleotides in a sequence-depended manner, thus increase their relative fitness. The formation of these peptide assemblies is dependent on the homochirality of the peptide monomers: while homochiral peptides (L-Phe-L-Phe and D-Phe-D-Phe) self-assemble rapidly in aqueous environment, heterochiral diastereoisomers (L-Phe-D-Phe and D-Phe-L-Phe) do not tend to self-assemble. This characteristic consists with the homochirality of all living matter. Finally, based on these findings, we propose a model for the role of short self-assembling peptides in the prebiotic molecular evolution and the origin of life.

Creating prebiotic sanctuary: self-assembling supramolecular Peptide structures bind and stabilize RNA.

PubMed

Carny, Ohad; Gazit, Ehud

2011-04-01

Any attempt to uncover the origins of life must tackle the known 'blind watchmaker problem'. That is to demonstrate the likelihood of the emergence of a prebiotic system simple enough to be formed spontaneously and yet complex enough to allow natural selection that will lead to Darwinistic evolution. Studies of short aromatic peptides revealed their ability to self-assemble into ordered and stable structures. The unique physical and chemical characteristics of these peptide assemblies point out to their possible role in the origins of life. We have explored mechanisms by which self-assembling short peptides and RNA fragments could interact together and go through a molecular co-evolution, using diphenylalanine supramolecular assemblies as a model system. The spontaneous formation of these self-assembling peptides under prebiotic conditions, through the salt-induced peptide formation (SIPF) pathway was demonstrated. These peptide assemblies possess the ability to bind and stabilize ribonucleotides in a sequence-depended manner, thus increase their relative fitness. The formation of these peptide assemblies is dependent on the homochirality of the peptide monomers: while homochiral peptides (L-Phe-L-Phe and D-Phe-D-Phe) self-assemble rapidly in aqueous environment, heterochiral diastereoisomers (L-Phe-D-Phe and D-Phe-L-Phe) do not tend to self-assemble. This characteristic consists with the homochirality of all living matter. Finally, based on these findings, we propose a model for the role of short self-assembling peptides in the prebiotic molecular evolution and the origin of life.

Tuning peptide self-assembly by an in-tether chiral center

PubMed Central

Hu, Kuan; Xiong, Wei; Li, Hu; Zhang, Pei-Yu; Yin, Feng; Zhang, Qianling; Jiang, Fan; Li, Zigang

2018-01-01

The self-assembly of peptides into ordered nanostructures is important for understanding both peptide molecular interactions and nanotechnological applications. However, because of the complexity and various self-assembling pathways of peptide molecules, design of self-assembling helical peptides with high controllability and tunability is challenging. We report a new self-assembling mode that uses in-tether chiral center-induced helical peptides as a platform for tunable peptide self-assembly with good controllability. It was found that self-assembling behavior was governed by in-tether substitutional groups, where chirality determined the formation of helical structures and aromaticity provided the driving force for self-assembly. Both factors were essential for peptide self-assembly to occur. Experiments and theoretical calculations indicate long-range crystal-like packing in the self-assembly, which was stabilized by a synergy of interpeptide π-π and π-sulfur interactions and hydrogen bond networks. In addition, the self-assembled peptide nanomaterials were demonstrated to be promising candidate materials for applications in biocompatible electrochemical supercapacitors.

Templated Formation of Luminescent Virus-like Particles by Tailor-Made Pt(II) Amphiphiles

PubMed Central

2018-01-01

Virus-like particles (VLPs) have been created from luminescent Pt(II) complex amphiphiles, able to form supramolecular structures in water solutions, that can be encapsulated or act as templates of cowpea chlorotic mottle virus capsid proteins. By virtue of a bottom-up molecular design, icosahedral and nonicosahedral (rod-like) VLPs have been constructed through diverse pathways, and a relationship between the molecular structure of the complexes and the shape and size of the VLPs has been observed. A deep insight into the mechanism for the templated formation of the differently shaped VLPs was achieved, by electron microscopy measurements (TEM and STEM) and bulk analysis (FPLC, DLS, photophysical investigations). Interestingly, the obtained VLPs can be visualized by their intense emission at room temperature, generated by the self-assembly of the Pt(II) complexes. The encapsulation of the luminescent species is further verified by their higher emission quantum yields inside the VLPs, which is due to the confinement effect of the protein cage. These hybrid materials demonstrate the potential of tailor-made supramolecular systems able to control the assembly of biological building blocks. PMID:29357236

Templated Formation of Luminescent Virus-like Particles by Tailor-Made Pt(II) Amphiphiles.

PubMed

Sinn, Stephan; Yang, Liulin; Biedermann, Frank; Wang, Di; Kübel, Christian; Cornelissen, Jeroen J L M; De Cola, Luisa

2018-02-14

Virus-like particles (VLPs) have been created from luminescent Pt(II) complex amphiphiles, able to form supramolecular structures in water solutions, that can be encapsulated or act as templates of cowpea chlorotic mottle virus capsid proteins. By virtue of a bottom-up molecular design, icosahedral and nonicosahedral (rod-like) VLPs have been constructed through diverse pathways, and a relationship between the molecular structure of the complexes and the shape and size of the VLPs has been observed. A deep insight into the mechanism for the templated formation of the differently shaped VLPs was achieved, by electron microscopy measurements (TEM and STEM) and bulk analysis (FPLC, DLS, photophysical investigations). Interestingly, the obtained VLPs can be visualized by their intense emission at room temperature, generated by the self-assembly of the Pt(II) complexes. The encapsulation of the luminescent species is further verified by their higher emission quantum yields inside the VLPs, which is due to the confinement effect of the protein cage. These hybrid materials demonstrate the potential of tailor-made supramolecular systems able to control the assembly of biological building blocks.

Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System.

PubMed

Chojnacka, Magdalena; Gornicka, Agnieszka; Oeljeklaus, Silke; Warscheid, Bettina; Chacinska, Agnieszka

2015-06-12

The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Complexin and Ca2+ stimulate SNARE-mediated membrane fusion

PubMed Central

Yoon, Tae-Young; Lu, Xiaobind; Diao, Jiajie; Lee, Soo-Min; Ha, Taekjip; Shin, Yeon-Kyun

2008-01-01

Ca2+-triggered, synchronized synaptic vesicle fusion underlies interneuronal communication. Complexin is a major binding partner of the SNARE complex, the core fusion machinery at the presynapse. The physiological data on complexin, however, have been at odds with each other, making delineation of its molecular function difficult. Here we report direct observation of two-faceted functions of complexin using the single-vesicle fluorescence fusion assay and EPR. We show that complexin I has two opposing effects on trans-SNARE assembly: inhibition of SNARE complex formation and stabilization of assembled SNARE complexes. Of note, SNARE-mediated fusion is markedly stimulated by complexin, and it is further accelerated by two orders of magnitude in response to an externally applied Ca2+ wave. We suggest that SNARE complexes, complexins and phospholipids collectively form a complex substrate for Ca2+ and Ca2+-sensing fusion effectors in neurotransmitter release. PMID:18552825

The Design, Synthesis, and Study of Solid-State Molecular Rotors: Structure/Function Relationships for Condensed-Phase Anisotropic Dynamics

NASA Astrophysics Data System (ADS)

Vogelsberg, Cortnie Sue

Amphidynamic crystals are an extremely promising platform for the development of artificial molecular machines and stimuli-responsive materials. In analogy to skeletal muscle, their function will rely upon the collective operation of many densely packed molecular machines (i.e. actin-bound myosin) that are self-assembled in a highly organized anisotropic medium. By choosing lattice-forming elements and moving "parts" with specific functionalities, individual molecular machines may be synthesized and self-assembled in order to carry out desirable functions. In recent years, efforts in the design of amphidynamic materials based on molecular gyroscopes and compasses have shown that a certain amount of free volume is essential to facilitate internal rotation and reorientation within a crystal. In order to further establish structure/function relationships to advance the development of increasingly complex molecular machinery, molecular rotors and a molecular "spinning" top were synthesized and incorporated into a variety of solid-state architectures with different degrees of periodicity, dimensionality, and free volume. Specifically, lamellar molecular crystals, hierarchically ordered periodic mesoporous organosilicas, and metal-organic frameworks were targeted for the development of solid-state molecular machines. Using an array of solid-state nuclear magnetic resonance spectroscopy techniques, the dynamic properties of these novel molecular machine assemblies were determined and correlated with their corresponding structural features. It was found that architecture type has a profound influence on functional dynamics. The study of layered molecular crystals, composed of either molecular rotors or "spinning" tops, probed functional dynamics within dense, highly organized environments. From their study, it was discovered that: 1) crystallographically distinct sites may be utilized to differentiate machine function, 2) halogen bonding interactions are sufficiently strong to direct an assembly of molecular machines, 3) the relative flexibility of the crystal environment proximate to a dynamic component may have a significant effect on its function, and, 4) molecular machines, which possess both solid-state photochemical reactivity and dynamics may show complex reaction kinetics if the correlation time of the dynamic process and the lifetime of the excited state occur on the same time scale and the dynamic moiety inherently participates as a reaction intermediate. The study of periodic mesoporous organosilica with hierarchical order probed molecular dynamics within 2D layers of molecular rotors, organized in only one dimension and with ca. 50% exposed to the mesopore free volume. From their study, it was discovered that: 1) molecular rotors, which comprise the layers of the mesopore walls, form a 2D rotational glass, 2) rotator dynamics within the 2D rotational glass undergo a transition to a 2D rotational fluid, and, 3) a 2D rotational glass transition may be exploited to develop hyper-sensitive thermally activated molecular machines. The study of a metal-organic framework assembled from molecular rotors probed dynamics in a periodic three-dimensional free-volume environment, without the presence of close contacts. From the study of this solid-state material, it was determined that: 1) the intrinsic electronic barrier is one of the few factors, which may affect functional dynamics in a true free-volume environment, and, 2) molecular machines with dynamic barriers <

Adaptive Correction from Virtually Complex Dynamic Libraries: The Role of Noncovalent Interactions in Structural Selection and Folding.

PubMed

Lafuente, Maria; Atcher, Joan; Solà, Jordi; Alfonso, Ignacio

2015-11-16

The hierarchical self-assembling of complex molecular systems is dictated by the chemical and structural information stored in their components. This information can be expressed through an adaptive process that determines the structurally fittest assembly under given environmental conditions. We have set up complex disulfide-based dynamic covalent libraries of chemically and topologically diverse pseudopeptidic compounds. We show how the reaction evolves from very complex mixtures at short reaction times to the almost exclusive formation of a major compound, through the establishment of intramolecular noncovalent interactions. Our experiments demonstrate that the systems evolve through error-check and error-correction processes. The nature of these interactions, the importance of the folding and the effects of the environment are also discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Clathrate colloidal crystals

NASA Astrophysics Data System (ADS)

Lin, Haixin; Lee, Sangmin; Sun, Lin; Spellings, Matthew; Engel, Michael; Glotzer, Sharon C.; Mirkin, Chad A.

2017-03-01

DNA-programmable assembly has been used to deliberately synthesize hundreds of different colloidal crystals spanning dozens of symmetries, but the complexity of the achieved structures has so far been limited to small unit cells. We assembled DNA-modified triangular bipyramids (~250-nanometer long edge, 177-nanometer short edge) into clathrate architectures. Electron microscopy images revealed that at least three different structures form as large single-domain architectures or as multidomain materials. Ordered assemblies, isostructural to clathrates, were identified with the help of molecular simulations and geometric analysis. These structures are the most sophisticated architectures made via programmable assembly, and their formation can be understood based on the shape of the nanoparticle building blocks and mode of DNA functionalization.

Supramolecular Differentiation for Construction of Anisotropic Fullerene Nanostructures by Time-Programmed Control of Interfacial Growth.

PubMed

Bairi, Partha; Minami, Kosuke; Hill, Jonathan P; Nakanishi, Waka; Shrestha, Lok Kumar; Liu, Chao; Harano, Koji; Nakamura, Eiichi; Ariga, Katsuhiko

2016-09-27

Supramolecular assembly can be used to construct a wide variety of ordered structures by exploiting the cumulative effects of multiple noncovalent interactions. However, the construction of anisotropic nanostructures remains subject to some limitations. Here, we demonstrate the preparation of anisotropic fullerene-based nanostructures by supramolecular differentiation, which is the programmed control of multiple assembly strategies. We have carefully combined interfacial assembly and local phase separation phenomena. Two fullerene derivatives, PhH and C12H, were together formed into self-assembled anisotropic nanostructures by using this approach. This technique is applicable for the construction of anisotropic nanostructures without requiring complex molecular design or complicated methodology.

Student Learning about Biomolecular Self-Assembly Using Two Different External Representations

PubMed Central

Höst, Gunnar E.; Larsson, Caroline; Olson, Arthur; Tibell, Lena A. E.

2013-01-01

Self-assembly is the fundamental but counterintuitive principle that explains how ordered biomolecular complexes form spontaneously in the cell. This study investigated the impact of using two external representations of virus self-assembly, an interactive tangible three-dimensional model and a static two-dimensional image, on student learning about the process of self-assembly in a group exercise. A conceptual analysis of self-assembly into a set of facets was performed to support study design and analysis. Written responses were collected in a pretest/posttest experimental design with 32 Swedish university students. A quantitative analysis of close-ended items indicated that the students improved their scores between pretest and posttest, with no significant difference between the conditions (tangible model/image). A qualitative analysis of an open-ended item indicated students were unfamiliar with self-assembly prior to the study. Students in the tangible model condition used the facets of self-assembly in their open-ended posttest responses more frequently than students in the image condition. In particular, it appears that the dynamic properties of the tangible model may support student understanding of self-assembly in terms of the random and reversible nature of molecular interactions. A tentative difference was observed in response complexity, with more multifaceted responses in the tangible model condition. PMID:24006395

Student learning about biomolecular self-assembly using two different external representations.

PubMed

Höst, Gunnar E; Larsson, Caroline; Olson, Arthur; Tibell, Lena A E

2013-01-01

Self-assembly is the fundamental but counterintuitive principle that explains how ordered biomolecular complexes form spontaneously in the cell. This study investigated the impact of using two external representations of virus self-assembly, an interactive tangible three-dimensional model and a static two-dimensional image, on student learning about the process of self-assembly in a group exercise. A conceptual analysis of self-assembly into a set of facets was performed to support study design and analysis. Written responses were collected in a pretest/posttest experimental design with 32 Swedish university students. A quantitative analysis of close-ended items indicated that the students improved their scores between pretest and posttest, with no significant difference between the conditions (tangible model/image). A qualitative analysis of an open-ended item indicated students were unfamiliar with self-assembly prior to the study. Students in the tangible model condition used the facets of self-assembly in their open-ended posttest responses more frequently than students in the image condition. In particular, it appears that the dynamic properties of the tangible model may support student understanding of self-assembly in terms of the random and reversible nature of molecular interactions. A tentative difference was observed in response complexity, with more multifaceted responses in the tangible model condition.

Synthetic polycations with controlled charge density and molecular weight as building blocks for biomaterials.

PubMed

Kleinberger, Rachelle M; Burke, Nicholas A D; Zhou, Christal; Stöver, Harald D H

2016-01-01

A series of polycations prepared by RAFT copolymerization of N-(3-aminopropyl)methacrylamide hydrochloride (APM) and N-(2-hydroxypropyl)methacrylamide, with molecular weights of 15 and 40 kDa, and APM content of 10-75 mol%, were tested as building blocks for electrostatically assembled hydrogels such as those used for cell encapsulation. Complexation and distribution of these copolymers within anionic calcium alginate gels, as well as cytotoxicity, cell attachment, and cell proliferation on surfaces grafted with the copolymers were found to depend on composition and molecular weight. Copolymers with lower cationic charge density and lower molecular weight showed less cytotoxicity and cell adhesion, and were more mobile within alginate gels. These findings aid in designing improved polyelectrolyte complexes for use as biomaterials.

Molecular interactions in self-assembled nano-structures of chitosan-sodium alginate based polyelectrolyte complexes.

PubMed

Wasupalli, Geeta Kumari; Verma, Devendra

2018-03-16

We report here the self-assembled structures of polyelectrolyte complexes (PECs) of polyanionic sodium alginate with the polycationic chitosan at room temperature. The PECs prepared at different pH values exhibited two distinct morphologies. The chitosan-alginate PECs self-assembled into the fibrous structure in a low pH range of pH3 to 7. The PECs obtained at high pH series around pH8 and above resulted in the formation of colloidal nanoparticles in the range of 120±9.48nm to 46.02±16.66nm. The zeta potential measurement showed that PECs prepared at lower pH (pH<6) exhibited nearly neutral surface charge, whereas PECs prepared at higher pH than 6 exhibited highly negative surface charge. The molecular interactions in nano-colloids and fibers were evaluated using FTIR analysis. The results attest that the ionic state of the chitosan and alginate plays an important role controlling the morphologies of the PECS. The present study has identified the enormous potential of the polyelectrolytes complexes to exploit shape by the alteration of ionic strength. These findings might be useful in the development of novel biomaterial. The produced fibers and nanocolloids could be applied as a biomaterial for tissue engineering and drug delivery. Copyright © 2017. Published by Elsevier B.V.

Molecular Engineering of Platinum(II) Terpyridine Complexes with Tetraphenylethylene-Modified Alkynyl Ligands: Supramolecular Assembly via Pt···Pt and/or π-π Stacking Interactions and the Formation of Various Superstructures.

PubMed

Cheng, Heung-Kiu; Yeung, Margaret Ching-Lam; Yam, Vivian Wing-Wah

2017-10-18

A series of platinum(II) terpyridine complexes with tetraphenylethylene-modified alkynyl ligands has been designed and synthesized. The introduction of the tetraphenylethylene motif has led to aggregation-induced emission (AIE) properties, which upon self-assembly led to the formation of metal-metal-to-ligand charge transfer (MMLCT) behavior stabilized by Pt···Pt and/or π-π interactions. Tuning the steric bulk or hydrophilicity through molecular engineering of the platinum(II) complexes has been found to alter their spectroscopic properties and result in interesting superstructures (including nanorods, nanospheres, nanowires, and nanoleaves) in the self-assembly process. The eye-catching color and emission changes upon varying the solvent compositions may have potential applications in chemosensing materials for the detection of microenvironment changes. Furthermore, the importance of the directional Pt···Pt and/or π-π interactions on the construction of distinctive superstructures has also been examined by UV-vis absorption and emission spectroscopy and transmission electron microscopy. This work represents the interplay of both inter- and intramolecular interactions as well as the energies of the two different chromophoric/luminophoric systems that may open up a new route for the development of platinum(II)-AIE hybrids as functional materials.

The life of plant mitochondrial complex I.

PubMed

Braun, Hans-Peter; Binder, Stefan; Brennicke, Axel; Eubel, Holger; Fernie, Alisdair R; Finkemeier, Iris; Klodmann, Jennifer; König, Ann-Christine; Kühn, Kristina; Meyer, Etienne; Obata, Toshihiro; Schwarzländer, Markus; Takenaka, Mizuki; Zehrmann, Anja

2014-11-01

The mitochondrial NADH dehydrogenase complex (complex I) of the respiratory chain has several remarkable features in plants: (i) particularly many of its subunits are encoded by the mitochondrial genome, (ii) its mitochondrial transcripts undergo extensive maturation processes (e.g. RNA editing, trans-splicing), (iii) its assembly follows unique routes, (iv) it includes an additional functional domain which contains carbonic anhydrases and (v) it is, indirectly, involved in photosynthesis. Comprising about 50 distinct protein subunits, complex I of plants is very large. However, an even larger number of proteins are required to synthesize these subunits and assemble the enzyme complex. This review aims to follow the complete "life cycle" of plant complex I from various molecular perspectives. We provide arguments that complex I represents an ideal model system for studying the interplay of respiration and photosynthesis, the cooperation of mitochondria and the nucleus during organelle biogenesis and the evolution of the mitochondrial oxidative phosphorylation system. Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Practical computational toolkits for dendrimers and dendrons structure design.

PubMed

Martinho, Nuno; Silva, Liana C; Florindo, Helena F; Brocchini, Steve; Barata, Teresa; Zloh, Mire

2017-09-01

Dendrimers and dendrons offer an excellent platform for developing novel drug delivery systems and medicines. The rational design and further development of these repetitively branched systems are restricted by difficulties in scalable synthesis and structural determination, which can be overcome by judicious use of molecular modelling and molecular simulations. A major difficulty to utilise in silico studies to design dendrimers lies in the laborious generation of their structures. Current modelling tools utilise automated assembly of simpler dendrimers or the inefficient manual assembly of monomer precursors to generate more complicated dendrimer structures. Herein we describe two novel graphical user interface toolkits written in Python that provide an improved degree of automation for rapid assembly of dendrimers and generation of their 2D and 3D structures. Our first toolkit uses the RDkit library, SMILES nomenclature of monomers and SMARTS reaction nomenclature to generate SMILES and mol files of dendrimers without 3D coordinates. These files are used for simple graphical representations and storing their structures in databases. The second toolkit assembles complex topology dendrimers from monomers to construct 3D dendrimer structures to be used as starting points for simulation using existing and widely available software and force fields. Both tools were validated for ease-of-use to prototype dendrimer structure and the second toolkit was especially relevant for dendrimers of high complexity and size.

Practical computational toolkits for dendrimers and dendrons structure design

NASA Astrophysics Data System (ADS)

Martinho, Nuno; Silva, Liana C.; Florindo, Helena F.; Brocchini, Steve; Barata, Teresa; Zloh, Mire

2017-09-01

Dendrimers and dendrons offer an excellent platform for developing novel drug delivery systems and medicines. The rational design and further development of these repetitively branched systems are restricted by difficulties in scalable synthesis and structural determination, which can be overcome by judicious use of molecular modelling and molecular simulations. A major difficulty to utilise in silico studies to design dendrimers lies in the laborious generation of their structures. Current modelling tools utilise automated assembly of simpler dendrimers or the inefficient manual assembly of monomer precursors to generate more complicated dendrimer structures. Herein we describe two novel graphical user interface toolkits written in Python that provide an improved degree of automation for rapid assembly of dendrimers and generation of their 2D and 3D structures. Our first toolkit uses the RDkit library, SMILES nomenclature of monomers and SMARTS reaction nomenclature to generate SMILES and mol files of dendrimers without 3D coordinates. These files are used for simple graphical representations and storing their structures in databases. The second toolkit assembles complex topology dendrimers from monomers to construct 3D dendrimer structures to be used as starting points for simulation using existing and widely available software and force fields. Both tools were validated for ease-of-use to prototype dendrimer structure and the second toolkit was especially relevant for dendrimers of high complexity and size.

Synthetic Molecular Machines for Active Self-Assembly: Prototype Algorithms, Designs, and Experimental Study

NASA Astrophysics Data System (ADS)

Dabby, Nadine L.

Computer science and electrical engineering have been the great success story of the twentieth century. The neat modularity and mapping of a language onto circuits has led to robots on Mars, desktop computers and smartphones. But these devices are not yet able to do some of the things that life takes for granted: repair a scratch, reproduce, regenerate, or grow exponentially fast--all while remaining functional. This thesis explores and develops algorithms, molecular implementations, and theoretical proofs in the context of "active self-assembly" of molecular systems. The long-term vision of active self-assembly is the theoretical and physical implementation of materials that are composed of reconfigurable units with the programmability and adaptability of biology's numerous molecular machines. En route to this goal, we must first find a way to overcome the memory limitations of molecular systems, and to discover the limits of complexity that can be achieved with individual molecules. One of the main thrusts in molecular programming is to use computer science as a tool for figuring out what can be achieved. While molecular systems that are Turing-complete have been demonstrated [Winfree, 1996], these systems still cannot achieve some of the feats biology has achieved. One might think that because a system is Turing-complete, capable of computing "anything," that it can do any arbitrary task. But while it can simulate any digital computational problem, there are many behaviors that are not "computations" in a classical sense, and cannot be directly implemented. Examples include exponential growth and molecular motion relative to a surface. Passive self-assembly systems cannot implement these behaviors because (a) molecular motion relative to a surface requires a source of fuel that is external to the system, and (b) passive systems are too slow to assemble exponentially-fast-growing structures. We call these behaviors "energetically incomplete" programmable behaviors. This class of behaviors includes any behavior where a passive physical system simply does not have enough physical energy to perform the specified tasks in the requisite amount of time. As we will demonstrate and prove, a sufficiently expressive implementation of an "active" molecular self-assembly approach can achieve these behaviors. Using an external source of fuel solves part of the problem, so the system is not "energetically incomplete." But the programmable system also needs to have sufficient expressive power to achieve the specified behaviors. Perhaps surprisingly, some of these systems do not even require Turing completeness to be sufficiently expressive. Building on a large variety of work by other scientists in the fields of DNA nanotechnology, chemistry and reconfigurable robotics, this thesis introduces several research contributions in the context of active self-assembly. We show that simple primitives such as insertion and deletion are able to generate complex and interesting results such as the growth of a linear polymer in logarithmic time and the ability of a linear polymer to treadmill. To this end we developed a formal model for active-self assembly that is directly implementable with DNA molecules. We show that this model is computationally equivalent to a machine capable of producing strings that are stronger than regular languages and, at most, as strong as context-free grammars. This is a great advance in the theory of active self-assembly as prior models were either entirely theoretical or only implementable in the context of macro-scale robotics. We developed a chain reaction method for the autonomous exponential growth of a linear DNA polymer. Our method is based on the insertion of molecules into the assembly, which generates two new insertion sites for every initial one employed. The building of a line in logarithmic time is a first step toward building a shape in logarithmic time. We demonstrate the first construction of a synthetic linear polymer that grows exponentially fast via insertion. We show that monomer molecules are converted into the polymer in logarithmic time via spectrofluorimetry and gel electrophoresis experiments. We also demonstrate the division of these polymers via the addition of a single DNA complex that competes with the insertion mechanism. This shows the growth of a population of polymers in logarithmic time. We characterize the DNA insertion mechanism that we utilize in Chapter 4. We experimentally demonstrate that we can control the kinetics of this reaction over at least seven orders of magnitude, by programming the sequences of DNA that initiate the reaction. In addition, we review co-authored work on programming molecular robots using prescriptive landscapes of DNA origami; this was the first microscopic demonstration of programming a molecular robot to walk on a 2-dimensional surface. We developed a snapshot method for imaging these random walking molecular robots and a CAPTCHA-like analysis method for difficult-to-interpret imaging data.

Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly.

PubMed

Thind, Anupriya Kaur; Wicker, Thomas; Šimková, Hana; Fossati, Dario; Moullet, Odile; Brabant, Cécile; Vrána, Jan; Doležel, Jaroslav; Krattinger, Simon G

2017-08-01

Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging. Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species. A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest. In order to accelerate gene cloning from any cropping line, we report 'targeted chromosome-based cloning via long-range assembly' (TACCA). TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage to assemble complex genomes. We applied TACCA to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months. Using this assembly we cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein.

High-speed superresolution imaging of the proteins in fission yeast clathrin-mediated endocytic actin patches

PubMed Central

Arasada, Rajesh; Sayyad, Wasim A.; Berro, Julien; Pollard, Thomas D.

2018-01-01

To internalize nutrients and cell surface receptors via clathrin-mediated endocytosis, cells assemble at least 50 proteins, including clathrin, clathrin-interacting proteins, actin filaments, and actin binding proteins, in a highly ordered and regulated manner. The molecular mechanism by which actin filament polymerization deforms the cell membrane is unknown, largely due to lack of knowledge about the organization of the regulatory proteins and actin filaments. We used high-speed superresolution localization microscopy of live fission yeast cells to improve the spatial resolution to ∼35 nm with 1-s temporal resolution. The nucleation promoting factors Wsp1p (WASp) and Myo1p (myosin-I) define two independent pathways that recruit Arp2/3 complex, which assembles two zones of actin filaments. Myo1p concentrates at the site of endocytosis and initiates a zone of actin filaments assembled by Arp2/3 complex. Wsp1p appears simultaneously at this site but subsequently moves away from the cell surface as it stimulates Arp2/3 complex to assemble a second zone of actin filaments. Cells lacking either nucleation-promoting factor assemble only one, stationary, zone of actin filaments. These observations support our two-zone hypothesis to explain endocytic tubule elongation and vesicle scission in fission yeast. PMID:29212877

Structure of the RZZ complex and molecular basis of its interaction with Spindly

PubMed Central

Mosalaganti, Shyamal; Keller, Jenny; Altenfeld, Anika; Rombaut, Pascaline; Petrovic, Arsen; Wohlgemuth, Sabine; Müller, Franziska; Herzog, Franz; Waldmann, Herbert

2017-01-01

Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules (MTs) during mitosis. The metazoan-specific ≈800-kD ROD–Zwilch–ZW10 (RZZ) complex builds a fibrous corona that assembles on mitotic kinetochores before MT attachment to promote chromosome alignment and robust spindle assembly checkpoint signaling. In this study, we combine biochemical reconstitutions, single-particle electron cryomicroscopy, cross-linking mass spectrometry, and structural modeling to build a complete model of human RZZ. We find that RZZ is structurally related to self-assembling cytosolic coat scaffolds that mediate membrane cargo trafficking, including Clathrin, Sec13–Sec31, and αβ’ε-COP. We show that Spindly, a dynein adaptor, is related to BicD2 and binds RZZ directly in a farnesylation-dependent but membrane-independent manner. Through a targeted chemical biology approach, we identify ROD as the Spindly farnesyl receptor. Our results suggest that RZZ is dynein’s cargo at human kinetochores. PMID:28320825

Virus assembly occurs following a pH- or Ca2+-triggered switch in the thermodynamic attraction between structural protein capsomeres.

PubMed

Chuan, Yap P; Fan, Yuan Y; Lua, Linda H L; Middelberg, Anton P J

2010-03-06

Viral self-assembly is of tremendous virological and biomedical importance. Although theoretical and crystallographic considerations suggest that controlled conformational change is a fundamental regulatory mechanism in viral assembly, direct proof that switching alters the thermodynamic attraction of self-assembling components has not been provided. Using the VP1 protein of polyomavirus, we report a new method to quantitatively measure molecular interactions under conditions of rapid protein self-assembly. We show, for the first time, that triggering virus capsid assembly through biologically relevant changes in Ca(2+) concentration, or pH, is associated with a dramatic increase in the strength of protein molecular attraction as quantified by the second virial coefficient (B(22)). B(22) decreases from -2.3 x 10(-4) mol ml g(-2) (weak protein-protein attraction) to -2.4 x 10(-3) mol ml g(-2) (strong protein attraction) for metastable and Ca(2+)-triggered self-assembling capsomeres, respectively. An assembly-deficient mutant (VP1CDelta63) is conversely characterized by weak protein-protein repulsion independently of chemical change sufficient to cause VP1 assembly. Concomitant switching of both VP1 assembly and thermodynamic attraction was also achieved by in vitro changes in ammonium sulphate concentration, consistent with protein salting-out behaviour. The methods and findings reported here provide new insight into viral assembly, potentially facilitating the development of new antivirals and vaccines, and will open the way to a more fundamental physico-chemical description of complex protein self-assembly systems.

9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments

PubMed Central

Dodonova, Svetlana O; Aderhold, Patrick; Kopp, Juergen; Ganeva, Iva; Röhling, Simone; Hagen, Wim J H; Sinning, Irmgard; Wieland, Felix; Briggs, John A G

2017-01-01

COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of βδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly. DOI: http://dx.doi.org/10.7554/eLife.26691.001 PMID:28621666

Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis

PubMed Central

2017-01-01

ABSTRACT Cells have an intrinsic ability to self-assemble and self-organize into complex and functional tissues and organs. By taking advantage of this ability, embryoids, organoids and gastruloids have recently been generated in vitro, providing a unique opportunity to explore complex embryological events in a detailed and highly quantitative manner. Here, we examine how such approaches are being used to answer fundamental questions in embryology, such as how cells self-organize and assemble, how the embryo breaks symmetry, and what controls timing and size in development. We also highlight how further improvements to these exciting technologies, based on the development of quantitative platforms to precisely follow and measure subcellular and molecular events, are paving the way for a more complete understanding of the complex events that help build the human embryo. PMID:28292844

DNA-Directed Assembly of Capture Tools for Constitutional Studies of Large Protein Complexes.

PubMed

Meyer, Rebecca; Faesen, Alex; Vogel, Katrin; Jeganathan, Sadasivam; Musacchio, Andrea; Niemeyer, Christof M

2015-06-10

Large supramolecular protein complexes, such as the molecular machinery involved in gene regulation, cell signaling, or cell division, are key in all fundamental processes of life. Detailed elucidation of structure and dynamics of such complexes can be achieved by reverse-engineering parts of the complexes in order to probe their interactions with distinctive binding partners in vitro. The exploitation of DNA nanostructures to mimic partially assembled supramolecular protein complexes in which the presence and state of two or more proteins are decisive for binding of additional building blocks is reported here. To this end, four-way DNA Holliday junction motifs bearing a fluorescein and a biotin tag, for tracking and affinity capture, respectively, are site-specifically functionalized with centromeric protein (CENP) C and CENP-T. The latter serves as baits for binding of the so-called KMN component, thereby mimicking early stages of the assembly of kinetochores, structures that mediate and control the attachment of microtubules to chromosomes in the spindle apparatus. Results from pull-down experiments are consistent with the hypothesis that CENP-C and CENP-T may bind cooperatively to the KMN network. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Exploitation of molecular mobilities for advanced organic optoelectronic and photonic nano-materials

NASA Astrophysics Data System (ADS)

Gray, Tomoko O.

Electro-optically active organic materials have shown great potential in advanced technologies such as ultrafast electro-optical switches for broadband communication, light-emitting diodes, and photovoltaic cells. Currently, the maturity of chemical synthesis enables a sophisticated integration of the active elements into complex macromolecules. Also, the structure-property relationships of the isolated single electrically/optically active elements are well established. Unfortunately, such correlations involving single molecule are not applicable to complex unstructured condensed systems, in which unique mesoscale properties and complex dynamics of super-/supra-molecular structures are present. Our current challenge arises, in particular, from a deficiency of appropriate characterization tools that close the gap between phenomenological measurements and theoretical models. This work addresses submolecular mobilities relevant for opto-electronic functionalities of photoluminescent polymers and non-linear optical (NLO) materials. Thereby, I will introduce novel nanoscale thermomechanical characterization tools that are based on scanning force microscopy. From nanoscale thermomechanical measurements sub-/super-molecular mobilities of novel optoelectronic materials can be inferred and to some degree controlled. For instance, we have explored interfacial constraints as a engineering tool to control molecular mobility. This will be illustrated with electroluminescent polymers, which are prone to undesired pi-pi aggregation due to the rod-like structure---intrinsic to all conjugated polymers. The nanoscale confinement is used to reduced chain mobility, and thus, hinders undesired aggregation, and consequently, yields superior spectral stability. From the nanomaterial design perspective, I will also address mobility control with targeted molecular designs. This involves two classes of novel NLO materials, side-chain dendronized polymers and self-assembling molecular glasses. The side-chain dendronized polymers are, due to the structural complexity, self-constrained systems. Our thermomechanical investigations identified that a local relaxation mode associated to the NLO side-chain is the critical design parameter in yielding high mobility to the active element. Relaxation processes of the self-assembling molecular glasses are discussed from a thermodynamic perspective involving both enthalpic and entropic contributions, considering the very special nature of interactions for the NLO molecular glasses, i.e., the formation and dissociation of phenyl/perfluorophenyl quadrupol pairs.

Photon Upconversion and Molecular Solar Energy Storage by Maximizing the Potential of Molecular Self-Assembly.

PubMed

Kimizuka, Nobuo; Yanai, Nobuhiro; Morikawa, Masa-Aki

2016-11-29

The self-assembly of functional molecules into ordered molecular assemblies and the fulfillment of potentials unique to their nanotomesoscopic structures have been one of the central challenges in chemistry. This Feature Article provides an overview of recent progress in the field of molecular self-assembly with the focus on the triplet-triplet annihilation-based photon upconversion (TTA-UC) and supramolecular storage of photon energy. On the basis of the integration of molecular self-assembly and photon energy harvesting, triplet energy migration-based TTA-UC has been achieved in varied molecular systems. Interestingly, some molecular self-assemblies dispersed in solution or organogels revealed oxygen barrier properties, which allowed TTA-UC even under aerated conditions. The elements of molecular self-assembly were also introduced to the field of molecular solar thermal fuel, where reversible photoliquefaction of ionic crystals to ionic liquids was found to double the molecular storage capacity with the simultaneous pursuit of switching ionic conductivity. A future prospect in terms of innovating molecular self-assembly toward molecular systems chemistry is also discussed.

Observation of the noncovalent assembly and disassembly pathways of the chaperone complex MtGimC by mass spectrometry

PubMed Central

Fändrich, Marcus; Tito, Mark A.; Leroux, Michel R.; Rostom, Adam A.; Hartl, F. Ulrich; Dobson, Christopher M.; Robinson, Carol V.

2000-01-01

We have analyzed a newly described archaeal GimC/prefoldin homologue, termed MtGimC, by using nanoflow electrospray coupled with time-of-flight MS. The molecular weight of the complex from Methanobacterium thermoautotrophicum corresponds to a well-defined hexamer of two α subunits and four β subunits. Dissociation of the complex within the gas phase reveals a quaternary arrangement of two central subunits, both α, and four peripheral β subunits. By constructing a thermally controlled nanoflow device, we have monitored the thermal stability of the complex by MS. The results of these experiments demonstrate that a significant proportion of the MtGimC hexamer remains intact under low-salt conditions at elevated temperatures. This finding is supported by data from CD spectroscopy, which show that at physiological salt concentrations, the complex remains stable at temperatures above 65°C. Mass spectrometric methods were developed to monitor in real time the assembly of the MtGimC hexamer from its component subunits. By using this methodology, the mass spectra recorded throughout the time course of the experiment showed the absence of any significantly populated intermediates, demonstrating that the assembly process is highly cooperative. Taken together, these data show that the complex is stable under the elevated temperatures that are appropriate for its hyperthermophile host and demonstrate that the assembly pathway leads exclusively to the hexamer, which is likely to be a structural unit in vivo. PMID:11087821

The molecular origins of specificity in the assembly of a multienzyme complex.

PubMed

Frank, René A W; Pratap, J Venkatesh; Pei, Xue Y; Perham, Richard N; Luisi, Ben F

2005-08-01

The pyruvate dehydrogenase (PDH) multienzyme complex is central to oxidative metabolism. We present the first crystal structure of a complex between pyruvate decarboxylase (E1) and the peripheral subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2). The interface is dominated by a "charge zipper" of networked salt bridges. Remarkably, the PSBD uses essentially the same zipper to alternately recognize the dihydrolipoyl dehydrogenase (E3) component of the PDH assembly. The PSBD achieves this dual recognition largely through the addition of a network of interfacial water molecules unique to the E1-PSBD complex. These structural comparisons illuminate our observations that the formation of this water-rich E1-E2 interface is largely enthalpy driven, whereas that of the E3-PSBD complex (from which water is excluded) is entropy driven. Interfacial water molecules thus diversify surface complementarity and contribute to avidity, enthalpically. Additionally, the E1-PSBD structure provides insight into the organization and active site coupling within the approximately 9 MDa PDH complex.

A method for multiprotein assembly in cells reveals independent action of kinesins in complex

PubMed Central

Norris, Stephen R.; Soppina, Virupakshi; Dizaji, Aslan S.; Schimert, Kristin I.; Sept, David; Cai, Dawen; Sivaramakrishnan, Sivaraj

2014-01-01

Teams of processive molecular motors are critical for intracellular transport and organization, yet coordination between motors remains poorly understood. Here, we develop a system using protein components to generate assemblies of defined spacing and composition inside cells. This system is applicable to studying macromolecular complexes in the context of cell signaling, motility, and intracellular trafficking. We use the system to study the emergent behavior of kinesin motors in teams. We find that two kinesin motors in complex act independently (do not help or hinder each other) and can alternate their activities. For complexes containing a slow kinesin-1 and fast kinesin-3 motor, the slow motor dominates motility in vitro but the fast motor can dominate on certain subpopulations of microtubules in cells. Both motors showed dynamic interactions with the complex, suggesting that motor–cargo linkages are sensitive to forces applied by the motors. We conclude that kinesin motors in complex act independently in a manner regulated by the microtubule track. PMID:25365993

Peptide-Based Molecular Hydrogels as Supramolecular Protein Mimics.

PubMed

Singh, Nishant; Kumar, Mohit; Miravet, Juan F; Ulijn, Rein V; Escuder, Beatriu

2017-01-23

This Minireview concerns recent advances in the design, synthesis, and application of low molecular-weight peptidic hydrogelators. The sequence-specific combinations of amino acid side chain functionalities combined with hydrogen bonding of amide backbones and hydrophobic (aromatic) capping groups give these peptidic molecules the intrinsic tendency to self-assemble. The most prevalent designs include N-capped amino acid residues, bolamphiphilic peptides, and amphipathic peptides. Factors such as hydrophobic effects, the Hofmeister effect, and tunable ionization influence their aggregation properties. The self-assembly of simple bio-inspired building blocks into higher organized structures allows comparisons to be drawn with proteins and their complex functionalities, providing preliminary insights into complex biological functions and also enabling their application in a wide range of fields including catalysis, biomedical applications, and mimicry of natural dissipative systems. The Minireview is concluded by a short summary and outlook, highlighting the advances and steps required to bridge the gaps in the understanding of such systems. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Ligand design for multidimensional magnetic materials: a metallosupramolecular perspective.

PubMed

Pardo, Emilio; Ruiz-García, Rafael; Cano, Joan; Ottenwaelder, Xavier; Lescouëzec, Rodrigue; Journaux, Yves; Lloret, Francesc; Julve, Miguel

2008-06-07

The aim and scope of this review is to show the general validity of the 'complex-as-ligand' approach for the rational design of metallosupramolecular assemblies of increasing structural and magnetic complexity. This is illustrated herein on the basis of our recent studies on oxamato complexes with transition metal ions looking for the limits of the research avenue opened by Kahn's pioneering research twenty years ago. The use as building blocks of mono-, di- and trinuclear metal complexes with a novel family of aromatic polyoxamato ligands allowed us to move further in the coordination chemistry-based approach to high-nuclearity coordination compounds and high-dimensionality coordination polymers. In order to do so, we have taken advantage of the new developments of metallosupramolecular chemistry and in particular, of the molecular-programmed self-assembly methods that exploit the coordination preferences of metal ions and specifically tailored ligands. The judicious choice of the oxamato metal building block (substitution pattern and steric requirements of the bridging ligand, as well as the electronic configuration and magnetic anisotropy of the metal ion) allowed us to control the overall structure and magnetic properties of the final multidimensional nD products (n = 0-3). These species exhibit interesting magnetic properties which are brand-new targets in the field of molecular magnetism, such as single-molecule or single-chain magnets, and the well-known class of molecule-based magnets. This unique family of molecule-based magnetic materials expands on the reported examples of nD species with cyanide and related oxalato and dithiooxalato analogues. Moreover, the development of new oxamato metal building blocks with potential photo or redox activity at the aromatic ligand counterpart will provide us with addressable, multifunctional molecular materials for future applications in molecular electronics and nanotechnology.

Targeting the Blind Spot of Polycationic Nanocarrier-Based siRNA Delivery

PubMed Central

Zheng, Mengyao; Pavan, Giovanni M.; Neeb, Manuel; Schaper, Andreas K.; Danani, Andrea; Klebe, Gerhard; Merkel, Olivia M.; Kissel, Thomas

2013-01-01

Polycationic nanocarriers attract increasing attention to the field of siRNA delivery. We investigated the self-assembly of siRNA vs pDNA with polycations, which are broadly used for nonviral gene and siRNA delivery. Although polyethyleneimine (PEI) was routinely adopted as siRNA carrier based on its efficacy in delivering pDNA, it has not been investigated yet why PEI efficiently delivers pDNA to cells but is controversially discussed in terms of efficacy for siRNA delivery. We are the first to investigate the self-assembly of PEI/siRNA vs PEI/pDNA and the steps of complexation and aggregation through different levels of hierarchy on the atomic and molecular scale with the novel synergistic use of molecular modeling, molecular dynamics simulation, isothermal titration calorimetry, and other characterization techniques. We are also the fist to elucidate atomic interactions, size, shape, stoichiometry, and association dynamics for polyplexes containing siRNA vs pDNA. Our investigation highlights differences in the hierarchical mechanism of formation of related polycation–siRNA and polycation–pDNA complexes. The results of fluorescence quenching assays indicated a biphasic behavior of siRNA binding with polycations where molecular reorganization of the siRNA within the polycations occurred at lower N/P ratios (nitrogen/phosphorus). Our results, for the first time, emphasize a biphasic behavior in siRNA complexation and the importance of low N/P ratios, which allow for excellent siRNA delivery efficiency. Our investigation highlights the formulation of siRNA complexes from a thermodynamic point of view and opens new perspectives to advance the rational design of new siRNA delivery systems. PMID:23036046

Modular Organization of Residue-Level Contacts Shapes the Selection Pressure on Individual Amino Acid Sites of Ribosomal Proteins.

PubMed

Mallik, Saurav; Kundu, Sudip

2017-04-01

Understanding the molecular evolution of macromolecular complexes in the light of their structure, assembly, and stability is of central importance. Here, we address how the modular organization of native molecular contacts shapes the selection pressure on individual residue sites of ribosomal complexes. The bacterial ribosomal complex is represented as a residue contact network where nodes represent amino acid/nucleotide residues and edges represent their van der Waals interactions. We find statistically overrepresented native amino acid-nucleotide contacts (OaantC, one amino acid contacts one or multiple nucleotides, internucleotide contacts are disregarded). Contact number is defined as the number of nucleotides contacted. Involvement of individual amino acids in OaantCs with smaller contact numbers is more random, whereas only a few amino acids significantly contribute to OaantCs with higher contact numbers. An investigation of structure, stability, and assembly of bacterial ribosome depicts the involvement of these OaantCs in diverse biophysical interactions stabilizing the complex, including high-affinity protein-RNA contacts, interprotein cooperativity, intersubunit bridge, packing of multiple ribosomal RNA domains, etc. Amino acid-nucleotide constituents of OaantCs with higher contact numbers are generally associated with significantly slower substitution rates compared with that of OaantCs with smaller contact numbers. This evolutionary rate heterogeneity emerges from the strong purifying selection pressure that conserves the respective amino acid physicochemical properties relevant to the stabilizing interaction with OaantC nucleotides. An analysis of relative molecular orientations of OaantC residues and their interaction energetics provides the biophysical ground of purifying selection conserving OaantC amino acid physicochemical properties. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Designing interfaces of hydrogenase-nanomaterial hybrids for efficient solar conversion.

PubMed

King, Paul W

2013-01-01

The direct conversion of sunlight into biofuels is an intriguing alternative to a continued reliance on fossil fuels. Natural photosynthesis has long been investigated both as a potential solution, and as a model for utilizing solar energy to drive a water-to-fuel cycle. The molecules and organizational structure provide a template to inspire the design of efficient molecular systems for photocatalysis. A clear design strategy is the coordination of molecular interactions that match kinetic rates and energetic levels to control the direction and flow of energy from light harvesting to catalysis. Energy transduction and electron-transfer reactions occur through interfaces formed between complexes of donor-acceptor molecules. Although the structures of several of the key biological complexes have been solved, detailed descriptions of many electron-transfer complexes are lacking, which presents a challenge to designing and engineering biomolecular systems for solar conversion. Alternatively, it is possible to couple the catalytic power of biological enzymes to light harvesting by semiconductor nanomaterials. In these molecules, surface chemistry and structure can be designed using ligands. The passivation effect of the ligand can also dramatically affect the photophysical properties of the semiconductor, and energetics of external charge-transfer. The length, degree of bond saturation (aromaticity), and solvent exposed functional groups of ligands can be manipulated to further tune the interface to control molecular assembly, and complex stability in photocatalytic hybrids. The results of this research show how ligand selection is critical to designing molecular interfaces that promote efficient self-assembly, charge-transfer and photocatalysis. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems. Copyright © 2013 Elsevier B.V. All rights reserved.

Assembling the bacterial segrosome.

PubMed

Hayes, Finbarr; Barillà, Daniela

2006-05-01

Genome segregation in prokaryotes is a highly ordered process that integrates with DNA replication, cytokinesis and other fundamental facets of the bacterial cell cycle. The segrosome is the nucleoprotein complex that mediates DNA segregation in bacteria, its assembly and organization is best understood for plasmid partition. The recent elucidation of structures of the ParB plasmid segregation protein bound to centromeric DNA, and of the tertiary structures of other segregation proteins, are key milestones in the path to deciphering the molecular basis of bacterial DNA segregation.

Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe–S Assembly Complex

PubMed Central

Fox, Nicholas G.; Das, Deepika; Chakrabarti, Mrinmoy; Lindahl, Paul A.; Barondeau, David P.

2015-01-01

Iron–sulfur (Fe–S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe–S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe–S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe–S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe–S assembly complex. Here the kinetics of Fe–S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe–S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe–S assembly complex. PMID:26016518

Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe-S Assembly Complex.

PubMed

Fox, Nicholas G; Das, Deepika; Chakrabarti, Mrinmoy; Lindahl, Paul A; Barondeau, David P

2015-06-30

Iron-sulfur (Fe-S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe-S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe-S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe-S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe-S assembly complex. Here the kinetics of Fe-S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe-S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe-S assembly complex.

Structure determination of an 11-subunit exosome in complex with RNA by molecular replacement

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

Makino, Debora Lika, E-mail: dmakino@biochem.mpg.de; Conti, Elena

The crystallographic steps towards the structure determination of a complete eukaryotic exosome complex bound to RNA are presented. Phasing of this 11-protein subunit complex was carried out via molecular replacement. The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3′ degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryoticmore » exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of a Saccharomyces cerevisiae 420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.« less

Assembly and function of the major histocompatibility complex (MHC) I peptide-loading complex are conserved across higher vertebrates.

PubMed

Hinz, Andreas; Jedamzick, Johanna; Herbring, Valentina; Fischbach, Hanna; Hartmann, Jessica; Parcej, David; Koch, Joachim; Tampé, Robert

2014-11-28

Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Superrepression through Altered Corepressor-Activated Protein:Protein Interactions.

PubMed

He, Chenlu; Custer, Gregory; Wang, Jingheng; Matysiak, Silvina; Beckett, Dorothy

2018-02-20

Small molecules regulate transcription in both eukaryotes and prokaryotes by either enhancing or repressing assembly of transcription regulatory complexes. For allosteric transcription repressors, superrepressor mutants can exhibit increased sensitivity to small molecule corepressors. However, because many transcription regulatory complexes assemble in multiple steps, the superrepressor phenotype can reflect changes in any or all of the individual assembly steps. Escherichia coli biotin operon repression complex assembly, which responds to input biotin concentration, occurs via three coupled equilibria, including corepressor binding, holorepressor dimerization, and binding of the dimer to DNA. A genetic screen has yielded superrepressor mutants that repress biotin operon transcription in vivo at biotin concentrations much lower than those required by the wild type repressor. In this work, isothermal titration calorimetry and sedimentation measurements were used to determine the superrepressor biotin binding and homodimerization properties. The results indicate that, although all variants exhibit biotin binding affinities similar to that measured for BirA wt , five of the six superrepressors show altered homodimerization energetics. Molecular dynamics simulations suggest that the altered dimerization results from perturbation of an electrostatic network that contributes to allosteric activation of BirA for dimerization. Modeling of the multistep repression complex assembly for these proteins reveals that the altered sensitivity of the transcription response to biotin concentration is readily explained solely by the altered superrepressor homodimerization energetics. These results highlight how coupled equilibria enable alterations in a transcription regulatory response to input signal through an indirect mechanism.

Structural DNA Nanotechnology: State of the Art and Future Perspective

PubMed Central

2015-01-01

Over the past three decades DNA has emerged as an exceptional molecular building block for nanoconstruction due to its predictable conformation and programmable intra- and intermolecular Watson–Crick base-pairing interactions. A variety of convenient design rules and reliable assembly methods have been developed to engineer DNA nanostructures of increasing complexity. The ability to create designer DNA architectures with accurate spatial control has allowed researchers to explore novel applications in many directions, such as directed material assembly, structural biology, biocatalysis, DNA computing, nanorobotics, disease diagnosis, and drug delivery. This Perspective discusses the state of the art in the field of structural DNA nanotechnology and presents some of the challenges and opportunities that exist in DNA-based molecular design and programming. PMID:25029570

Indanedione based binary chromophore supramolecular systems as a NLO active polymer composites

NASA Astrophysics Data System (ADS)

Rutkis, M.; Tokmakovs, A.; Jecs, E.; Kreicberga, J.; Kampars, V.; Kokars, V.

2010-06-01

Novel route to obtain EO material is proposed by supramolecular assembly of neutral-ground-state (NGS) and zwitterionic (ZWI) NLO chromophores in binary chromophore organic glass (BCOG) host-guest system. On a basis of our Langeven Dynamics (LD) molecular modeling combined with quantum chemical calculations, we have shown that anticipated enhancement NLO efficiency of BCOG material is possible via electrostatic supramolecular assembly of NGS with ZWI chromophore in antiparallel manner. Binding energy of such complex could be more dependent on molecular compatibility of components and local (atomic) charge distribution, then overall molecular dipole moments. According to our LD simulations these supramolecular bind structures of NGS and ZWI chromophores can sustain thermally assisted electrical field poling. For the one of experimentally investigated systems, build from dimethylaminobenzylidene 1,3-indanedione containing host and zwitterionic indanedione-1,3 pyridinium betaine as a guest, almost twofold enhancement of NLO efficiency was observed.

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein-peptide system.

PubMed

Inostroza-Brito, Karla E; Collin, Estelle; Siton-Mendelson, Orit; Smith, Katherine H; Monge-Marcet, Amàlia; Ferreira, Daniela S; Rodríguez, Raúl Pérez; Alonso, Matilde; Rodríguez-Cabello, José Carlos; Reis, Rui L; Sagués, Francesc; Botto, Lorenzo; Bitton, Ronit; Azevedo, Helena S; Mata, Alvaro

2015-11-01

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein-peptide system

NASA Astrophysics Data System (ADS)

Inostroza-Brito, Karla E.; Collin, Estelle; Siton-Mendelson, Orit; Smith, Katherine H.; Monge-Marcet, Amàlia; Ferreira, Daniela S.; Rodríguez, Raúl Pérez; Alonso, Matilde; Rodríguez-Cabello, José Carlos; Reis, Rui L.; Sagués, Francesc; Botto, Lorenzo; Bitton, Ronit; Azevedo, Helena S.; Mata, Alvaro

2015-11-01

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.

Vertebrate Presynaptic Active Zone Assembly: a Role Accomplished by Diverse Molecular and Cellular Mechanisms.

PubMed

Torres, Viviana I; Inestrosa, Nibaldo C

2018-06-01

Among all the biological systems in vertebrates, the central nervous system (CNS) is the most complex, and its function depends on specialized contacts among neurons called synapses. The assembly and organization of synapses must be exquisitely regulated for a normal brain function and network activity. There has been a tremendous effort in recent decades to understand the molecular and cellular mechanisms participating in the formation of new synapses and their organization, maintenance, and regulation. At the vertebrate presynapses, proteins such as Piccolo, Bassoon, RIM, RIM-BPs, CAST/ELKS, liprin-α, and Munc13 are constant residents and participate in multiple and dynamic interactions with other regulatory proteins, which define network activity and normal brain function. Here, we review the function of these active zone (AZ) proteins and diverse factors involved in AZ assembly and maintenance, with an emphasis on axonal trafficking of precursor vesicles, protein homo- and hetero-oligomeric interactions as a mechanism of AZ trapping and stabilization, and the role of F-actin in presynaptic assembly and its modulation by Wnt signaling.

Expanding the scale of molecular biophysics.

PubMed

Levine, Herbert

2016-10-07

Here, I argue that some of the secrets of complex biological function rely on assemblies of many heterogeneous proteins that together enable sophisticated sensing and actuating processes. Evolution seems to delight in making these structures and in continually elaborating upon their capabilities. Developing tools that can go beyond the few protein limit, both on the experimental frontier and from a theoretical, conceptual framework, should be an extremely high priority for the next generation of molecular biophysicists.

Vitamin D receptor (VDR) promoter targeting through a novel chromatin remodeling complex.

PubMed

Kato, Shigeaki; Fujiki, Ryoji; Kitagawa, Hirochika

2004-05-01

We have purified nuclear complexes for Vitamin D receptor (VDR), and identified one of them as a novel ATP-dependent chromatine remodeling containing Williams syndrome transcription factor (WSTF), that is supposed to be responsible for Williams syndrome. This complex (WSTF including nucleosome assembly complex (WINAC)) exhibited an ATP-dependent chromatin remodeling activity in vitro. Transient expression assays revealed that WINAC potentiates ligand-induced function of VDR in gene activation and repression. Thus, this study describes a molecular basis of the VDR function on chromosomal DNA through chromatine remodeling.

Bacterial flagella and Type III secretion: case studies in the evolution of complexity.

PubMed

Pallen, M J; Gophna, U

2007-01-01

Bacterial flagella at first sight appear uniquely sophisticated in structure, so much so that they have even been considered 'irreducibly complex' by the intelligent design movement. However, a more detailed analysis reveals that these remarkable pieces of molecular machinery are the product of processes that are fully compatible with Darwinian evolution. In this chapter we present evidence for such processes, based on a review of experimental studies, molecular phylogeny and microbial genomics. Several processes have played important roles in flagellar evolution: self-assembly of simple repeating subunits, gene duplication with subsequent divergence, recruitment of elements from other systems ('molecular bricolage'), and recombination. We also discuss additional tentative new assignments of homology (FliG with MgtE, FliO with YscJ). In conclusion, rather than providing evidence of intelligent design, flagellar and non-flagellar Type III secretion systems instead provide excellent case studies in the evolution of complex systems from simpler components.

DNAzyme-Based Logic Gate-Mediated DNA Self-Assembly.

PubMed

Zhang, Cheng; Yang, Jing; Jiang, Shuoxing; Liu, Yan; Yan, Hao

2016-01-13

Controlling DNA self-assembly processes using rationally designed logic gates is a major goal of DNA-based nanotechnology and programming. Such controls could facilitate the hierarchical engineering of complex nanopatterns responding to various molecular triggers or inputs. Here, we demonstrate the use of a series of DNAzyme-based logic gates to control DNA tile self-assembly onto a prescribed DNA origami frame. Logic systems such as "YES," "OR," "AND," and "logic switch" are implemented based on DNAzyme-mediated tile recognition with the DNA origami frame. DNAzyme is designed to play two roles: (1) as an intermediate messenger to motivate downstream reactions and (2) as a final trigger to report fluorescent signals, enabling information relay between the DNA origami-framed tile assembly and fluorescent signaling. The results of this study demonstrate the plausibility of DNAzyme-mediated hierarchical self-assembly and provide new tools for generating dynamic and responsive self-assembly systems.

Caenorhabditis elegans Evolves a New Architecture for the Multi-aminoacyl-tRNA Synthetase Complex*

PubMed Central

Havrylenko, Svitlana; Legouis, Renaud; Negrutskii, Boris; Mirande, Marc

2011-01-01

MARS is an evolutionary conserved supramolecular assembly of aminoacyl-tRNA synthetases found in eukaryotes. This complex was thought to be ubiquitous in the deuterostome and protostome clades of bilaterians because similar complexes were isolated from arthropods and vertebrates. However, several features of the component enzymes suggested that in the nematode Caenorhabditis elegans, a species grouped with arthropods in modern phylogeny, this complex might not exist, or should display a significantly different structural organization. C. elegans was also taken as a model system to study in a multicellular organism amenable to experimental approaches, the reason for existence of these supramolecular entities. Here, using a proteomic approach, we have characterized the components of MARS in C. elegans. We show that this organism evolved a specific structural organization of this complex, which contains several bona fide components of the MARS complexes known so far, but also displays significant variations. These data highlight molecular evolution events that took place after radiation of bilaterians. Remarkably, it shows that expansion of MARS assembly in metazoans is not linear, but is the result of additions but also of subtractions along evolution. We then undertook an experimental approach, using inactivation of the endogenous copy of methionyl-tRNA synthetase by RNAi and expression of transgenic variants, to understand the role in complex assembly and the in vivo functionality, of the eukaryotic-specific domains appended to aminoacyl-tRNA synthetases. We show that rescue of the worms and assembly of transgenic variants into MARS rest on the presence of these appended domains. PMID:21685384

Caenorhabditis elegans evolves a new architecture for the multi-aminoacyl-tRNA synthetase complex.

PubMed

Havrylenko, Svitlana; Legouis, Renaud; Negrutskii, Boris; Mirande, Marc

2011-08-12

MARS is an evolutionary conserved supramolecular assembly of aminoacyl-tRNA synthetases found in eukaryotes. This complex was thought to be ubiquitous in the deuterostome and protostome clades of bilaterians because similar complexes were isolated from arthropods and vertebrates. However, several features of the component enzymes suggested that in the nematode Caenorhabditis elegans, a species grouped with arthropods in modern phylogeny, this complex might not exist, or should display a significantly different structural organization. C. elegans was also taken as a model system to study in a multicellular organism amenable to experimental approaches, the reason for existence of these supramolecular entities. Here, using a proteomic approach, we have characterized the components of MARS in C. elegans. We show that this organism evolved a specific structural organization of this complex, which contains several bona fide components of the MARS complexes known so far, but also displays significant variations. These data highlight molecular evolution events that took place after radiation of bilaterians. Remarkably, it shows that expansion of MARS assembly in metazoans is not linear, but is the result of additions but also of subtractions along evolution. We then undertook an experimental approach, using inactivation of the endogenous copy of methionyl-tRNA synthetase by RNAi and expression of transgenic variants, to understand the role in complex assembly and the in vivo functionality, of the eukaryotic-specific domains appended to aminoacyl-tRNA synthetases. We show that rescue of the worms and assembly of transgenic variants into MARS rest on the presence of these appended domains.

Chemically programmed self-sorting of gelator networks.

PubMed

Morris, Kyle L; Chen, Lin; Raeburn, Jaclyn; Sellick, Owen R; Cotanda, Pepa; Paul, Alison; Griffiths, Peter C; King, Stephen M; O'Reilly, Rachel K; Serpell, Louise C; Adams, Dave J

2013-01-01

Controlling the order and spatial distribution of self-assembly in multicomponent supramolecular systems could underpin exciting new functional materials, but it is extremely challenging. When a solution of different components self-assembles, the molecules can either coassemble, or self-sort, where a preference for like-like intermolecular interactions results in coexisting, homomolecular assemblies. A challenge is to produce generic and controlled 'one-pot' fabrication methods to form separate ordered assemblies from 'cocktails' of two or more self-assembling species, which might have relatively similar molecular structures and chemistry. Self-sorting in supramolecular gel phases is hence rare. Here we report the first example of the pH-controlled self-sorting of gelators to form self-assembled networks in water. Uniquely, the order of assembly can be predefined. The assembly of each component is preprogrammed by the pK(a) of the gelator. This pH-programming method will enable higher level, complex structures to be formed that cannot be accessed by simple thermal gelation.

DNA nanotechnology and fluorescence applications.

PubMed

Schlichthaerle, Thomas; Strauss, Maximilian T; Schueder, Florian; Woehrstein, Johannes B; Jungmann, Ralf

2016-06-01

Structural DNA nanotechnology allow researchers to use the unique molecular recognition properties of DNA strands to construct nanoscale objects with almost arbitrary complexity in two and three dimensions. Abstracted as molecular breadboards, DNA nanostructures enable nanometer-precise placement of guest molecules such as proteins, fluorophores, or nanoparticles. These assemblies can be used to study biological phenomena with unprecedented control over number, spacing, and molecular identity. Here, we give a general introduction to structural DNA nanotechnology and more specifically discuss applications of DNA nanostructures in the field of fluorescence and plasmonics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evolution of an ancient protein function involved in organized multicellularity in animals

PubMed Central

Anderson, Douglas P; Whitney, Dustin S; Hanson-Smith, Victor; Woznica, Arielle; Campodonico-Burnett, William; Volkman, Brian F; King, Nicole; Thornton, Joseph W; Prehoda, Kenneth E

2016-01-01

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which – the evolution of GKPID’s capacity to bind the cortical marker protein – can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals. DOI: http://dx.doi.org/10.7554/eLife.10147.001 PMID:26740169

HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites

PubMed Central

Pujol, François M.; Laketa, Vibor; Schmidt, Florian; Mukenhirn, Markus; Müller, Barbara; Boulant, Steeve; Grimm, Dirk; Keppler, Oliver T.

2016-01-01

ABSTRACT The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process. IMPORTANCE CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention. PMID:27170757

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.

H-Bonding Assisted Self-Assembly of Anionic and Neutral Ligand on Metal: A Comprehensive Strategy To Mimic Ditopic Ligands in Olefin Polymerization.

PubMed

Mote, Nilesh R; Patel, Ketan; Shinde, Dinesh R; Gaikwad, Shahaji R; Koshti, Vijay S; Gonnade, Rajesh G; Chikkali, Samir H

2017-10-16

Self-assembly of two neutral ligands on a metal to mimic bidentate ligand coordination has been frequently encountered in the recent past, but self-assembly of an anionic ligand on a metal template alongside a neutral ligand remains an elusive target. Such a self-assembly is hampered by additional complexity, wherein a highly negatively charged anion can form intermolecular hydrogen bonding with the supramolecular motif, leaving no scope for self-assembly with neutral ligand. Presented here is the self-association of anionic ligand 3-ureidobenzoic acid (2a) and neutral ligand 1-(3-(diphenylphosphanyl)phenyl)urea (1a) on a metal template to yield metal complex [{COOC 6 H 4 NH(CO)NH 2 }{Ph 2 PC 6 H 4 NH(CO)NH 2 }PdMeDMSO] (4a). The identity of 4a was established by NMR and mass spectroscopy. Along the same lines, 3-(3-phenylureido)benzoic acid (2b) and 1-(3-(diphenylphosphanyl)phenyl)-3-phenylurea (1b) self-assemble on a metal template to produce palladium complex [{COOC 6 H 4 NH(CO)NHPh}{Ph 2 PC 6 H 4 NH(CO)NHPh}PdMePy] (5c). The existence of 5c was confirmed by Job plot, 1-2D NMR spectroscopy, deuterium labeling, IR spectroscopy, UV-vis spectroscopy, model complex synthesis, and DFT calculations. These solution and gas phase investigations authenticated the presence of intramolecular hydrogen bonding between hydrogen's of 1b and carbonyl oxygen of 2b. The generality of the supramolecular approach has been validated by preparing six complexes from four monodentate ligands, and their synthetic utility was demonstrated in ethylene polymerization. Complex 4a was found to be the most active, leading to the production of highly branched polyethylene with a molecular weight of 55700 g/mol and melting temperature of 112 °C.

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery

PubMed Central

Ranatunga, Wasantha; Gakh, Oleksandr; Galeano, Belinda K.; Smith, Douglas Y.; Söderberg, Christopher A. G.; Al-Karadaghi, Salam; Thompson, James R.; Isaya, Grazia

2016-01-01

The biosynthesis of Fe-S clusters is a vital process involving the delivery of elemental iron and sulfur to scaffold proteins via molecular interactions that are still poorly defined. We reconstituted a stable, functional complex consisting of the iron donor, Yfh1 (yeast frataxin homologue 1), and the Fe-S cluster scaffold, Isu1, with 1:1 stoichiometry, [Yfh1]24·[Isu1]24. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional reconstruction of this complex at a resolution of ∼17 Å. In addition, via chemical cross-linking, limited proteolysis, and mass spectrometry, we identified protein-protein interaction surfaces within the complex. The data together reveal that [Yfh1]24·[Isu1]24 is a roughly cubic macromolecule consisting of one symmetric Isu1 trimer binding on top of one symmetric Yfh1 trimer at each of its eight vertices. Furthermore, molecular modeling suggests that two subunits of the cysteine desulfurase, Nfs1, may bind symmetrically on top of two adjacent Isu1 trimers in a manner that creates two putative [2Fe-2S] cluster assembly centers. In each center, conserved amino acids known to be involved in sulfur and iron donation by Nfs1 and Yfh1, respectively, are in close proximity to the Fe-S cluster-coordinating residues of Isu1. We suggest that this architecture is suitable to ensure concerted and protected transfer of potentially toxic iron and sulfur atoms to Isu1 during Fe-S cluster assembly. PMID:26941001

In vitro assembly of semi-artificial molecular machine and its use for detection of DNA damage.

PubMed

Minchew, Candace L; Didenko, Vladimir V

2012-01-11

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc). This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage. Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks), fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO(4) blunt-ended break (DNase I-type breaks), if T4 DNA ligase is present in the solution. When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO(4) blunt-ended breaks, the ligase reacts with 5'PO(4) DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO(4) blunt-ended DNA breaks. This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues. Copyright © 2012 Journal of Visualized Experiments

Antiviral RNA Recognition and Assembly by RLR Family Innate Immune Sensors

PubMed Central

Bruns, Annie M.; Horvath, Curt M.

2014-01-01

Virus-encoded molecular signatures, such as cytosolic double-stranded or otherwise biochemically distinct RNA species, trigger cellular antiviral signaling. Cytoplasmic proteins recognize these non-self RNAs and activate signal transduction pathways that drive the expression of virus-induced genes, including the primary antiviral cytokine, IFNβ, and diverse direct and indirect antiviral effectors [1–4]. One important group of cytosolic RNA sensors known as the RIG-I like receptors (RLRs) is comprised of three proteins that are similar in structure and function. The RLR proteins, RIG-I, MDA5, and LGP2, share the ability to recognize nucleic acid signatures produced by virus infections and activate antiviral signaling. Emerging evidence indicates that RNA detection by RLRs culminates in the assembly of dynamic multimeric ribonucleoprotein (RNP) complexes. These RNPs can act as signaling platforms that are capable of propagating and amplifying antiviral signaling responses. Despite their common domain structures and similar abilities to induce antiviral responses, the RLRs differ in their enzymatic properties, their intrinsic abilities to recognize RNA, and their ability to assemble into filamentous complexes. This molecular specialization has enabled the RLRs to recognize and respond to diverse virus infections, and to mediate both unique and overlapping functions in immune regulation [5, 6]. PMID:25081315

Microrheological Characterization of Collagen Systems: From Molecular Solutions to Fibrillar Gels

PubMed Central

Shayegan, Marjan; Forde, Nancy R.

2013-01-01

Collagen is the most abundant protein in the extracellular matrix (ECM), where its structural organization conveys mechanical information to cells. Using optical-tweezers-based microrheology, we investigated mechanical properties both of collagen molecules at a range of concentrations in acidic solution where fibrils cannot form and of gels of collagen fibrils formed at neutral pH, as well as the development of microscale mechanical heterogeneity during the self-assembly process. The frequency scaling of the complex shear modulus even at frequencies of ∼10 kHz was not able to resolve the flexibility of collagen molecules in acidic solution. In these solutions, molecular interactions cause significant transient elasticity, as we observed for 5 mg/ml solutions at frequencies above ∼200 Hz. We found the viscoelasticity of solutions of collagen molecules to be spatially homogeneous, in sharp contrast to the heterogeneity of self-assembled fibrillar collagen systems, whose elasticity varied by more than an order of magnitude and in power-law behavior at different locations within the sample. By probing changes in the complex shear modulus over 100-minute timescales as collagen self-assembled into fibrils, we conclude that microscale heterogeneity appears during early phases of fibrillar growth and continues to develop further during this growth phase. Experiments in which growing fibrils dislodge microspheres from an optical trap suggest that fibril growth is a force-generating process. These data contribute to understanding how heterogeneities develop during self-assembly, which in turn can help synthesis of new materials for cellular engineering. PMID:23936454

Computational complexity of algorithms for sequence comparison, short-read assembly and genome alignment.

PubMed

Baichoo, Shakuntala; Ouzounis, Christos A

A multitude of algorithms for sequence comparison, short-read assembly and whole-genome alignment have been developed in the general context of molecular biology, to support technology development for high-throughput sequencing, numerous applications in genome biology and fundamental research on comparative genomics. The computational complexity of these algorithms has been previously reported in original research papers, yet this often neglected property has not been reviewed previously in a systematic manner and for a wider audience. We provide a review of space and time complexity of key sequence analysis algorithms and highlight their properties in a comprehensive manner, in order to identify potential opportunities for further research in algorithm or data structure optimization. The complexity aspect is poised to become pivotal as we will be facing challenges related to the continuous increase of genomic data on unprecedented scales and complexity in the foreseeable future, when robust biological simulation at the cell level and above becomes a reality. Copyright © 2017 Elsevier B.V. All rights reserved.

Dissecting the Calcium-Induced Differentiation of Human Primary Keratinocytes Stem Cells by Integrative and Structural Network Analyses

PubMed Central

Toufighi, Kiana; Yang, Jae-Seong; Luis, Nuno Miguel; Aznar Benitah, Salvador; Lehner, Ben; Serrano, Luis; Kiel, Christina

2015-01-01

The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55%) are composed of non-dynamic and dynamic gene products (‘di-chromatic’), 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation. PMID:25946651

Modeling Molecular Machinery

ERIC Educational Resources Information Center

Hunter, Christine

2015-01-01

Imagine a microscopic world filled with tiny motors, ratchets, switches, and pumps controlled by complex signaling and feedback systems. Now imagine that these parts can assemble themselves. This is the world presented to students in the protein structure unit of a genetic engineering course. Students learn how protein folding gives rise to the…

Autophagosomal membranes assemble at ER-plasma membrane contact sites.

PubMed

Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

2017-01-01

The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

Assembly of Multi-tRNA Synthetase Complex via Heterotetrameric Glutathione Transferase-homology Domains.

PubMed

Cho, Ha Yeon; Maeng, Seo Jin; Cho, Hyo Je; Choi, Yoon Seo; Chung, Jeong Min; Lee, Sangmin; Kim, Hoi Kyoung; Kim, Jong Hyun; Eom, Chi-Yong; Kim, Yeon-Gil; Guo, Min; Jung, Hyun Suk; Kang, Beom Sik; Kim, Sunghoon

2015-12-04

Many multicomponent protein complexes mediating diverse cellular processes are assembled through scaffolds with specialized protein interaction modules. The multi-tRNA synthetase complex (MSC), consisting of nine different aminoacyl-tRNA synthetases and three non-enzymatic factors (AIMP1-3), serves as a hub for many signaling pathways in addition to its role in protein synthesis. However, the assembly process and structural arrangement of the MSC components are not well understood. Here we show the heterotetrameric complex structure of the glutathione transferase (GST) domains shared among the four MSC components, methionyl-tRNA synthetase (MRS), glutaminyl-prolyl-tRNA synthetase (EPRS), AIMP2 and AIMP3. The MRS-AIMP3 and EPRS-AIMP2 using interface 1 are bridged via interface 2 of AIMP3 and EPRS to generate a unique linear complex of MRS-AIMP3:EPRS-AIMP2 at the molar ratio of (1:1):(1:1). Interestingly, the affinity at interface 2 of AIMP3:EPRS can be varied depending on the occupancy of interface 1, suggesting the dynamic nature of the linear GST tetramer. The four components are optimally arranged for maximal accommodation of additional domains and proteins. These characteristics suggest the GST tetramer as a unique and dynamic structural platform from which the MSC components are assembled. Considering prevalence of the GST-like domains, this tetramer can also provide a tool for the communication of the MSC with other GST-containing cellular factors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Synthesis, characterization, molecular docking and biological studies of self assembled transition metal dithiocarbamates of substituted pyrrole-2-carboxaldehyde.

PubMed

Nami, Shahab A A; Ullah, Irfan; Alam, Mahboob; Lee, Dong-Ung; Sarikavakli, Nursabah

2016-07-01

A series of self assembled 3d transition metal dithiocarbamate, M(pdtc) [where M=Mn(II), Fe(II), Co(II), Ni(II) and Cu(II)] have been synthesized and spectroscopically characterized. The bidentate dithiocarbamate ligand Na2pdtc (Disodium-1,4-phenyldiaminobis (pyrrole-1-sulfino)dithioate) was prepared by insertion reaction of carbondisulfide with Schiff base, N,N'-bis-(1H-pyrrol-2-ylmethylene)-benzene-1,4-diamine (L1) in basic medium. The simple substitution reaction between the metal halide and Na2pdtc yielded the title complexes in moderate yields. However, the in situ procedure gives high yield with the formation of single product as evident by TLC. Elemental analysis, IR, (1)H and (13)C NMR spectra, UV-vis., magnetic susceptibility and conductance measurements were done to characterize the complexes, M(pdtc). All the evidences suggest that the complexes have tetrahedral geometry excepting Cu(II) which is found to be square planar. A symmetrical bidentate coordination of the dithiocarbamato moiety has been observed in all the complexes. The conductivity data show that the complexes are non-electrolyte in nature. The anti-oxidant activity of the ligand, Na2pdtc and its transition metal complexes, M(pdtc) have been carried out using DPPH and Cu(pdtc) was found to be most effective. The anti-microbial activity of the Na2pdtc and M(pdtc) complexes have been carried out and on this basis the molecular docking study of the most effective complex, Cu(pdtc) has also been reported. Copyright © 2016 Elsevier B.V. All rights reserved.

High Molecular Weight Forms of Mammalian Respiratory Chain Complex II

PubMed Central

Nůsková, Hana; Holzerová, Eliška; Vrbacký, Marek; Pecina, Petr; Hejzlarová, Kateřina; Kľučková, Katarína; Rohlena, Jakub; Neuzil, Jiri; Houštěk, Josef

2013-01-01

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase. PMID:23967256

Exploring the science of thinking independently together: Faraday Discussion Volume 204 - Complex Molecular Surfaces and Interfaces, Sheffield, UK, July 2017.

PubMed

Samperi, M; Hirsch, B E; Diaz Fernandez, Y A

2017-11-23

The 2017 Faraday Discussion on Complex Molecular Surfaces and Interfaces brought together theoreticians and experimentalists from both physical and chemical backgrounds to discuss the relevant applied and fundamental research topics within the broader field of chemical surface analysis and characterization. Main discussion topics from the meeting included the importance of "disordered" two-dimensional (2D) molecular structures and the utility of kinetically trapped states. An emerging need for new experimental tools to address dynamics and kinetic pathways involved in self-assembled systems, as well as the future prospects and current limitations of in silico studies were also discussed. The following article provides a brief overview of the work presented and the challenges discussed during the meeting.

Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials

PubMed Central

2015-01-01

In this review we intend to provide a relatively comprehensive summary of the work of supramolecular hydrogelators after 2004 and to put emphasis particularly on the applications of supramolecular hydrogels/hydrogelators as molecular biomaterials. After a brief introduction of methods for generating supramolecular hydrogels, we discuss supramolecular hydrogelators on the basis of their categories, such as small organic molecules, coordination complexes, peptides, nucleobases, and saccharides. Following molecular design, we focus on various potential applications of supramolecular hydrogels as molecular biomaterials, classified by their applications in cell cultures, tissue engineering, cell behavior, imaging, and unique applications of hydrogelators. Particularly, we discuss the applications of supramolecular hydrogelators after they form supramolecular assemblies but prior to reaching the critical gelation concentration because this subject is less explored but may hold equally great promise for helping address fundamental questions about the mechanisms or the consequences of the self-assembly of molecules, including low molecular weight ones. Finally, we provide a perspective on supramolecular hydrogelators. We hope that this review will serve as an updated introduction and reference for researchers who are interested in exploring supramolecular hydrogelators as molecular biomaterials for addressing the societal needs at various frontiers. PMID:26646318

Sequential and ordered assembly of a large DNA repair complex on undamaged chromatin

PubMed Central

Ziani, Salim; Nagy, Zita; Alekseev, Sergey; Soutoglou, Evi; Egly, Jean-Marc

2014-01-01

In nucleotide excision repair (NER), damage recognition by XPC-hHR23b is described as a critical step in the formation of the preincision complex (PInC) further composed of TFIIH, XPA, RPA, XPG, and ERCC1-XPF. To obtain new molecular insights into the assembly of the PInC, we analyzed its formation independently of DNA damage by using the lactose operator/repressor reporter system. We observed a sequential and ordered self-assembly of the PInC operating upon immobilization of individual NER factors on undamaged chromatin and mimicking that functioning on a bona fide NER substrate. We also revealed that the recruitment of the TFIIH subunit TTDA, involved in trichothiodystrophy group A disorder (TTD-A), was key in the completion of the PInC. TTDA recruits XPA through its first 15 amino acids, depleted in some TTD-A patients. More generally, these results show that proteins forming large nuclear complexes can be recruited sequentially on chromatin in the absence of their natural DNA target and with no reciprocity in their recruitment. PMID:25154395

Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs.

PubMed

Machado-Pinilla, Rosario; Liger, Dominique; Leulliot, Nicolas; Meier, U Thomas

2012-10-01

The AAA+ ATPases pontin and reptin function in a staggering array of cellular processes including chromatin remodeling, transcriptional regulation, DNA damage repair, and assembly of macromolecular complexes, such as RNA polymerase II and small nucleolar (sno) RNPs. However, the molecular mechanism for all of these AAA+ ATPase associated activities is unknown. Here we document that, during the biogenesis of H/ACA RNPs (including telomerase), the assembly factor SHQ1 holds the pseudouridine synthase NAP57/dyskerin in a viselike grip, and that pontin and reptin (as components of the R2TP complex) are required to pry NAP57 from SHQ1. Significantly, the NAP57 domain captured by SHQ1 harbors most mutations underlying X-linked dyskeratosis congenita (X-DC) implicating the interface between the two proteins as a target of this bone marrow failure syndrome. Homing in on the essential first steps of H/ACA RNP biogenesis, our findings provide the first insight into the mechanism of action of pontin and reptin in the assembly of macromolecular complexes.

Mechanism of the AAA+ ATPases pontin and reptin in the biogenesis of H/ACA RNPs

PubMed Central

Machado-Pinilla, Rosario; Liger, Dominique; Leulliot, Nicolas; Meier, U. Thomas

2012-01-01

The AAA+ ATPases pontin and reptin function in a staggering array of cellular processes including chromatin remodeling, transcriptional regulation, DNA damage repair, and assembly of macromolecular complexes, such as RNA polymerase II and small nucleolar (sno) RNPs. However, the molecular mechanism for all of these AAA+ ATPase associated activities is unknown. Here we document that, during the biogenesis of H/ACA RNPs (including telomerase), the assembly factor SHQ1 holds the pseudouridine synthase NAP57/dyskerin in a viselike grip, and that pontin and reptin (as components of the R2TP complex) are required to pry NAP57 from SHQ1. Significantly, the NAP57 domain captured by SHQ1 harbors most mutations underlying X-linked dyskeratosis congenita (X-DC) implicating the interface between the two proteins as a target of this bone marrow failure syndrome. Homing in on the essential first steps of H/ACA RNP biogenesis, our findings provide the first insight into the mechanism of action of pontin and reptin in the assembly of macromolecular complexes. PMID:22923768

Molecular basis of APC/C regulation by the spindle assembly checkpoint

PubMed Central

Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

2016-01-01

In the dividing eukaryotic cell the spindle assembly checkpoint (SAC) ensures each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), a multimeric assembly that inhibits the APC/C, delaying chromosome segregation. Here, using cryo-electron microscopy we determined the near-atomic resolution structure of an APC/C-MCC complex (APC/CMCC). We reveal how degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit (Cdc20APC/C) responsible for substrate interactions. BubR1 also obstructs binding of UbcH10 (APC/C’s initiating E2) to repress APC/C ubiquitination activity. Conformational variability of the complex allows for UbcH10 association, and we show from a structure of APC/CMCC in complex with UbcH10 how the Cdc20 subunit intrinsic to the MCC (Cdc20MCC) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced. PMID:27509861

Quantifying quality in DNA self-assembly

PubMed Central

Wagenbauer, Klaus F.; Wachauf, Christian H.; Dietz, Hendrik

2014-01-01

Molecular self-assembly with DNA is an attractive route for building nanoscale devices. The development of sophisticated and precise objects with this technique requires detailed experimental feedback on the structure and composition of assembled objects. Here we report a sensitive assay for the quality of assembly. The method relies on measuring the content of unpaired DNA bases in self-assembled DNA objects using a fluorescent de-Bruijn probe for three-base ‘codons’, which enables a comparison with the designed content of unpaired DNA. We use the assay to measure the quality of assembly of several multilayer DNA origami objects and illustrate the use of the assay for the rational refinement of assembly protocols. Our data suggests that large and complex objects like multilayer DNA origami can be made with high strand integration quality up to 99%. Beyond DNA nanotechnology, we speculate that the ability to discriminate unpaired from paired nucleic acids in the same macromolecule may also be useful for analysing cellular nucleic acids. PMID:24751596

Whole-cell biocomputing

NASA Technical Reports Server (NTRS)

Simpson, M. L.; Sayler, G. S.; Fleming, J. T.; Applegate, B.

2001-01-01

The ability to manipulate systems on the molecular scale naturally leads to speculation about the rational design of molecular-scale machines. Cells might be the ultimate molecular-scale machines and our ability to engineer them is relatively advanced when compared with our ability to control the synthesis and direct the assembly of man-made materials. Indeed, engineered whole cells deployed in biosensors can be considered one of the practical successes of molecular-scale devices. However, these devices explore only a small portion of cellular functionality. Individual cells or self-organized groups of cells perform extremely complex functions that include sensing, communication, navigation, cooperation and even fabrication of synthetic nanoscopic materials. In natural systems, these capabilities are controlled by complex genetic regulatory circuits, which are only partially understood and not readily accessible for use in engineered systems. Here, we focus on efforts to mimic the functionality of man-made information-processing systems within whole cells.

The water-hydrophobic interface: neutral and charged solute adsorption at fluorocarbon and hydrocarbon self-assembled monolayers (SAMs).

PubMed

Hopkins, Adam J; Richmond, Geraldine L

2013-03-01

Adsorption of small molecular solutes in an aqueous solution to a soft hydrophobic surface is a topic relevant to many fields. In biological and industrial systems, the interfacial environment is often complex, containing an array of salts and organic compounds in the solution phase. Additionally, the surface itself can have a complex structure that can interact in unpredictable ways with small solutes in its vicinity. In this work, we studied model adsorption processes on hydrocarbon and fluorocarbon self-assembled monolayers by using vibrational sum frequency spectroscopy, with methanol and butylammonium chloride as adsorbates. The results indicate that differences in surface functionality have a significant impact on the organization of adsorbed organic species at hydrophobic surfaces.

Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis.

PubMed

Kalendar, Ruslan; Lee, David; Schulman, Alan H

2011-08-01

The polymerase chain reaction is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. We have developed and tested efficient tools for PCR primer and probe design, which also predict oligonucleotide properties based on experimental studies of PCR efficiency. The tools provide comprehensive facilities for designing primers for most PCR applications and their combinations, including standard, multiplex, long-distance, inverse, real-time, unique, group-specific, bisulphite modification assays, Overlap-Extension PCR Multi-Fragment Assembly, as well as a programme to design oligonucleotide sets for long sequence assembly by ligase chain reaction. The in silico PCR primer or probe search includes comprehensive analyses of individual primers and primer pairs. It calculates the melting temperature for standard and degenerate oligonucleotides including LNA and other modifications, provides analyses for a set of primers with prediction of oligonucleotide properties, dimer and G-quadruplex detection, linguistic complexity, and provides a dilution and resuspension calculator. Copyright © 2011 Elsevier Inc. All rights reserved.

Host-Guest Chemistry in Integrated Porous Space Formed by Molecular Self-Assembly at Liquid-Solid Interfaces.

PubMed

Iritani, Kohei; Tahara, Kazukuni; De Feyter, Steven; Tobe, Yoshito

2017-05-16

Host-guest chemistry in two-dimensional (2D) space, that is, physisorbed monolayers of a single atom or a single molecular thickness on surfaces, has become a subject of intense current interest because of perspectives for various applications in molecular-scale electronics, selective sensors, and tailored catalysis. Scanning tunneling microscopy has been used as a powerful tool for the visualization of molecules in real space on a conducting substrate surface. For more than a decade, we have been investigating the self-assembly of a series of triangle-shaped phenylene-ethynylene macrocycles called dehydrobenzo[12]annulenes (DBAs). These molecules are substituted with six alkyl chains and are capable of forming hexagonal porous 2D molecular networks via van der Waals interactions between interdigitated alkyl chains at the interface of organic solvents and graphite. The dimension of the nanoporous space or nanowell formed by the self-assembly of DBAs can be controlled from 1.6 to 4.7 nm by simply changing the alkyl chain length from C 6 to C 20 . Single molecules as well as homoclusters and heteroclusters are capable of coadsorbing within the host matrix using shape- and size-complementarity principles. Moreover, on the basis of the versatility of the DBA molecules that allows chemical modification of the alkyl chain terminals, we were able to decorate the interior space of the nanoporous networks with functional groups such as azobenzenedicarboxylic acid for photoresponsive guest adsorption/desorption or fluoroalkanes and tetraethylene glycol groups for selective guest binding by electrostatic interactions and zinc-porphyrin units for complexation with a guest by charge-transfer interactions. In this Feature Article, we describe the general aspects of molecular self-assembly at liquid/solid interfaces, followed by the formation of programmed porous molecular networks using rationally designed molecular building blocks. We focus on our own work involving host-guest chemistry in integrated nanoporous space that is modified for specific purposes.

LINCing complex functions at the nuclear envelope

PubMed Central

Rothballer, Andrea; Schwartz, Thomas U.; Kutay, Ulrike

2013-01-01

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. PMID:23324460

The devil is in the details: comparison between COP9 signalosome (CSN) and the LID of the 26S proteasome.

PubMed

Meister, Cindy; Gulko, Miriam Kolog; Köhler, Anna M; Braus, Gerhard H

2016-02-01

The COP9 signalosome (CSN) and the proteasomal LID are conserved macromolecular complexes composed of at least eight subunits with molecular weights of approximately 350 kDa. CSN and LID are part of the ubiquitin–proteasome pathway and cleave isopeptide linkages of lysine side chains on target proteins. CSN cleaves the isopeptide bond of ubiquitin-like protein Nedd8 from cullins, whereas the LID cleaves ubiquitin from target proteins sentenced for degradation. CSN and LID are structurally and functionally similar but the order of the assembly pathway seems to be different. The assembly differs in at least the last subunit joining the pre-assembled subcomplex. This review addresses the similarities and differences in structure, function and assembly of CSN and LID.

Design of Bioinorganic Materials at the Interface of Coordination and Biosupramolecular Chemistry.

PubMed

Maity, Basudev; Ueno, Takafumi

2017-04-01

Protein assemblies have recently become known as potential molecular scaffolds for applications in materials science and bio-nanotechnology. Efforts to design protein assemblies for construction of protein-based hybrid materials with metal ions, metal complexes, nanomaterials and proteins now represent a growing field with a common aim of providing novel functions and mimicking natural functions. However, the important roles of protein assemblies in coordination and biosupramolecular chemistry have not been systematically investigated and characterized. In this personal account, we focus on our recent progress in rational design of protein assemblies using bioinorganic chemistry for (1) exploration of unnatural reactions, (2) construction of functional protein architectures, and (3) in vivo applications. © 2017 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices.

PubMed

Linko, Veikko; Ora, Ari; Kostiainen, Mauri A

2015-10-01

DNA molecules can be assembled into custom predesigned shapes via hybridization of sequence-complementary domains. The folded structures have high spatial addressability and a tremendous potential to serve as platforms and active components in a plethora of bionanotechnological applications. DNA is a truly programmable material, and its nanoscale engineering thus opens up numerous attractive possibilities to develop novel methods for therapeutics. The tailored molecular devices could be used in targeting cells and triggering the cellular actions in the biological environment. In this review we focus on the DNA-based assemblies - primarily DNA origami nanostructures - that could perform complex tasks in cells and serve as smart drug-delivery vehicles in, for example, cancer therapy, prodrug medication, and enzyme replacement therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular dynamics simulations of electrostatics and hydration distributions around RNA and DNA motifs

NASA Astrophysics Data System (ADS)

Marlowe, Ashley E.; Singh, Abhishek; Semichaevsky, Andrey V.; Yingling, Yaroslava G.

2009-03-01

Nucleic acid nanoparticles can self-assembly through the formation of complementary loop-loop interactions or stem-stem interactions. Presence and concentration of ions can significantly affect the self-assembly process and the stability of the nanostructure. In this presentation we use explicit molecular dynamics simulations to examine the variations in cationic distributions and hydration environment around DNA and RNA helices and loop-loop interactions. Our simulations show that the potassium and sodium ionic distributions are different around RNA and DNA motifs which could be indicative of ion mediated relative stability of loop-loop complexes. Moreover in RNA loop-loop motifs ions are consistently present and exchanged through a distinct electronegative channel. We will also show how we used the specific RNA loop-loop motif to design a RNA hexagonal nanoparticle.

Structure of a bacterial virus DNA-injection protein complex reveals a decameric assembly with a constricted molecular channel

DOE PAGES

Zhao, Haiyan; Speir, Jeffrey A.; Matsui, Tsutomu; ...

2016-02-16

The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. Themore » assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. As a result, the gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.« less

Structure of a bacterial virus DNA-injection protein complex reveals a decameric assembly with a constricted molecular channel

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

Zhao, Haiyan; Speir, Jeffrey A.; Matsui, Tsutomu

The multi-layered cell envelope structure of Gram-negative bacteria represents significant physical and chemical barriers for short-tailed phages to inject phage DNA into the host cytoplasm. Here we show that a DNA-injection protein of bacteriophage Sf6, gp12, forms a 465-kDa, decameric assembly in vitro. The electron microscopic structure of the gp12 assembly shows a ~150-Å, mushroom-like architecture consisting of a crown domain and a tube-like domain, which embraces a 25-Å-wide channel that could precisely accommodate dsDNA. The constricted channel suggests that gp12 mediates rapid, uni-directional injection of phage DNA into host cells by providing a molecular conduit for DNA translocation. Themore » assembly exhibits a 10-fold symmetry, which may be a common feature among DNA-injection proteins of P22-like phages and may suggest a symmetry mismatch with respect to the 6-fold symmetric phage tail. As a result, the gp12 monomer is highly flexible in solution, supporting a mechanism for translocation of the protein through the conduit of the phage tail toward the host cell envelope, where it assembles into a DNA-injection device.« less

Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex*

PubMed Central

Soundararajan, Rama; Ziera, Tim; Koo, Eric; Ling, Karen; Wang, Jian; Borden, Steffen A.; Pearce, David

2012-01-01

Hormone regulation of ion transport in the kidney tubules is essential for fluid and electrolyte homeostasis in vertebrates. A large body of evidence has suggested that transporters and channels exist in multiprotein regulatory complexes; however, relatively little is known about the composition of these complexes or their assembly. The epithelial sodium channel (ENaC) in particular is tightly regulated by the salt-regulatory hormone aldosterone, which acts at least in part by increasing expression of the serine-threonine kinase SGK1. Here we show that aldosterone induces the formation of a 1.0–1.2-MDa plasma membrane complex, which includes ENaC, SGK1, and the ENaC inhibitor Nedd4-2, a key target of SGK1. We further show that this complex contains the PDZ domain-containing protein connector enhancer of kinase suppressor of Ras isoform 3 (CNK3). CNK3 physically interacts with ENaC, Nedd4-2, and SGK1; enhances the interactions among them; and stimulates ENaC function in a PDZ domain-dependent, aldosterone-induced manner. These results strongly suggest that CNK3 is a molecular scaffold, which coordinates the assembly of a multiprotein ENaC-regulatory complex and hence plays a central role in Na+ homeostasis. PMID:22851176

Surfactant mediated polyelectrolyte self-assembly

DOE PAGES

Goswami, Monojoy; Borreguero Calvo, Jose M.; Pincus, Phillip A.; ...

2015-11-25

Self-assembly and dynamics of polyelectrolyte (PE) surfactant complex (PES) is investigated using molecular dynamics simulations. The complexation is systematically studied for five different PE backbone charge densities. At a fixed surfactant concentration the PES complexation exhibits pearl-necklace to agglomerated double spherical structures with a PE chain decorating the surfactant micelles. The counterions do not condense on the complex, but are released in the medium with a random distribution. The relaxation dynamics for three different length scales, polymer chain, segmental and monomer, show distinct features of the charge and neutral species; the counterions are fastest followed by the PE chain andmore » surfactants. The surfactant heads and tails have the slowest relaxation due to their restricted movement inside the agglomerated structure. At the shortest length scale, all the charge and neutral species show similar relaxation dynamics confirming Rouse behavior at monomer length scales. Overall, the present study highlights the structure-property relationship for polymer-surfactant complexation. These results will help improve the understanding of PES complex and should aid in the design of better materials for future applications.« less

Mutation screening of 75 candidate genes in 152 complex I deficiency cases identifies pathogenic variants in 16 genes including NDUFB9.

PubMed

Haack, Tobias B; Madignier, Florence; Herzer, Martina; Lamantea, Eleonora; Danhauser, Katharina; Invernizzi, Federica; Koch, Johannes; Freitag, Martin; Drost, Rene; Hillier, Ingo; Haberberger, Birgit; Mayr, Johannes A; Ahting, Uwe; Tiranti, Valeria; Rötig, Agnes; Iuso, Arcangela; Horvath, Rita; Tesarova, Marketa; Baric, Ivo; Uziel, Graziella; Rolinski, Boris; Sperl, Wolfgang; Meitinger, Thomas; Zeviani, Massimo; Freisinger, Peter; Prokisch, Holger

2012-02-01

Mitochondrial complex I deficiency is the most common cause of mitochondrial disease in childhood. Identification of the molecular basis is difficult given the clinical and genetic heterogeneity. Most patients lack a molecular definition in routine diagnostics. A large-scale mutation screen of 75 candidate genes in 152 patients with complex I deficiency was performed by high-resolution melting curve analysis and Sanger sequencing. The causal role of a new disease allele was confirmed by functional complementation assays. The clinical phenotype of patients carrying mutations was documented using a standardised questionnaire. Causative mutations were detected in 16 genes, 15 of which had previously been associated with complex I deficiency: three mitochondrial DNA genes encoding complex I subunits, two mitochondrial tRNA genes and nuclear DNA genes encoding six complex I subunits and four assembly factors. For the first time, a causal mutation is described in NDUFB9, coding for a complex I subunit, resulting in reduction in NDUFB9 protein and both amount and activity of complex I. These features were rescued by expression of wild-type NDUFB9 in patient-derived fibroblasts. Mutant NDUFB9 is a new cause of complex I deficiency. A molecular diagnosis related to complex I deficiency was established in 18% of patients. However, most patients are likely to carry mutations in genes so far not associated with complex I function. The authors conclude that the high degree of genetic heterogeneity in complex I disorders warrants the implementation of unbiased genome-wide strategies for the complete molecular dissection of mitochondrial complex I deficiency.

Guiding principles for peptide nanotechnology through directed discovery.

PubMed

Lampel, A; Ulijn, R V; Tuttle, T

2018-05-21

Life's diverse molecular functions are largely based on only a small number of highly conserved building blocks - the twenty canonical amino acids. These building blocks are chemically simple, but when they are organized in three-dimensional structures of tremendous complexity, new properties emerge. This review explores recent efforts in the directed discovery of functional nanoscale systems and materials based on these same amino acids, but that are not guided by copying or editing biological systems. The review summarises insights obtained using three complementary approaches of searching the sequence space to explore sequence-structure relationships for assembly, reactivity and complexation, namely: (i) strategic editing of short peptide sequences; (ii) computational approaches to predicting and comparing assembly behaviours; (iii) dynamic peptide libraries that explore the free energy landscape. These approaches give rise to guiding principles on controlling order/disorder, complexation and reactivity by peptide sequence design.

Flexible Stoichiometry and Asymmetry of the PIDDosome Core Complex by Heteronuclear NMR Spectroscopy and Mass Spectrometry

PubMed Central

Nematollahi, Lily A.; Garza-Garcia, Acely; Bechara, Chérine; Esposito, Diego; Morgner, Nina; Robinson, Carol V.; Driscoll, Paul C.

2015-01-01

Homotypic death domain (DD)–DD interactions are important in the assembly of oligomeric signaling complexes such as the PIDDosome that acts as a platform for activation of caspase-2-dependent apoptotic signaling. The structure of the PIDDosome core complex exhibits an asymmetric three-layered arrangement containing five PIDD-DDs in one layer, five RAIDD-DDs in a second layer and an additional two RAIDD-DDs. We addressed complex formation between PIDD-DD and RAIDD-DD in solution using heteronuclear nuclear magnetic resonance (NMR) spectroscopy, nanoflow electrospray ionization mass spectrometry and size-exclusion chromatography with multi-angle light scattering. The DDs assemble into complexes displaying molecular masses in the range 130–158 kDa and RAIDD-DD:PIDD-DD stoichiometries of 5:5, 6:5 and 7:5. These data suggest that the crystal structure is representative of only the heaviest species in solution and that two RAIDD-DDs are loosely attached to the 5:5 core. Two-dimensional 1H,15N-NMR experiments exhibited signal loss upon complexation consistent with the formation of high-molecular-weight species. 13C-Methyl-transverse relaxation optimized spectroscopy measurements of the PIDDosome core exhibit signs of differential line broadening, cross-peak splitting and chemical shift heterogeneity that reflect the presence of non-equivalent sites at interfaces within an asymmetric complex. Experiments using a mutant RAIDD-DD that forms a monodisperse 5:5 complex with PIDD-DD show that the spectroscopic signature derives from the quasi- but non-exact equivalent environments of each DD. Since this characteristic was previously demonstrated for the complex between the DDs of CD95 and FADD, the NMR data for this system are consistent with the formation of a structure homologous to the PIDDosome core. PMID:25528640

Molecular Dynamics Study of Surfactant Self-Assembly on Single-Walled Carbon Nanotubes (SWCNTs)

NASA Astrophysics Data System (ADS)

Phelan, Frederick, Jr.

2015-03-01

Single-walled carbon nanotubes (SWNCTs) are materials with structural, electronic and optical properties that make them attractive for a myriad of advanced technology applications. Increased adaptation of these materials requires advancement in separation techniques which enables them to be sorted with increased reliability into monodisperse fractions with respect to length and chirality. Most separation techniques currently in use rely on dispersion of tubes in aqueous solution using surfactants. This results in a colloidal mixture in which tubes are packed and individually dispersed in a surfactant shell. Understanding the structure and properties of the SWCNT-surfactant complex at the molecular level, and how this is affected by chirality, will help to improve separations processes. In this work, we study the structure and properties of SWCNT-surfactant colloidal complexes using all-atom molecular dynamics. Self-assembled structures are computed for a number of combinations SWCNT/surfactant, and also, co-surfactant mixtures for the bile salt surfactant sodium deoxycholate (DOC) and the anionic surfactant sodium dodecyl sulfate (SDS). From the radial distribution function we estimate the size of the SWCNT hydration layer, and use that information to compute the buoyant densities of unfilled tubes for a number of concentrations. Estimates of the change in hydrodynamic radius with increased surfactant packing and the binding energies of the individual surfactants are also obtained.

ATOMIC RESOLUTION CRYO ELECTRON MICROSCOPY OF MACROMOLECULAR COMPLEXES

PubMed Central

ZHOU, Z. HONG

2013-01-01

Single-particle cryo electron microscopy (cryoEM) is a technique for determining three-dimensional (3D) structures from projection images of molecular complexes preserved in their “native,” noncrystalline state. Recently, atomic or near-atomic resolution structures of several viruses and protein assemblies have been determined by single-particle cryoEM, allowing ab initio atomic model building by following the amino acid side chains or nucleic acid bases identifiable in their cryoEM density maps. In particular, these cryoEM structures have revealed extended arms contributing to molecular interactions that are otherwise not resolved by the conventional structural method of X-ray crystallography at similar resolutions. High-resolution cryoEM requires careful consideration of a number of factors, including proper sample preparation to ensure structural homogeneity, optimal configuration of electron imaging conditions to record high-resolution cryoEM images, accurate determination of image parameters to correct image distortions, efficient refinement and computation to reconstruct a 3D density map, and finally appropriate choice of modeling tools to construct atomic models for functional interpretation. This progress illustrates the power of cryoEM and ushers it into the arsenal of structural biology, alongside conventional techniques of X-ray crystallography and NMR, as a major tool (and sometimes the preferred one) for the studies of molecular interactions in supramolecular assemblies or machines. PMID:21501817

Programmable self-assembly of three-dimensional nanostructures from 10,000 unique components

NASA Astrophysics Data System (ADS)

Ong, Luvena L.; Hanikel, Nikita; Yaghi, Omar K.; Grun, Casey; Strauss, Maximilian T.; Bron, Patrick; Lai-Kee-Him, Josephine; Schueder, Florian; Wang, Bei; Wang, Pengfei; Kishi, Jocelyn Y.; Myhrvold, Cameron; Zhu, Allen; Jungmann, Ralf; Bellot, Gaetan; Ke, Yonggang; Yin, Peng

2017-12-01

Nucleic acids (DNA and RNA) are widely used to construct nanometre-scale structures with ever increasing complexity, with possible application in fields such as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early kilodalton-scale examples containing typically tens of unique DNA strands. The introduction of DNA origami, which uses many staple strands to fold one long scaffold strand into a desired structure, has provided access to megadalton-scale nanostructures that contain hundreds of unique DNA strands. Even larger DNA origami structures are possible, but manufacturing and manipulating an increasingly long scaffold strand remains a challenge. An alternative and more readily scalable approach involves the assembly of DNA bricks, which each consist of four short binding domains arranged so that the bricks can interlock. This approach does not require a scaffold; instead, the short DNA brick strands self-assemble according to specific inter-brick interactions. First-generation bricks used to create three-dimensional structures are 32 nucleotides long, consisting of four eight-nucleotide binding domains. Protocols have been designed to direct the assembly of hundreds of distinct bricks into well formed structures, but attempts to create larger structures have encountered practical challenges and had limited success. Here we show that DNA bricks with longer, 13-nucleotide binding domains make it possible to self-assemble 0.1-1-gigadalton, three-dimensional nanostructures from tens of thousands of unique components, including a 0.5-gigadalton cuboid containing about 30,000 unique bricks and a 1-gigadalton rotationally symmetric tetramer. We also assembled a cuboid that contains around 10,000 bricks and about 20,000 uniquely addressable, 13-base-pair ‘voxels’ that serves as a molecular canvas for three-dimensional sculpting. Complex, user-prescribed, three-dimensional cavities can be produced within this molecular canvas, enabling the creation of shapes such as letters, a helicoid and a teddy bear. We anticipate that with further optimization of structure design, strand synthesis and assembly procedure even larger structures could be accessible, which could be useful for applications such as positioning functional components.

Molecular recognition on a cavitand-functionalized silicon surface.

PubMed

Biavardi, Elisa; Favazza, Maria; Motta, Alessandro; Fragalà, Ignazio L; Massera, Chiara; Prodi, Luca; Montalti, Marco; Melegari, Monica; Condorelli, Guglielmo G; Dalcanale, Enrico

2009-06-03

A Si(100) surface featuring molecular recognition properties was obtained by covalent functionalization with a tetraphosphonate cavitand (Tiiii), able to complex positively charged species. Tiiii cavitand was grafted onto the Si by photochemical hydrosilylation together with 1-octene as a spatial spectator. The recognition properties of the Si-Tiiii surface were demonstrated through two independent analytical techniques, namely XPS and fluorescence spectroscopy, during the course of reversible complexation-guest exchange-decomplexation cycles with specifically designed ammonium and pyridinium salts. Control experiments employing a Si(100) surface functionalized with a structurally similar, but complexation inactive, tetrathiophosphonate cavitand (TSiiii) demonstrated no recognition events. This provides evidence for the complexation properties of the Si-Tiiii surface, ruling out the possibility of nonspecific interactions between the substrate and the guests. The residual Si-O(-) terminations on the surface replace the guests' original counterions, thus stabilizing the complex ion pairs. These results represent a further step toward the control of self-assembly of complex supramolecular architectures on surfaces.

Design of ferrocene-dipeptide bioorganometallic conjugates to induce chirality-organized structures.

PubMed

Moriuchi, Toshiyuki; Hirao, Toshikazu

2010-07-20

The highly ordered molecular assemblies in proteins can have a variety of functions, as observed in enzymes, receptors, and the like. Synthetic scientists are constructing bioinspired systems by harnessing the self-assembling properties of short peptides. Secondary structures such as alpha-helices, beta-sheets, and beta-turns are important in protein folding, which is mostly directed and stabilized by hydrogen bonding and the hydrophobic interactions of side chains. The design of secondary structure mimics that are composed of short peptides has attracted much attention, both for gaining fundamental insight into the factors affecting protein folding and for developing pharmacologically useful compounds, artificial receptors, asymmetric catalysts, and new materials. Ferrocenes are an organometallic scaffold with a central reverse-turn unit based on the inter-ring spacing of about 3.3 A, which is a suitable distance for hydrogen bonding between attached peptide strands. The conjugation of organometallic compounds with biomolecules such as amino acids, peptides, and DNA should provide novel systems that reflect properties of both the ferrocene and the biologically derived moieties. In this Account, we focus on recent advances in the design of ferrocene-peptide bioconjugates, which help illustrate the peptidomimetic basis for protein folding and the means of constructing highly ordered molecular assemblies. Ferrocene-peptide bioconjugates are constructed to form chirality-organized structures in both solid and solution states. The ferrocene serves as a reliable organometallic scaffold for the construction of protein secondary structures via intramolecular hydrogen bonding: the attached dipeptide strands are constrained within the appropriate dimensions. The introduction of the chiral dipeptide chains into the ferrocene scaffold induces the conformational enantiomerization of the ferrocenyl moiety; the chirality-organized structure results from intramolecular hydrogen bonding. The configuration and sequence of the amino acids are instrumental in the process. Regulation of the directionality and specificity of hydrogen bonding is a key component in the design of various molecular assemblies. Ferrocene-peptide bioconjugates also have a strong tendency to self-assemble through the contributions of available hydrogen-bonding donors in the solid state. Some ferrocene-peptide bioconjugates bearing only one dipeptide chain exhibit a helically ordered molecular assembly through a network of intermolecular (rather than intramolecular) hydrogen bonds. The propensity to form the chiral helicity appears to be controlled by the chirality of the dipeptide chains. Organization of host molecules is a useful strategy for forming artificial receptors. The conformationally regulated ferrocene-peptide bioconjugate provides the chirality-organized binding site for size-selective and chiral recognition of dicarboxylic acids through multipoint hydrogen bonds. Metal ions serve a variety of purposes in proteins, including structural stabilization for biological function. The complexation of ferrocene-peptide bioconjugates with palladium(II) compounds not only stabilizes the chirality conformational regulation but also induces conformational regulation of the dipeptide chain through complexation and intramolecular chirality organization. Construction of the chirality-organized ferrocene-peptide bioconjugates is also achieved by metal-directed assembly. These varied examples amply demonstrate the value of ferrocene-peptide bioconjugates in asserting architectural control over highly ordered molecular assemblies.

Molecular engineering of lanthanide ion chelating phospholipids generating assemblies with a switched magnetic susceptibility.

PubMed

Isabettini, Stéphane; Massabni, Sarah; Hodzic, Arnel; Durovic, Dzana; Kohlbrecher, Joachim; Ishikawa, Takashi; Fischer, Peter; Windhab, Erich J; Walde, Peter; Kuster, Simon

2017-08-09

Lanthanide ion (Ln 3+ ) chelating amphiphiles are powerful molecules for tailoring the magnetic response of polymolecular assemblies. Mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-diethylene triaminepentaacetate (DMPE-DTPA) complexed to Ln 3+ deliver highly magnetically responsive bicelles. Their magnetic properties are readily tuned by changing the bicellar size or the magnetic susceptibility Δχ of the bilayer lipids. The former technique is intrinsically bound to the region of the phase diagram guarantying the formation of bicelles. Methods aiming towards manipulating the Δχ of the bilayer are comparatively more robust, flexible and lacking. Herein, we synthesized a new Ln 3+ chelating phospholipid using glutamic acid as a backbone: DMPE-Glu-DTPA. The chelate polyhedron was specifically engineered to alter the Δχ, whilst remaining geometrically similar to DMPE-DTPA. Planar asymmetric assemblies hundreds of nanometers in size were achieved presenting unprecedented magnetic alignments. The DMPE-Glu-DTPA/Ln 3+ complex switched the Δχ, achieving perpendicular alignment of assemblies containing Dy 3+ and parallel alignment of those containing Tm 3+ . Moreover, samples with chelated Yb 3+ were more alignable than the Tm 3+ chelating counterparts. Such a possibility has never been demonstrated for planar Ln 3+ chelating polymolecular assemblies. The physico-chemical properties of these novel assemblies were further studied by monitoring the alignment behavior at different temperatures and by including 16 mol% of cholesterol (Chol-OH) in the phospholipid bilayer. The DMPE-Glu-DTPA/Ln 3+ complex and the resulting assemblies are promising candidates for applications in numerous fields including pharmaceutical technologies, structural characterization of membrane biomolecules by NMR spectroscopy, as contrasting agents for magnetic resonance imaging, and for the development of smart optical gels.

Dramatic Increase in the Signal and Sensitivity of Detection via Self-Assembly of Branched DNA

PubMed Central

Kim, Kyung-Tae; Chae, Chi-Bom

2011-01-01

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA. PMID:21870112

Interfacing DNA nanodevices with biology: challenges, solutions and perspectives

NASA Astrophysics Data System (ADS)

Vinther, Mathias; Kjems, Jørgen

2016-08-01

The cellular machinery performs millions of complex reactions with extreme precision at nanoscale. From studying these reactions, scientists have become inspired to build artificial nanosized molecular devices with programmed functions. One of the fundamental tools in designing and creating these nanodevices is molecular self-assembly. In nature, deoxyribonucleic acid (DNA) is inarguably one of the most remarkable self-assembling molecules. Governed by the Watson-Crick base-pairing rules, DNA assembles with a structural reliability and predictability based on sequence composition unlike any other complex biological polymer. This consistency has enabled rational design of hundreds of two- and three-dimensional shapes with a molecular precision and homogeneity not preceded by any other known technology at the nanometer scale. During the last two decades, DNA nanotechnology has undergone a rapid evolution pioneered by the work of Nadrian Seeman (Kallenbach et al 1983 Nature 205 829-31). Especially the introduction of the versatile DNA Origami technique by Rothemund (2006 Nature 440 297-302) led to an efflorescence of new DNA-based self-assembled nanostructures (Andersen et al 2009 Nature 459 73-6, Douglas et al 2009 Nature 459 414-8, Dietz et al 2009 Science 325 725-30, Han et al 2011 Science 332 342-6, Iinuma et al 2014 Science 344 65-9), and variations of this technique have contributed to an increasing repertoire of DNA nanostructures (Wei et al 2012 Nature 485 623-6, Ke et al 2012 Science 338 1177-83, Benson et al 2015 Nature 523 441-4, Zhang et al 2015 Nat. Nanotechnol. 10 779-84, Scheible et al 2015 Small 11 5200-5). These advances have naturally triggered the question: What can these DNA nanostructures be used for? One of the leading proposals of use for DNA nanotechnology has been in biology and biomedicine acting as a molecular ‘nanorobot’ or smart drug interacting with the cellular machinery. In this review, we will explore and examine the perspective of DNA nanotechnology for such use. We summarize which requirements DNA nanostructures must fulfil to function in cellular environments and inside living organisms. In addition, we highlight recent advances in interfacing DNA nanostructures with biology.

PRISM-EM: template interface-based modelling of multi-protein complexes guided by cryo-electron microscopy density maps.

PubMed

Kuzu, Guray; Keskin, Ozlem; Nussinov, Ruth; Gursoy, Attila

2016-10-01

The structures of protein assemblies are important for elucidating cellular processes at the molecular level. Three-dimensional electron microscopy (3DEM) is a powerful method to identify the structures of assemblies, especially those that are challenging to study by crystallography. Here, a new approach, PRISM-EM, is reported to computationally generate plausible structural models using a procedure that combines crystallographic structures and density maps obtained from 3DEM. The predictions are validated against seven available structurally different crystallographic complexes. The models display mean deviations in the backbone of <5 Å. PRISM-EM was further tested on different benchmark sets; the accuracy was evaluated with respect to the structure of the complex, and the correlation with EM density maps and interface predictions were evaluated and compared with those obtained using other methods. PRISM-EM was then used to predict the structure of the ternary complex of the HIV-1 envelope glycoprotein trimer, the ligand CD4 and the neutralizing protein m36.

Exploring the Molecular Growth of Two Gigantic Half‐Closed Polyoxometalate Clusters {Mo180} and {Mo130Ce6}

PubMed Central

Xuan, Weimin; Pow, Robert; Long, De‐Liang

2017-01-01

Abstract Understanding the process of the self‐assembly of gigantic polyoxometalates and their subsequent molecular growth, by the addition of capping moieties onto the oxo‐frameworks, is critical for the development of the designed assembly of complex high‐nuclearity cluster species, yet such processes remain far from being understood. Herein we describe the molecular growth from {Mo150} and {Mo120Ce6} to afford two half‐closed gigantic molybdenum blue clusters {Mo180} (1) and {Mo130Ce6} (2), respectively. Compound 1 features a hat‐shaped structure with the parent wheel‐shaped {Mo150} being capped by a {Mo30} unit on one side. Similarly, 2 exhibits an elliptical lanthanide‐doped wheel {Mo120Ce6} that is sealed by a {Mo10} unit on one side. Moreover, the observation of the parent uncapped {Mo150} and {Mo120Ce6} clusters as minor products during the synthesis of 1 and 2 strongly suggests that the molecular growth process can be initialized from {Mo150} and {Mo120Ce6} in solution, respectively. PMID:28508585

Self-assembly programming of DNA polyominoes.

PubMed

Ong, Hui San; Syafiq-Rahim, Mohd; Kasim, Noor Hayaty Abu; Firdaus-Raih, Mohd; Ramlan, Effirul Ikhwan

2016-10-20

Fabrication of functional DNA nanostructures operating at a cellular level has been accomplished through molecular programming techniques such as DNA origami and single-stranded tiles (SST). During implementation, restrictive and constraint dependent designs are enforced to ensure conformity is attainable. We propose a concept of DNA polyominoes that promotes flexibility in molecular programming. The fabrication of complex structures is achieved through self-assembly of distinct heterogeneous shapes (i.e., self-organised optimisation among competing DNA basic shapes) with total flexibility during the design and assembly phases. In this study, the plausibility of the approach is validated using the formation of multiple 3×4 DNA network fabricated from five basic DNA shapes with distinct configurations (monomino, tromino and tetrominoes). Computational tools to aid the design of compatible DNA shapes and the structure assembly assessment are presented. The formations of the desired structures were validated using Atomic Force Microscopy (AFM) imagery. Five 3×4 DNA networks were successfully constructed using combinatorics of these five distinct DNA heterogeneous shapes. Our findings revealed that the construction of DNA supra-structures could be achieved using a more natural-like orchestration as compared to the rigid and restrictive conventional approaches adopted previously. Copyright © 2016 Elsevier B.V. All rights reserved.

Human Pyruvate Dehydrogenase Complex E2 and E3BP Core Subunits: New Models and Insights from Molecular Dynamics Simulations.

PubMed

Hezaveh, Samira; Zeng, An-Ping; Jandt, Uwe

2016-05-19

Targeted manipulation and exploitation of beneficial properties of multienzyme complexes, especially for the design of novel and efficiently structured enzymatic reaction cascades, require a solid model understanding of mechanistic principles governing the structure and functionality of the complexes. This type of system-level and quantitative knowledge has been very scarce thus far. We utilize the human pyruvate dehydrogenase complex (hPDC) as a versatile template to conduct corresponding studies. Here we present new homology models of the core subunits of the hPDC, namely E2 and E3BP, as the first time effort to elucidate the assembly of hPDC core based on molecular dynamic simulation. New models of E2 and E3BP were generated and validated at atomistic level for different properties of the proteins. The results of the wild type dimer simulations showed a strong hydrophobic interaction between the C-terminal and the hydrophobic pocket which is the main driving force in the intertrimer binding and the core self-assembly. On the contrary, the C-terminal truncated versions exhibited a drastic loss of hydrophobic interaction leading to a dimeric separation. This study represents a significant step toward a model-based understanding of structure and function of large multienzyme systems like PDC for developing highly efficient biocatalyst or bioreaction cascades.

Generic concept to program the time domain of self-assemblies with a self-regulation mechanism.

PubMed

Heuser, Thomas; Steppert, Ann-Kathrin; Lopez, Catalina Molano; Zhu, Baolei; Walther, Andreas

2015-04-08

Nature regulates complex structures in space and time via feedback loops, kinetically controlled transformations, and under energy dissipation to allow non-equilibrium processes. Although man-made static self-assemblies realize excellent control over hierarchical structures via molecular programming, managing their temporal destiny by self-regulation is a largely unsolved challenge. Herein, we introduce a generic concept to control the time domain by programming the lifetimes of switchable self-assemblies in closed systems. We conceive dormant deactivators that, in combination with fast promoters, enable a unique kinetic balance to establish an autonomously self-regulating, transient pH-state, whose duration can be programmed over orders of magnitude-from minutes to days. Coupling this non-equilibrium state to pH-switchable self-assemblies allows predicting their assembly/disassembly fate in time, similar to a precise self-destruction mechanism. We demonstrate a platform approach by programming self-assembly lifetimes of block copolymers, nanoparticles, and peptides, enabling dynamic materials with a self-regulation functionality.

Lipid membrane-assisted condensation and assembly of amphiphilic Janus particles

DOE PAGES

Chambers, Mariah; Mallory, Stewart Anthony; Malone, Heather; ...

2016-01-01

Amphiphilic Janus particles self-assemble into complex metastructures, but little is known about how their assembly might be modified by weak interactions with a nearby biological membrane surface. Here, we report an integrated experimental and molecular dynamics simulation study to investigate the self-assembly of amphiphilic Janus particles on a lipid membrane. We created an experimental system in which Janus particles are allowed to self-assemble in the same medium where zwitterionic lipids form giant unilamellar vesicles (GUVs). Janus particles spontaneously concentrated on the inner leaflet of the GUVs. They exhibited biased orientation and heterogeneous rotational dynamics as revealed by single particle rotationalmore » tracking. The combined experimental and simulation results show that Janus particles concentrate on the lipid membranes due to weak particle–lipid attraction, whereas the biased orientation of particles is driven predominantly by inter-particle interactions. Furthermore, this study demonstrates the potential of using lipid membranes to influence the self-assembly of Janus particles.« less

Light-induced nonadiabatic dynamics in molecular assemblies and nanostructures

NASA Astrophysics Data System (ADS)

Mitric, Roland

The combination of mixed quantum-classical dynamics with efficient electronic structure methods was developed in order to simulate the light-induced processes in complex molecules, multichromophoric aggregates and metallic nanostructures. We will demonstrate how the combination of nonadiabatic dynamics with experimental pump-probe techniques such as time-resolved photoelectron imaging (TRPEI) allows to fully resolve the mechanism of excited state relaxation through conical intersections in several prototype organic- and biomolecules. Specifically, the role of the solvent in the excited state relaxation in microsolvated and fully solvated systems will be addressed. Currently there is growing evidence that nonadiabatic relaxation processes also play a fundamental role in determining the efficiency of excitonic transfer or charge injection in multichromophoric assemblies. Since such systems are currently out of the reach of the state-of-the-art quantum chemistry a development of even more efficient quantum chemical approaches is necessary in order to describe the excited state dynamics in such assemblies. For this purpose we have recently developed long-range corrected time-dependent density functional tight binding (LC-TDDFTB) nonadiabatic dynamics and combined it with the QM/MM approach in order to simulate exciton relaxation in complex systems. The applications of the method to the investigation of the optical properties and dynamics in multichromophoric assemblies including stacked pi-conjugated organic chromophores, model molecular crystals as well as self-organized dye aggregates will be presented. Finally, we will address exciton transport dynamics coupled with the light propagation in hybrid exciton-plasmon nanostructures, which represent promising materials fort the development of novel light-harvesting systems.

Co-self-assembly of cationic microparticles to deliver pEGFP-ZNF580 for promoting the transfection and migration of endothelial cells

PubMed Central

Feng, Yakai; Guo, Mengyang; Liu, Wen; Hao, Xuefang; Lu, Wei; Ren, Xiangkui; Shi, Changcan; Zhang, Wencheng

2017-01-01

The gene transfection efficiency of polyethylenimine (PEI) varies with its molecular weight. Usually, high molecular weight of PEI means high gene transfection, as well as high cytotoxicity in gene delivery in vivo. In order to enhance the transfection efficiency and reduce the cytotoxicity of PEI-based gene carriers, a novel cationic gene carrier was developed by co-self-assembly of cationic copolymers. First, a star-shaped copolymer poly(3(S)-methyl-morpholine-2,5-dione-co-lactide) (P(MMD-co-LA)) was synthesized using D-sorbitol as an initiator, and the cationic copolymer (P(MMD-co-LA)-g-PEI) was obtained after grafting low-molecular weight PEI. Then, by co-self-assembly of this cationic copolymer and a diblock copolymer methoxy-poly(ethylene glycol) (mPEG)-b-P(MMD-co-LA), microparticles (MPs) were formed. The core of MPs consisted of a biodegradable block of P(MMD-co-LA), and the shell was formed by mPEG and PEI blocks. Finally, after condensation of pEGFP-ZNF580 by these MPs, the plasmids were protected from enzymatic hydrolysis effectively. The result indicated that pEGFP-ZNF580-loaded MP complexes were suitable for cellular uptake and gene transfection. When the mass ratio of mPEG-b-P(MMD-co-LA) to P(MMD-co-LA)-g-PEI reached 3/1, the cytotoxicity of the complexes was very low at low concentration (20 μg mL−1). Additionally, pEGFP-ZNF580 could be transported into endothelial cells (ECs) effectively via the complexes of MPs/pEGFP-ZNF580. Wound-healing assay showed that the transfected ECs recovered in 24 h. Cationic MPs designed in the present study could be used as an applicable gene carrier for the endothelialization of artificial blood vessels. PMID:28053529

Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers.

PubMed

Zhang, Yongli

2017-07-01

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are universal molecular engines that drive membrane fusion. Particularly, synaptic SNAREs mediate fast calcium-triggered fusion of neurotransmitter-containing vesicles with plasma membranes for synaptic transmission, the basis of all thought and action. During membrane fusion, complementary SNAREs located on two apposed membranes (often called t- and v-SNAREs) join together to assemble into a parallel four-helix bundle, releasing the energy to overcome the energy barrier for fusion. A long-standing hypothesis suggests that SNAREs act like a zipper to draw the two membranes into proximity and thereby force them to fuse. However, a quantitative test of this SNARE zippering hypothesis was hindered by difficulties to determine the energetics and kinetics of SNARE assembly and to identify the relevant folding intermediates. Here, we first review different approaches that have been applied to study SNARE assembly and then focus on high-resolution optical tweezers. We summarize the folding energies, kinetics, and pathways of both wild-type and mutant SNARE complexes derived from this new approach. These results show that synaptic SNAREs assemble in four distinct stages with different functions: slow N-terminal domain association initiates SNARE assembly; a middle domain suspends and controls SNARE assembly; and rapid sequential zippering of the C-terminal domain and the linker domain directly drive membrane fusion. In addition, the kinetics and pathway of the stagewise assembly are shared by other SNARE complexes. These measurements prove the SNARE zippering hypothesis and suggest new mechanisms for SNARE assembly regulated by other proteins. © 2017 The Protein Society.

Haplotype assembly in polyploid genomes and identical by descent shared tracts.

PubMed

Aguiar, Derek; Istrail, Sorin

2013-07-01

Genome-wide haplotype reconstruction from sequence data, or haplotype assembly, is at the center of major challenges in molecular biology and life sciences. For complex eukaryotic organisms like humans, the genome is vast and the population samples are growing so rapidly that algorithms processing high-throughput sequencing data must scale favorably in terms of both accuracy and computational efficiency. Furthermore, current models and methodologies for haplotype assembly (i) do not consider individuals sharing haplotypes jointly, which reduces the size and accuracy of assembled haplotypes, and (ii) are unable to model genomes having more than two sets of homologous chromosomes (polyploidy). Polyploid organisms are increasingly becoming the target of many research groups interested in the genomics of disease, phylogenetics, botany and evolution but there is an absence of theory and methods for polyploid haplotype reconstruction. In this work, we present a number of results, extensions and generalizations of compass graphs and our HapCompass framework. We prove the theoretical complexity of two haplotype assembly optimizations, thereby motivating the use of heuristics. Furthermore, we present graph theory-based algorithms for the problem of haplotype assembly using our previously developed HapCompass framework for (i) novel implementations of haplotype assembly optimizations (minimum error correction), (ii) assembly of a pair of individuals sharing a haplotype tract identical by descent and (iii) assembly of polyploid genomes. We evaluate our methods on 1000 Genomes Project, Pacific Biosciences and simulated sequence data. HapCompass is available for download at http://www.brown.edu/Research/Istrail_Lab/. Supplementary data are available at Bioinformatics online.

Two-stages of chiral selectivity in the molecular self-assembly of tryptophan

NASA Astrophysics Data System (ADS)

Guisinger, Nathan

Both chirality and molecular assembly are essential and key components to life. In this study we explore the molecular assembly of the amino acid tryptophan (both L- and D- chiralities) on Cu(111). Our investigation utilizes low temperature scanning tunneling microscopy to observe resulting assemblies at the molecular scale. We find that depositing a racemic mixture of both L- and D- tryptophan results in the assembly of basic 6 molecule ``Lego'' structures that are enantiopure. These enantiopure ``Legos'' further assemble into 1-dimensional chains one block at a time. These resulting chains are also enantiopure with chiral selectivity occurring at two stages of assembly. Utilizing scanning tunneling spectroscopy we are able to probe the electronic structure of the chiral Legos that give insight into the root of the observed selectivity. Two-stages of chiral selectivity in the molecular self-assembly of tryptophan.

In Vitro Formation of Plant RNA-Induced Silencing Complexes Using an Extract of Evacuolated Tobacco Protoplasts.

PubMed

Iki, Taichiro; Ishikawa, Masayuki; Yoshikawa, Manabu

2017-01-01

Small RNA-mediated gene silencing is involved in a variety of biological processes among many eukaryotic organisms. The silencing effector, generally referred to as RNA-induced silencing complex (RISC), comprises an ARGONAUTE (AGO) protein and a small single-stranded guide RNA in its core. RISCs recognize target genes containing sequences complementary to the guide RNA and repress their expression transcriptionally or posttranscriptionally. In vitro systems that recapitulate RISC assembly are useful not only to decipher the molecular mechanisms underlying the assembly process itself but also to dissect the downstream silencing pathways mediated by RISCs. Here, we describe a method for in vitro plant RISC assembly, which relies on an extract of evacuolated protoplasts derived from Nicotiana tabacum BY-2 suspension-cultured cells. In this extract, synthetic duplexes of small RNAs are incorporated into AGO proteins that are synthesized by in vitro translation, and then duplex unwinding and selective strand elimination result in formation of mature RISCs.

Optical properties of azobenzene-functionalized self-assembled monolayers: Intermolecular coupling and many-body interactions

NASA Astrophysics Data System (ADS)

Cocchi, Caterina; Moldt, Thomas; Gahl, Cornelius; Weinelt, Martin; Draxl, Claudia

2016-12-01

In a joint theoretical and experimental work, the optical properties of azobenzene-functionalized self-assembled monolayers (SAMs) are studied at different molecular packing densities. Our results, based on density-functional and many-body perturbation theory, as well as on differential reflectance (DR) spectroscopy, shed light on the microscopic mechanisms ruling photo-absorption in these systems. While the optical excitations are intrinsically excitonic in nature, regardless of the molecular concentration, in densely packed SAMs intermolecular coupling and local-field effects are responsible for a sizable weakening of the exciton binding strength. Through a detailed analysis of the character of the electron-hole pairs, we show that distinct excitations involved in the photo-isomerization at low molecular concentrations are dramatically broadened by intermolecular interactions. Spectral shifts in the calculated DR spectra are in good agreement with the experimental results. Our findings represent an important step forward to rationalize the excited-state properties of these complex materials.

HIV-1 Vif promotes the formation of high molecular mass APOBEC3G complexes

PubMed Central

Goila-Gaur, Ritu; Khan, Mohammad A.; Miyagi, Eri; Kao, Sandra; Opi, Sandrine; Takeuchi, Hiroaki; Strebel, Klaus

2008-01-01

HIV-1 Vif inhibits the antiviral activity of APOBEC3G (APO3G) by inducing proteasomal degradation. Here, we studied the effects of Vif on APO3G in vitro. In this system, Vif did not cause APO3G degradation. Instead, Vif induced changes in APO3G that affected immunoprecipitation of the native protein. This effect required wt Vif and was reversed by heat-denaturation of APO3G. Sucrose gradient analysis demonstrated that wt Vif induced the gradual transition of APO3G translated in vitro or expressed in HeLa cells from a low molecular mass conformation to puromycin-sensitive high molecular mass (HMM) complexes. In the absence of Vif or the presence of biologically inactive Vif APO3G failed to form HMM complexes. Our results expose a novel function of Vif that promotes the assembly of APO3G into presumably packaging-incompetent HMM complexes and may explain how Vif can overcome the APO3G-imposed block to HIV replication under conditions of no or inefficient APO3G degradation. PMID:18023836

Interaction of the iron–sulfur cluster assembly protein IscU with the Hsc66/Hsc20 molecular chaperone system of Escherichia coli

PubMed Central

Hoff, Kevin G.; Silberg, Jonathan J.; Vickery, Larry E.

2000-01-01

The iscU gene in bacteria is located in a gene cluster encoding proteins implicated in iron–sulfur cluster assembly and an hsc70-type (heat shock cognate) molecular chaperone system, iscSUA-hscBA. To investigate possible interactions between these systems, we have overproduced and purified the IscU protein from Escherichia coli and have studied its interactions with the hscA and hscB gene products Hsc66 and Hsc20. IscU and its iron–sulfur complex (IscU–Fe/S) stimulated the basal steady-state ATPase activity of Hsc66 weakly in the absence of Hsc20 but, in the presence of Hsc20, increased the ATPase activity up to 480-fold. Hsc20 also decreased the apparent Km for IscU stimulation of Hsc66 ATPase activity, and surface plasmon resonance studies revealed that Hsc20 enhances binding of IscU to Hsc66. Surface plasmon resonance and isothermal titration calorimetry further showed that IscU and Hsc20 form a complex, and Hsc20 may thereby aid in the targeting of IscU to Hsc66. These results establish a direct and specific role for the Hsc66/Hsc20 chaperone system in functioning with isc gene components for the assembly of iron–sulfur cluster proteins. PMID:10869428

Hydrogen bonded binary molecular adducts derived from exobidentate N-donor ligand with dicarboxylic acids: Acid⋯imidazole hydrogen-bonding interactions in neutral and ionic heterosynthons

NASA Astrophysics Data System (ADS)

Kathalikkattil, Amal Cherian; Damodaran, Subin; Bisht, Kamal Kumar; Suresh, Eringathodi

2011-01-01

Four new binary molecular compounds between a flexible exobidentate N-heterocycle and a series of dicarboxylic acids have been synthesized. The N-donor 1,4-bis(imidazol-1-ylmethyl)benzene (bix) was reacted with flexible and rigid dicarboxylic acids viz., cyclohexane-1,4-dicarboxylic acid (H 2chdc), naphthalene-1,4-dicarboxylic acid (H 2npdc) and 1H-pyrazole-3,5-dicarboxylic acid (H 2pzdc), generating four binary molecular complexes. X-ray crystallographic investigation of the molecular adducts revealed the primary intermolecular interactions carboxylic acid⋯amine (via O-H⋯N) as well as carboxylate⋯protonated amine (via N-H +⋯O -) within the binary compounds, generating layered and two-dimensional sheet type H-bonded networks involving secondary weak interactions (C-H⋯O) including the solvent of crystallization. Depending on the differences in p Ka values of the selected base/acid (Δp Ka), diverse H-bonded supramolecular assemblies could be premeditated. This study demonstrates the H-bonding interactions between imidazole/imidazolium cation and carboxylic acid/carboxylate anion in providing sufficient driving force for the directed assembly of binary molecular complexes. In the two-component solid form of hetero synthons involving bix and dicarboxylic acid, only H 2chdc exist as cocrystal with bix, while all the other three compounds crystallized exclusively as salt, in agreement with the Δp Ka values predicted for the formation of salts/cocrystals from the base and acid used in the synthesis of supramolecular solids.

Disentangling polydispersity in the PCNA−p15PAF complex, a disordered, transient and multivalent macromolecular assembly

PubMed Central

Cordeiro, Tiago N.; Chen, Po-chia; De Biasio, Alfredo; Sibille, Nathalie; Blanco, Francisco J.; Hub, Jochen S.; Crehuet, Ramon

2017-01-01

Abstract The intrinsically disordered p15PAF regulates DNA replication and repair when interacting with the Proliferating Cell Nuclear Antigen (PCNA) sliding clamp. As many interactions between disordered proteins and globular partners involved in signaling and regulation, the complex between p15PAF and trimeric PCNA is of low affinity, forming a transient complex that is difficult to characterize at a structural level due to its inherent polydispersity. We have determined the structure, conformational fluctuations, and relative population of the five species that coexist in solution by combining small-angle X-ray scattering (SAXS) with molecular modelling. By using explicit ensemble descriptions for the individual species, built using integrative approaches and molecular dynamics (MD) simulations, we collectively interpreted multiple SAXS profiles as population-weighted thermodynamic mixtures. The analysis demonstrates that the N-terminus of p15PAF penetrates the PCNA ring and emerges on the back face. This observation substantiates the role of p15PAF as a drag regulating PCNA processivity during DNA repair. Our study reveals the power of ensemble-based approaches to decode structural, dynamic, and thermodynamic information from SAXS data. This strategy paves the way for deciphering the structural bases of flexible, transient and multivalent macromolecular assemblies involved in pivotal biological processes. PMID:28180305

Polymerization of the tubulin-colchicine complex: relation to microtubule assembly.

PubMed

Andreu, J M; Wagenknecht, T; Timasheff, S N

1983-03-29

The polymerization of purified tubulin-colchicine complex, which results in polymers different from microtubules under microtubule-promoting conditions, has been characterized. It proceeds as a nucleated condensation polymerization, requires Mg2+, and is inhibited by small concentrations of Ca2+. Polymerization requires GTP binding, but GDP is inhibitory. The GTPase activity proceeds, but it is unlinked to polymerization. The thermodynamic characteristics of the growth reaction, namely, the apparent changes of free energy, enthalpy, entropy, heat capacity, and preferential interaction with H+ and Mg2+, are very similar to those of microtubule assembly. It is proposed that the interactions responsible for the two types of polymerization are very similar and that the molecular mechanism of microtubule inhibition by colchicine may consist in a drug-induced distortion of the normal protomer bonding geometry.

A photo-cross-linking approach to monitor folding and assembly of newly synthesized proteins in a living cell.

PubMed

Miyazaki, Ryoji; Myougo, Naomi; Mori, Hiroyuki; Akiyama, Yoshinori

2018-01-12

Many proteins form multimeric complexes that play crucial roles in various cellular processes. Studying how proteins are correctly folded and assembled into such complexes in a living cell is important for understanding the physiological roles and the qualitative and quantitative regulation of the complex. However, few methods are suitable for analyzing these rapidly occurring processes. Site-directed in vivo photo-cross-linking is an elegant technique that enables analysis of protein-protein interactions in living cells with high spatial resolution. However, the conventional site-directed in vivo photo-cross-linking method is unsuitable for analyzing dynamic processes. Here, by combining an improved site-directed in vivo photo-cross-linking technique with a pulse-chase approach, we developed a new method that can analyze the folding and assembly of a newly synthesized protein with high spatiotemporal resolution. We demonstrate that this method, named the pulse-chase and in vivo photo-cross-linking experiment (PiXie), enables the kinetic analysis of the formation of an Escherichia coli periplasmic (soluble) protein complex (PhoA). We also used our new technique to investigate assembly/folding processes of two membrane complexes (SecD-SecF in the inner membrane and LptD-LptE in the outer membrane), which provided new insights into the biogenesis of these complexes. Our PiXie method permits analysis of the dynamic behavior of various proteins and enables examination of protein-protein interactions at the level of individual amino acid residues. We anticipate that our new technique will have valuable utility for studies of protein dynamics in many organisms. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis

PubMed Central

Borek, Weronika E.; Groocock, Lynda M.; Samejima, Itaru; Zou, Juan; de Lima Alves, Flavia; Rappsilber, Juri; Sawin, Kenneth E.

2015-01-01

Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation ‘switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation. PMID:26243668

Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction.

PubMed

Bule, Pedro; Pires, Virgínia M R; Alves, Victor D; Carvalho, Ana Luísa; Prates, José A M; Ferreira, Luís M A; Smith, Steven P; Gilbert, Harry J; Noach, Ilit; Bayer, Edward A; Najmudin, Shabir; Fontes, Carlos M G A

2018-05-03

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

Molecular Studies of Complex Soil Organic Matter Interactions with Metal Ions and Mineral Surfaces using Classical Molecular Dynamics and Quantum Chemistry Methods

NASA Astrophysics Data System (ADS)

Andersen, A.; Govind, N.; Laskin, A.

2017-12-01

Mineral surfaces have been implicated as potential protectors of soil organic matter (SOM) against decomposition and ultimate mineralization to small molecules which can provide nutrients for plants and soil microbes and can also contribute to the Earth's elemental cycles. SOM is a complex mixture of organic molecules of biological origin at varying degrees of decomposition and can, itself, self-assemble in such a way as to expose some biomolecule types to biotic and abiotic attack while protecting other biomolecule types. The organization of SOM and SOM with mineral surfaces and solvated metal ions is driven by an interplay of van der Waals and electrostatic interactions leading to partitioning of hydrophilic (e.g. sugars) and hydrophobic (e.g., lipids) SOM components that can be bridged with amphiphilic molecules (e.g., proteins). Classical molecular dynamics simulations can shed light on assemblies of organic molecules alone or complexation with mineral surfaces. The role of chemical reactions is also an important consideration in potential chemical changes of the organic species such as oxidation/reduction, degradation, chemisorption to mineral surfaces, and complexation with solvated metal ions to form organometallic systems. For the study of chemical reactivity, quantum chemistry methods can be employed and combined with structural insight provided by classical MD simulations. Moreover, quantum chemistry can also simulate spectroscopic signatures based on chemical structure and is a valuable tool in interpreting spectra from, notably, x-ray absorption spectroscopy (XAS). In this presentation, we will discuss our classical MD and quantum chemistry findings on a model SOM system interacting with mineral surfaces and solvated metal ions.

Leigh syndrome associated with mitochondrial complex I deficiency due to a novel mutation in the NDUFS1 gene.

PubMed

Martín, Miguel A; Blázquez, Alberto; Gutierrez-Solana, Luis G; Fernández-Moreira, Daniel; Briones, Paz; Andreu, Antoni L; Garesse, Rafael; Campos, Yolanda; Arenas, Joaquín

2005-04-01

Mutations in the nuclear-encoded subunits of complex I of the mitochondrial respiratory chain are a recognized cause of Leigh syndrome (LS). Recently, 6 mutations in the NDUFS1 gene were identified in 3 families. To describe a Spanish family with LS, complex I deficiency in muscle, and a novel mutation in the NDUFS1 gene. Using molecular genetic approaches, we identified the underlying molecular defect in a patient with LS with a complex I defect. The proband was a child who displayed the clinical features of LS. Muscle biochemistry results showed a complex I defect of the mitochondrial respiratory chain. Sequencing analysis of the mitochondrial DNA-encoded ND genes, the nuclear DNA-encoded NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS6, NDUFS7, NDUFS8, and NDUFAB1 genes, and the complex I assembly factor CIA30 gene revealed a novel homozygous L231V mutation (c.691C-->G) in the NDUFS1 gene. The parents were heterozygous carriers of the L231V mutation. Identifying nuclear mutations as a cause of respiratory chain disorders will enhance the possibility of prenatal diagnosis and help us understand how molecular defects can lead to complex I deficiency.

Ground-State Electronic Structure of RC-LH1 and LH2 Pigment Assemblies of Purple Bacteria via the EBF-MO Method.

PubMed

Shrestha, Kushal; Jakubikova, Elena

2015-08-20

Light-harvesting antennas are protein-pigment complexes that play a crucial role in natural photosynthesis. The antenna complexes absorb light and transfer energy to photosynthetic reaction centers where charge separation occurs. This work focuses on computational studies of the electronic structure of the pigment networks of light-harvesting complex I (LH1), LH1 with the reaction center (RC-LH1), and light-harvesting complex II (LH2) found in purple bacteria. As the pigment networks of LH1, RC-LH1, and LH2 contain thousands of atoms, conventional density functional theory (DFT) and ab initio calculations of these systems are not computationally feasible. Therefore, we utilize DFT in conjunction with the energy-based fragmentation with molecular orbitals method and a semiempirical approach employing the extended Hückel model Hamiltonian to determine the electronic properties of these pigment assemblies. Our calculations provide a deeper understanding of the electronic structure of natural light-harvesting complexes, especially their pigment networks, which could assist in rational design of artificial photosynthetic devices.

Nanostructures formed by cyclodextrin covered procainamide through supramolecular self assembly - Spectral and molecular modeling study

NASA Astrophysics Data System (ADS)

Rajendiran, N.; Mohandoss, T.; Sankaranarayanan, R. K.

2015-02-01

Inclusion complexation behavior of procainamide (PCA) with two cyclodextrins (α-CD and β-CD) were analyzed by absorption, fluorescence, scanning electron microscope (SEM), transmission electron microscope (TEM), Raman image, FT-IR, differential scanning colorimeter (DSC), Powder X ray diffraction (XRD) and 1H NMR. Blue shift was observed in β-CD whereas no significant spectral shift observed in α-CD. The inclusion complex formation results suggest that water molecules also present in the inside of the CD cavity. The present study revealed that the phenyl ring of the PCA drug is entrapped in the CD cavity. Cyclodextrin studies show that PCA forms 1:2 inclusion complex with α-CD and β-CD. PCA:α-CD complex form nano-sized particles (46 nm) and PCA:β-CD complex form self-assembled to micro-sized tubular structures. The shape-shifting of 2D nanosheets into 1D microtubes by simple rolling mechanism were analysed by micro-Raman and TEM images. Thermodynamic parameters (ΔH, ΔG and ΔS) of inclusion process were determined from semiempirical PM3 calculations.

The oncoprotein Ski acts as an antagonist of transforming growth factor-beta signaling by suppressing Smad2 phosphorylation.

PubMed

Prunier, Celine; Pessah, Marcia; Ferrand, Nathalie; Seo, Su Ryeon; Howe, Philip; Atfi, Azeddine

2003-07-11

The phosphorylation of Smad2 and Smad3 by the transforming growth factor (TGF)-beta-activated receptor kinases and their subsequent heterodimerization with Smad4 and translocation to the nucleus form the basis for a model how Smad proteins work to transmit TGF-beta signals. The transcriptional activity of Smad2-Smad4 or Smad3-Smad4 complexes can be limited by the corepressor Ski, which is believed to interact with Smad complexes on TGF-beta-responsive promoters and represses their ability to activate TGF-beta target genes by assembling on DNA a repressor complex containing histone deacetylase. Here we show that Ski can block TGF-beta signaling by interfering with the phosphorylation of Smad2 and Smad3 by the activated TGF-beta type I receptor. Furthermore, we demonstrate that overexpression of Ski induces the assembly of Smad2-Smad4 and Smad3-Smad4 complexes independent of TGF-beta signaling. The ability of Ski to engage Smad proteins in nonproductive complexes provides new insights into the molecular mechanism used by Ski for disabling TGF-beta signaling.

High-performance mussel-inspired adhesives of reduced complexity.

PubMed

Ahn, B Kollbe; Das, Saurabh; Linstadt, Roscoe; Kaufman, Yair; Martinez-Rodriguez, Nadine R; Mirshafian, Razieh; Kesselman, Ellina; Talmon, Yeshayahu; Lipshutz, Bruce H; Israelachvili, Jacob N; Waite, J Herbert

2015-10-19

Despite the recent progress in and demand for wet adhesives, practical underwater adhesion remains limited or non-existent for diverse applications. Translation of mussel-inspired wet adhesion typically entails catechol functionalization of polymers and/or polyelectrolytes, and solution processing of many complex components and steps that require optimization and stabilization. Here we reduced the complexity of a wet adhesive primer to synthetic low-molecular-weight catecholic zwitterionic surfactants that show very strong adhesion (∼50 mJ m(-2)) and retain the ability to coacervate. This catecholic zwitterion adheres to diverse surfaces and self-assembles into a molecularly smooth, thin (<4 nm) and strong glue layer. The catecholic zwitterion holds particular promise as an adhesive for nanofabrication. This study significantly simplifies bio-inspired themes for wet adhesion by combining catechol with hydrophobic and electrostatic functional groups in a small molecule.

Self-organisation of dodeca-dendronized fullerene into supramolecular discs and helical columns containing a nanowire-like core.

PubMed

Guerra, Sebastiano; Iehl, Julien; Holler, Michel; Peterca, Mihai; Wilson, Daniela A; Partridge, Benjamin E; Zhang, Shaodong; Deschenaux, Robert; Nierengarten, Jean-François; Percec, Virgil

2015-06-01

Twelve chiral and achiral self-assembling dendrons have been grafted onto a [60]fullerene hexa-adduct core by copper-catalyzed alkyne azide "click" cycloaddition. The structure adopted by these compounds was determined by the self-assembling peripheral dendrons. These twelve dendrons mediate the self-organisation of the dendronized [60]fullerene into a disc-shaped structure containing the [60]fullerene in the centre. The fullerene-containing discs self-organise into helical supramolecular columns with a fullerene nanowire-like core, forming a 2D columnar hexagonal periodic array. These unprecedented supramolecular structures and their assemblies are expected to provide new developments in chiral complex molecular systems and their application to organic electronics and solar cells.

Gigadalton-scale shape-programmable DNA assemblies

NASA Astrophysics Data System (ADS)

Wagenbauer, Klaus F.; Sigl, Christian; Dietz, Hendrik

2017-12-01

Natural biomolecular assemblies such as molecular motors, enzymes, viruses and subcellular structures often form by self-limiting hierarchical oligomerization of multiple subunits. Large structures can also assemble efficiently from a few components by combining hierarchical assembly and symmetry, a strategy exemplified by viral capsids. De novo protein design and RNA and DNA nanotechnology aim to mimic these capabilities, but the bottom-up construction of artificial structures with the dimensions and complexity of viruses and other subcellular components remains challenging. Here we show that natural assembly principles can be combined with the methods of DNA origami to produce gigadalton-scale structures with controlled sizes. DNA sequence information is used to encode the shapes of individual DNA origami building blocks, and the geometry and details of the interactions between these building blocks then control their copy numbers, positions and orientations within higher-order assemblies. We illustrate this strategy by creating planar rings of up to 350 nanometres in diameter and with atomic masses of up to 330 megadaltons, micrometre-long, thick tubes commensurate in size to some bacilli, and three-dimensional polyhedral assemblies with sizes of up to 1.2 gigadaltons and 450 nanometres in diameter. We achieve efficient assembly, with yields of up to 90 per cent, by using building blocks with validated structure and sufficient rigidity, and an accurate design with interaction motifs that ensure that hierarchical assembly is self-limiting and able to proceed in equilibrium to allow for error correction. We expect that our method, which enables the self-assembly of structures with sizes approaching that of viruses and cellular organelles, can readily be used to create a range of other complex structures with well defined sizes, by exploiting the modularity and high degree of addressability of the DNA origami building blocks used.

Gigadalton-scale shape-programmable DNA assemblies.

PubMed

Wagenbauer, Klaus F; Sigl, Christian; Dietz, Hendrik

2017-12-06

Natural biomolecular assemblies such as molecular motors, enzymes, viruses and subcellular structures often form by self-limiting hierarchical oligomerization of multiple subunits. Large structures can also assemble efficiently from a few components by combining hierarchical assembly and symmetry, a strategy exemplified by viral capsids. De novo protein design and RNA and DNA nanotechnology aim to mimic these capabilities, but the bottom-up construction of artificial structures with the dimensions and complexity of viruses and other subcellular components remains challenging. Here we show that natural assembly principles can be combined with the methods of DNA origami to produce gigadalton-scale structures with controlled sizes. DNA sequence information is used to encode the shapes of individual DNA origami building blocks, and the geometry and details of the interactions between these building blocks then control their copy numbers, positions and orientations within higher-order assemblies. We illustrate this strategy by creating planar rings of up to 350 nanometres in diameter and with atomic masses of up to 330 megadaltons, micrometre-long, thick tubes commensurate in size to some bacilli, and three-dimensional polyhedral assemblies with sizes of up to 1.2 gigadaltons and 450 nanometres in diameter. We achieve efficient assembly, with yields of up to 90 per cent, by using building blocks with validated structure and sufficient rigidity, and an accurate design with interaction motifs that ensure that hierarchical assembly is self-limiting and able to proceed in equilibrium to allow for error correction. We expect that our method, which enables the self-assembly of structures with sizes approaching that of viruses and cellular organelles, can readily be used to create a range of other complex structures with well defined sizes, by exploiting the modularity and high degree of addressability of the DNA origami building blocks used.

In Touch with Molecules: Improving Student Learning with Innovative Molecular Models

ERIC Educational Resources Information Center

Davenport, Jodi; Silberglitt, Matt; Olson, Arthur

2013-01-01

How do viruses self-assemble? Why do DNA bases pair the way they do? What factors determine whether strands of proteins fold into sheets or helices? Why does handedness matter? A deep understanding of core issues in biology requires students to understand both complex spatial structures of molecules and the interactions involved in dynamic…

In the Middle of a Chain Interaction.

PubMed

Kinsella, Sinéad; Prehn, Jochen H M

2016-10-20

In this issue of Molecular Cell, Fu et al. (2016) present a detailed structural analysis of death-inducing signaling complex (DISC) assembly and regulation through flexible caspase-8 interactions with cFLIP L , cFLIP S , and the viral inhibitor MC159, thereby identifying novel apoptosis control mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Light-emitting self-assembled peptide nucleic acids exhibit both stacking interactions and Watson-Crick base pairing.

PubMed

Berger, Or; Adler-Abramovich, Lihi; Levy-Sakin, Michal; Grunwald, Assaf; Liebes-Peer, Yael; Bachar, Mor; Buzhansky, Ludmila; Mossou, Estelle; Forsyth, V Trevor; Schwartz, Tal; Ebenstein, Yuval; Frolow, Felix; Shimon, Linda J W; Patolsky, Fernando; Gazit, Ehud

2015-04-01

The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offer chemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecular recognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form ordered nanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are short DNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesized and assayed for their ability to self-associate. Only three guanine-containing di-PNAs-CG, GC and GG-could form ordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discrete architectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stacking interactions and Watson-Crick base pairing. The assemblies were also found to exhibit optical properties including voltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

Probing the Effect of Molecular Nonuniformity in Directed Self-Assembly of Diblock Copolymers in Nanoconfined Space.

PubMed

Pitet, Louis M; Alexander-Moonen, Els; Peeters, Emiel; Druzhinina, Tamara S; Wuister, Sander F; Lynd, Nathaniel A; Meijer, E W

2015-10-27

Various complex self-assembled morphologies of lamellar- and cylinder-forming block copolymers comprising poly(dimethylsiloxane)-b-polylactide (PDMS-b-PLA) confined in cylindrical channels were generated. Combining top-down lithography with bottom-up block copolymer self-assembly grants access to morphologies that are otherwise inaccessible with the bulk materials. Channel diameter (D) was systematically varied with four diblock copolymers having different compositions and bulk domain spacing (L0), corresponding to a range of frustration ratios (D/L0 from 2 to 4). Excessive packing frustration imposed by the channels leads to contorted domains. The resulting morphologies depend strongly on both D/L0 and copolymer composition. Under several circumstances, mixtures of complex morphologies were observed, which hypothetically arise from the severe sensitivity to D/L0 combined with the inherent compositional/molar mass dispersities associated with the nonuniform synthetic materials and silicon templates. Stochastic calculations offer compelling support for the hypothesis, and tractable pathways toward solving this apparent conundrum are proposed. The materials hold great promise for next-generation nanofabrication to address several emerging technologies, offering significantly enhanced versatility to basic diblock copolymers as templates for fabricating complex nanoscale objects.

How curvature-generating proteins build scaffolds on membrane nanotubes

PubMed Central

Evergren, Emma; Golushko, Ivan; Prévost, Coline; Renard, Henri-François; Johannes, Ludger; McMahon, Harvey T.; Lorman, Vladimir; Voth, Gregory A.; Bassereau, Patricia

2016-01-01

Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein–membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30–40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes. PMID:27655892

The molecular assembly of the ionic liquid/aliphatic carboxylic acid/aliphatic amine as effective and safety transdermal permeation enhancers.

PubMed

Kubota, Koji; Shibata, Akira; Yamaguchi, Toshikazu

2016-04-30

In spite of numerous advantages, transdermal drug delivery systems are unfeasible for most drugs because of the barrier effect of the stratum corneum. Ionic liquids were recently used to enhance transdermal drug delivery by improving drug solubility. In the present study, safe and effective ionic liquids for transdermal absorption were obtained as salts generated by a neutralization reaction between highly biocompatible aliphatic carboxylic acids (octanoic acid or isostearic acid) and aliphatic amines (diisopropanolamine or triisopropanolamine) (Medrx Co., Ltd., 2009). The mechanism of skin permeability enhancement by ionic liquids was investigated by hydrophilic phenol red and hydrophobic tulobuterol. Further, the skin permeation enhancing effect was remarkably superior in the acid excess state rather than the neutralization state. Infrared absorption spectrum analysis confirmed that ionic liquids/aliphatic carboxylic acid/aliphatic amine are coexisting at all mixing states. In the acid excess state, ionic liquids interact with aliphatic carboxylic acids via hydrogen bonds. Thus, the skin permeation enhancing effect is not caused by the ionic liquid alone. The "liquid salt mixture," referred to as a complex of ingredients coexisting with ionic liquids, forms a molecular assembly incorporating hydrophilic drug. This molecular assembly was considered an effective and safety enhancer of transdermal drug permeation. Copyright © 2016. Published by Elsevier B.V.

Cross dimerization of amyloid-β and αsynuclein proteins in aqueous environment: a molecular dynamics simulations study.

PubMed

Jose, Jaya C; Chatterjee, Prathit; Sengupta, Neelanjana

2014-01-01

Self-assembly of the intrinsically unstructured proteins, amyloid beta (Aβ) and alpha synclein (αSyn), are associated with Alzheimer's Disease, and Parkinson's and Lewy Body Diseases, respectively. Importantly, pathological overlaps between these neurodegenerative diseases, and the possibilities of interactions between Aβ and αSyn in biological milieu emerge from several recent clinical reports and in vitro studies. Nevertheless, there are very few molecular level studies that have probed the nature of spontaneous interactions between these two sequentially dissimilar proteins and key characteristics of the resulting cross complexes. In this study, we have used atomistic molecular dynamics simulations to probe the possibility of cross dimerization between αSyn1-95 and Aβ1-42, and thereby gain insights into their plausible early assembly pathways in aqueous environment. Our analyses indicate a strong probability of association between the two sequences, with inter-protein attractive electrostatic interactions playing dominant roles. Principal component analysis revealed significant heterogeneity in the strength and nature of the associations in the key interaction modes. In most, the interactions of repeating Lys residues, mainly in the imperfect repeats 'KTKEGV' present in αSyn1-95 were found to be essential for cross interactions and formation of inter-protein salt bridges. Additionally, a hydrophobicity driven interaction mode devoid of salt bridges, where the non-amyloid component (NAC) region of αSyn1-95 came in contact with the hydrophobic core of Aβ1-42 was observed. The existence of such hetero complexes, and therefore hetero assembly pathways may lead to polymorphic aggregates with variations in pathological attributes. Our results provide a perspective on development of therapeutic strategies for preventing pathogenic interactions between these proteins.

Immune activation with peptide assemblies carrying Lewis y tumor-associated carbohydrate antigen.

PubMed

Yamazaki, Yuji; Watabe, Naoki; Obata, Hiroaki; Hara, Eri; Ohmae, Masashi; Kimura, Shunsaku

2017-02-01

Molecular assemblies varying morphologies in a wide range from spherical micelle, nanosheet, curved sheet, nanotube and vesicle were prepared and loaded with Lewis y (Le y ) tumor-associated carbohydrate antigen on the assembly surface. The molecular assemblies were composed of poly(sarcosine) m -block-poly(L-lactic acid) 30 (m = 15 or 50, Lactosome), poly(sarcosine) m -block-(D/L-Leu-Aib) n (m = 22 or 30, n = 6 or 8) and their combinations. The molecular assemblies carrying Le y on the surface were administered in BALB/c nu/nu mice. The major epitopes of the molecular assemblies are commonly Le y and poly(sarcosine). IgM productions upon administrations of the molecular assemblies were assayed by ELISA, showing that anti-poly(sarcosine) IgM was highly produced by Lactosome of spherical micelle but with a negligible amount of anti-Le y IgM. On the other hand, the nanosheet of the interdigitated monolayer triggered the production of anti-Le y IgM but with less anti-poly(sarcosine) IgM production. Taken together, IgM specificity differs according to the molecular environment of the epitopes in the molecular assemblies. The antigenicity of poly(sarcosine) was augmented in polymeric micelle providing loose environment for B cells to penetrate in, whereas a high density of Le y on the molecular assembly was required for anti-Le y IgM production. The antigenicity of Le y is therefore dependent on the molecular assemblies on which Le y is displayed on the surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

A Library of the Nanoscale Self-Assembly of Amino Acids on Metal Surfaces

NASA Astrophysics Data System (ADS)

Iski, Erin; Yitamben, Esmeralda; Guisinger, Nathan

2012-02-01

The investigation of the hierarchical self-assembly of amino acids on surfaces represents a unique test-bed for the origin of enantio-favoritism in biology and the transmission of chirality from single molecules to complete surface layers. These chiral systems, in particular the assembly of isoleucine and alanine on Cu(111), represent a direct link to the understanding of certain biological processes, specifically the preference for some amino acids to form alpha helices vs. beta-pleated sheets in the secondary structure of proteins. Low temperature, ultra-high vacuum, scanning tunneling microscopy (LT UHV-STM) is used to study the hierarchical self-assembly of different amino acids on a Cu(111) single crystal in an effort to build a library of their two-dimensional structure with molecular-scale resolution for enhanced protein and peptide studies. Both enantiopure and racemic structures are studied in order to elucidate how chirality can affect the self-assembly of the amino acids. In some cases, density functional theory (DFT) models can be used to confirm the experimental structure. The advent of such a library with fully resolved, two-dimensional structures at different molecular coverages would address some of the complex questions surrounding the preferential formation of alpha helices vs. beta-pleated sheets in proteins and lead to a better understanding of the key role played by these amino acids in protein sequencing.

High Performance Parallel Computational Nanotechnology

NASA Technical Reports Server (NTRS)

Saini, Subhash; Craw, James M. (Technical Monitor)

1995-01-01

At a recent press conference, NASA Administrator Dan Goldin encouraged NASA Ames Research Center to take a lead role in promoting research and development of advanced, high-performance computer technology, including nanotechnology. Manufacturers of leading-edge microprocessors currently perform large-scale simulations in the design and verification of semiconductor devices and microprocessors. Recently, the need for this intensive simulation and modeling analysis has greatly increased, due in part to the ever-increasing complexity of these devices, as well as the lessons of experiences such as the Pentium fiasco. Simulation, modeling, testing, and validation will be even more important for designing molecular computers because of the complex specification of millions of atoms, thousands of assembly steps, as well as the simulation and modeling needed to ensure reliable, robust and efficient fabrication of the molecular devices. The software for this capacity does not exist today, but it can be extrapolated from the software currently used in molecular modeling for other applications: semi-empirical methods, ab initio methods, self-consistent field methods, Hartree-Fock methods, molecular mechanics; and simulation methods for diamondoid structures. In as much as it seems clear that the application of such methods in nanotechnology will require powerful, highly powerful systems, this talk will discuss techniques and issues for performing these types of computations on parallel systems. We will describe system design issues (memory, I/O, mass storage, operating system requirements, special user interface issues, interconnects, bandwidths, and programming languages) involved in parallel methods for scalable classical, semiclassical, quantum, molecular mechanics, and continuum models; molecular nanotechnology computer-aided designs (NanoCAD) techniques; visualization using virtual reality techniques of structural models and assembly sequences; software required to control mini robotic manipulators for positional control; scalable numerical algorithms for reliability, verifications and testability. There appears no fundamental obstacle to simulating molecular compilers and molecular computers on high performance parallel computers, just as the Boeing 777 was simulated on a computer before manufacturing it.

Mechanisms of amyloid formation revealed by solution NMR

PubMed Central

Karamanos, Theodoros K.; Kalverda, Arnout P.; Thompson, Gary S.; Radford, Sheena E.

2015-01-01

Amyloid fibrils are proteinaceous elongated aggregates involved in more than fifty human diseases. Recent advances in electron microscopy and solid state NMR have allowed the characterization of fibril structures to different extents of refinement. However, structural details about the mechanism of fibril formation remain relatively poorly defined. This is mainly due to the complex, heterogeneous and transient nature of the species responsible for assembly; properties that make them difficult to detect and characterize in structural detail using biophysical techniques. The ability of solution NMR spectroscopy to investigate exchange between multiple protein states, to characterize transient and low-population species, and to study high molecular weight assemblies, render NMR an invaluable technique for studies of amyloid assembly. In this article we review state-of-the-art solution NMR methods for investigations of: (a) protein dynamics that lead to the formation of aggregation-prone species; (b) amyloidogenic intrinsically disordered proteins; and (c) protein–protein interactions on pathway to fibril formation. Together, these topics highlight the power and potential of NMR to provide atomic level information about the molecular mechanisms of one of the most fascinating problems in structural biology. PMID:26282197

Paranemic Crossover DNA: There and Back Again.

PubMed

Wang, Xing; Chandrasekaran, Arun Richard; Shen, Zhiyong; Ohayon, Yoel P; Wang, Tong; Kizer, Megan E; Sha, Ruojie; Mao, Chengde; Yan, Hao; Zhang, Xiaoping; Liao, Shiping; Ding, Baoquan; Chakraborty, Banani; Jonoska, Natasha; Niu, Dong; Gu, Hongzhou; Chao, Jie; Gao, Xiang; Li, Yuhang; Ciengshin, Tanashaya; Seeman, Nadrian C

2018-06-18

Over the past 35 years, DNA has been used to produce various nanometer-scale constructs, nanomechanical devices, and walkers. Construction of complex DNA nanostructures relies on the creation of rigid DNA motifs. Paranemic crossover (PX) DNA is one such motif that has played many roles in DNA nanotechnology. Specifically, PX cohesion has been used to connect topologically closed molecules, to assemble a three-dimensional object, and to create two-dimensional DNA crystals. Additionally, a sequence-dependent nanodevice based on conformational change between PX and its topoisomer, JX 2 , has been used in robust nanoscale assembly lines, as a key component in a DNA transducer, and to dictate polymer assembly. Furthermore, the PX motif has recently found a new role directly in basic biology, by possibly serving as the molecular structure for double-stranded DNA homology recognition, a prominent feature of molecular biology and essential for many crucial biological processes. This review discusses the many attributes and usages of PX-DNA-its design, characteristics, applications, and potential biological relevance-and aims to accelerate the understanding of PX-DNA motif in its many roles and manifestations.

Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery*

PubMed Central

Gakh, Oleksandr; Ranatunga, Wasantha; Smith, Douglas Y.; Ahlgren, Eva-Christina; Al-Karadaghi, Salam; Thompson, James R.; Isaya, Grazia

2016-01-01

Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN42–210 (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with ∼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN42–210]24·[NFS1]24·[ISD11]24·[ISCU]24 complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN42–210]24·[ISCU]24 sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN42–210 trimer at each of its eight vertices. Binding of 12 [NFS1]2·[ISD11]2 sub-complexes to the surface results in a globular macromolecule with a diameter of ∼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN42–210 to ISCU. PMID:27519411

Four-color single molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes

PubMed Central

DeRocco, Vanessa C.; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A.; Weninger, Keith

2010-01-01

To allow studies of conformational changes within multi-molecular complexes, we present a simultaneous, 4-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera based, wide-field detection. We further demonstrate labeling histidine-tagged proteins non-covalently with tris-Nitrilotriacetic acid (tris-NTA) conjugated dyes to achieve single molecule detection. We combine these methods to co-localize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes. PMID:21091445

Structure and function of archaeal prefoldin, a co-chaperone of group II chaperonin.

PubMed

Ohtaki, Akashi; Noguchi, Keiichi; Yohda, Masafumi

2010-01-01

Molecular chaperones are key cellular components involved in the maintenance of protein homeostasis and other unrelated functions. Prefoldin is a chaperone that acts as a co-factor of group II chaperonins in eukaryotes and archaea. It assists proper folding of protein by capturing nonnative proteins and delivering it to the group II chaperonin. Eukaryotic prefoldin is a multiple subunit complex composed of six different polypeptide chains. Archaeal prefoldin, on the other hand, is a heterohexameric complex composed of two alpha and four beta subunits, and forms a double beta barrel assembly with six long coiled coils protruding from it like a jellyfish with six tentacles. Based on the structural information of the archaeal prefoldin, substrate recognition and prefoldin-chaperonin binding mechanisms have been investigated. In this paper, we review a series of studies on the molecular mechanisms of archaeal PFD function. Particular emphasis will be placed on the molecular structures revealed by X-ray crystallography and molecular dynamics induced by binding to nonnative protein substrates.

Programmable disorder in random DNA tilings

NASA Astrophysics Data System (ADS)

Tikhomirov, Grigory; Petersen, Philip; Qian, Lulu

2017-03-01

Scaling up the complexity and diversity of synthetic molecular structures will require strategies that exploit the inherent stochasticity of molecular systems in a controlled fashion. Here we demonstrate a framework for programming random DNA tilings and show how to control the properties of global patterns through simple, local rules. We constructed three general forms of planar network—random loops, mazes and trees—on the surface of self-assembled DNA origami arrays on the micrometre scale with nanometre resolution. Using simple molecular building blocks and robust experimental conditions, we demonstrate control of a wide range of properties of the random networks, including the branching rules, the growth directions, the proximity between adjacent networks and the size distribution. Much as combinatorial approaches for generating random one-dimensional chains of polymers have been used to revolutionize chemical synthesis and the selection of functional nucleic acids, our strategy extends these principles to random two-dimensional networks of molecules and creates new opportunities for fabricating more complex molecular devices that are organized by DNA nanostructures.

Adsorption and self-assembly of bio-organic molecules at model surfaces: A route towards increased complexity

NASA Astrophysics Data System (ADS)

Costa, Dominique; Pradier, Claire-Marie; Tielens, Frederik; Savio, Letizia

2015-12-01

Understanding the bio-physical-chemical interactions at nanostructured biointerfaces and the assembly mechanisms of so-called hybrid nano-composites is nowadays a key issue for nanoscience in view of the many possible applications foreseen. The contribution of surface science in this field is noteworthy since, using a bottom-up approach, it allows the investigation of the fundamental processes at the basis of complex interfacial phenomena and thus it helps to unravel the elementary mechanisms governing them. Nowadays it is well demonstrated that a wide variety of different molecular assemblies can form upon adsorption of small biomolecules at surfaces. The geometry of such self-organized structures can often be tuned by a careful control of the experimental conditions during the deposition process. Indeed an impressive number of studies exists (both experimental and - to a lesser extent - theoretical), which demonstrates the ability of molecular self-assembly to create different structural motifs in a more or less predictable manner, by tuning the molecular building blocks as well as the metallic substrate. In this frame, amino acids and small peptides at surfaces are key, basic, systems to be studied. The amino acids structure is simple enough to serve as a model for the chemisorption of biofunctional molecules, but their adsorption at surfaces has applications in surface functionalization, in enantiospecific catalysis, biosensing, shape control of nanoparticles or in emerging fields such as "green" corrosion inhibition. In this paper we review the most recent advances in this field. We shall start from the adsorption of amino acids at metal surfaces and we will evolve then in the direction of more complex systems, in the light of the latest improvements of surface science techniques and of computational methods. On one side, we will focus on amino acids adsorption at oxide surfaces, on the other on peptide adsorption both at metal and oxide substrates. Particular attention will be drawn to the added value provided by the combination of several experimental surface science techniques and to the precious contribution of advanced complementary computational methods to resolve the details of systems of increased complexity. Finally, some hints on experiments performed in presence of water and then characterized in UHV and on the related theoretical work will be presented. This is a further step towards a better approximation of real biological systems. However, since the methods employed are often not typical of surface science, this topic is not developed in detail.

Role of small subunit in mediating assembly of red-type form I Rubisco.

PubMed

Joshi, Jidnyasa; Mueller-Cajar, Oliver; Tsai, Yi-Chin C; Hartl, F Ulrich; Hayer-Hartl, Manajit

2015-01-09

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme involved in photosynthetic carbon fixation, converting atmospheric CO2 to organic compounds. Form I Rubisco is a cylindrical complex composed of eight large (RbcL) subunits that are capped by four small subunits (RbcS) at the top and four at the bottom. Form I Rubiscos are phylogenetically divided into green- and red-type. Some red-type enzymes have catalytically superior properties. Thus, understanding their folding and assembly is of considerable biotechnological interest. Folding of the green-type RbcL subunits in cyanobacteria is mediated by the GroEL/ES chaperonin system, and assembly to holoenzyme requires specialized chaperones such as RbcX and RAF1. Here, we show that the red-type RbcL subunits in the proteobacterium Rhodobacter sphaeroides also fold with GroEL/ES. However, assembly proceeds in a chaperone-independent manner. We find that the C-terminal β-hairpin extension of red-type RbcS, which is absent in green-type RbcS, is critical for efficient assembly. The β-hairpins of four RbcS subunits form an eight-stranded β-barrel that protrudes into the central solvent channel of the RbcL core complex. The two β-barrels stabilize the complex through multiple interactions with the RbcL subunits. A chimeric green-type RbcS carrying the C-terminal β-hairpin renders the assembly of a cyanobacterial Rubisco independent of RbcX. Our results may facilitate the engineering of crop plants with improved growth properties expressing red-type Rubisco. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems

PubMed Central

Kohn, Kurt W.

1999-01-01

Eventually to understand the integrated function of the cell cycle regulatory network, we must organize the known interactions in the form of a diagram, map, and/or database. A diagram convention was designed capable of unambiguous representation of networks containing multiprotein complexes, protein modifications, and enzymes that are substrates of other enzymes. To facilitate linkage to a database, each molecular species is symbolically represented only once in each diagram. Molecular species can be located on the map by means of indexed grid coordinates. Each interaction is referenced to an annotation list where pertinent information and references can be found. Parts of the network are grouped into functional subsystems. The map shows how multiprotein complexes could assemble and function at gene promoter sites and at sites of DNA damage. It also portrays the richness of connections between the p53-Mdm2 subsystem and other parts of the network. PMID:10436023

Protonated sugars: vibrational spectroscopy and conformational structure of protonated O-methyl α-D-galactopyranoside

NASA Astrophysics Data System (ADS)

Rudić, Svemir; Xie, Hong-bin; Gerber, R. Benny; Simons, John P.

2012-08-01

'Bridging' protons provide a common structural motif in biological assemblies such as proton wires and proton-bound dimers. Here we present a 'proof-of-principle' computational and vibrational spectroscopic investigation of an 'intra-molecular proton-bound dimer,' O-methyl α-D-galactopyranoside (αMeGal-H+), generated in the gas phase through photo-ionisation of its complex with phenol in a molecular beam. Its vibrational spectrum corresponds well with a classical molecular dynamics simulation conducted 'on-the-fly' and also with the lowest-energy structures predicted by DFT and ab initio calculations. They reveal proton-bound structures that bridge neighbouring pairs of oxygen atoms, preferentially O6 and O4, linked together within the carbohydrate scaffold. Motivated by the possibility of an entry into the microscopic mechanism of its acid (or enzyme)-catalysed hydrolysis, we also report the corresponding predictions for its singly hydrated complex.

Perspective: On the importance of hydrodynamic interactions in the subcellular dynamics of macromolecules

PubMed Central

Skolnick, Jeffrey

2016-01-01

An outstanding challenge in computational biophysics is the simulation of a living cell at molecular detail. Over the past several years, using Stokesian dynamics, progress has been made in simulating coarse grained molecular models of the cytoplasm. Since macromolecules comprise 20%-40% of the volume of a cell, one would expect that steric interactions dominate macromolecular diffusion. However, the reduction in cellular diffusion rates relative to infinite dilution is due, roughly equally, to steric and hydrodynamic interactions, HI, with nonspecific attractive interactions likely playing rather a minor role. HI not only serve to slow down long time diffusion rates but also cause a considerable reduction in the magnitude of the short time diffusion coefficient relative to that at infinite dilution. More importantly, the long range contribution of the Rotne-Prager-Yamakawa diffusion tensor results in temporal and spatial correlations that persist up to microseconds and for intermolecular distances on the order of protein radii. While HI slow down the bimolecular association rate in the early stages of lipid bilayer formation, they accelerate the rate of large scale assembly of lipid aggregates. This is suggestive of an important role for HI in the self-assembly kinetics of large macromolecular complexes such as tubulin. Since HI are important, questions as to whether continuum models of HI are adequate as well as improved simulation methodologies that will make simulations of more complex cellular processes practical need to be addressed. Nevertheless, the stage is set for the molecular simulations of ever more complex subcellular processes. PMID:27634243

Molecular self-assembly on surfaces

NASA Astrophysics Data System (ADS)

Mateo-Marti, E.; Pradier, C. M.

2012-09-01

The aim of the present research is to study the interaction of biomolecules, among them single amino acids, on metallic and mineral surfaces, and their chemical reactivity by means of powerful surface science techniques. Therefore, the use of simple biomolecules gives fundamental and significant information, including an adequate control of biomolecule-surface interactions, which will be unattainable to develop with more complex molecules. Furthermore, these studies are focussed on the catalytic properties of different surfaces that could be involved in molecular self-organization processes and the formation of prebiotic organic compounds.

Building bridges between cellular and molecular structural biology.

PubMed

Patwardhan, Ardan; Brandt, Robert; Butcher, Sarah J; Collinson, Lucy; Gault, David; Grünewald, Kay; Hecksel, Corey; Huiskonen, Juha T; Iudin, Andrii; Jones, Martin L; Korir, Paul K; Koster, Abraham J; Lagerstedt, Ingvar; Lawson, Catherine L; Mastronarde, David; McCormick, Matthew; Parkinson, Helen; Rosenthal, Peter B; Saalfeld, Stephan; Saibil, Helen R; Sarntivijai, Sirarat; Solanes Valero, Irene; Subramaniam, Sriram; Swedlow, Jason R; Tudose, Ilinca; Winn, Martyn; Kleywegt, Gerard J

2017-07-06

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures.

Let's push things forward: disruptive technologies and the mechanics of tissue assembly.

PubMed

Varner, Victor D; Nelson, Celeste M

2013-09-01

Although many of the molecular mechanisms that regulate tissue assembly in the embryo have been delineated, the physical forces that couple these mechanisms to actual changes in tissue form remain unclear. Qualitative studies suggest that mechanical loads play a regulatory role in development, but clear quantitative evidence has been lacking. This is partly owing to the complex nature of these problems - embryonic tissues typically undergo large deformations and exhibit evolving, highly viscoelastic material properties. Still, despite these challenges, new disruptive technologies are enabling study of the mechanics of tissue assembly in unprecedented detail. Here, we present novel experimental techniques that enable the study of each component of these physical problems: kinematics, forces, and constitutive properties. Specifically, we detail advances in light sheet microscopy, optical coherence tomography, traction force microscopy, fluorescence force spectroscopy, microrheology and micropatterning. Taken together, these technologies are helping elucidate a more quantitative understanding of the mechanics of tissue assembly.

Let's push things forward: disruptive technologies and the mechanics of tissue assembly

PubMed Central

Varner, Victor D.; Nelson, Celeste M.

2013-01-01

Although many of the molecular mechanisms that regulate tissue assembly in the embryo have been delineated, the physical forces that couple these mechanisms to actual changes in tissue form remain unclear. Qualitative studies suggest that mechanical loads play a regulatory role in development, but clear quantitative evidence has been lacking. This is partly owing to the complex nature of these problems – embryonic tissues typically undergo large deformations and exhibit evolving, highly viscoelastic material properties. Still, despite these challenges, new disruptive technologies are enabling study of the mechanics of tissue assembly in unprecedented detail. Here, we present novel experimental techniques that enable the study of each component of these physical problems: kinematics, forces, and constitutive properties. Specifically, we detail advances in light sheet microscopy, optical coherence tomography, traction force microscopy, fluorescence force spectroscopy, microrheology and micropatterning. Taken together, these technologies are helping elucidate a more quantitative understanding of the mechanics of tissue assembly. PMID:23907401

Chemical cross-linking of the urease complex from Helicobacter pylori and analysis by Fourier transform ion cyclotron resonance mass spectrometry and molecular modeling

NASA Astrophysics Data System (ADS)

Carlsohn, Elisabet; Ångström, Jonas; Emmett, Mark R.; Marshall, Alan G.; Nilsson, Carol L.

2004-05-01

Chemical cross-linking of proteins is a well-established method for structural mapping of small protein complexes. When combined with mass spectrometry, cross-linking can reveal protein topology and identify contact sites between the peptide surfaces. When applied to surface-exposed proteins from pathogenic organisms, the method can reveal structural details that are useful in vaccine design. In order to investigate the possibilities of applying cross-linking on larger protein complexes, we selected the urease enzyme from Helicobacter pylori as a model. This membrane-associated protein complex consists of two subunits: [alpha] (26.5 kDa) and [beta] (61.7 kDa). Three ([alpha][beta]) heterodimers form a trimeric ([alpha][beta])3 assembly which further associates into a unique dodecameric 1.1 MDa complex composed of four ([alpha][beta])3 units. Cross-linked peptides from trypsin-digested urease complex were analyzed by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and molecular modeling. Two potential cross-linked peptides (present in the cross-linked sample but undetectable in [alpha], [beta], and native complex) were assigned. Molecular modeling of urease [alpha][beta] complex and trimeric urease units ([alpha][beta])3 revealed a linkage site between the [alpha]-subunit and the [beta]-subunit, and an internal cross-linkage in the [beta]-subunit.

In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA

PubMed Central

La Rocca, Gaspare; Olejniczak, Scott H.; González, Alvaro J.; Briskin, Daniel; Vidigal, Joana A.; Spraggon, Lee; DeMatteo, Raymond G.; Radler, Megan R.; Lindsten, Tullia; Ventura, Andrea; Tuschl, Thomas; Leslie, Christina S.; Thompson, Craig B.

2015-01-01

MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3′ untranslated region (UTR) of target mRNAs. In cell lines, Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase–RAC-alpha serine/threonine-protein kinase–mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling. PMID:25568082

Layer-by-Layer Molecular Assemblies for Dye-Sensitized Photoelectrosynthesis Cells Prepared by Atomic Layer Deposition

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

Wang, Degao; Sheridan, Matthew V.; Shan, Bing

2017-08-30

In a Dye Sensitized Photoelectrosynthesis Cell (DSPEC) the relative orientation of catalyst and chromophore play important roles. Here we introduce a new, robust, Atomic Layer Deposition (ALD) procedure for the preparation of assemblies on wide bandgap semiconductors. In the procedure, phosphonated metal complex precursors react with metal ion bridging to an external chromophore or catalyst to give assemblies bridged by Al(III), Sn(IV), Ti(IV), or Zr(IV) metal oxide units as bridges. The procedure has been extended to chromophore-catalyst assemblies for water oxidation catalysis. A SnO2 bridged assembly on SnO2/TiO2 core/shell electrodes undergoes water splitting with an incident photon conversion efficiency (IPCE)more » of 17.1% at 440 nm. Reduction of water at a Ni(II)-based catalyst on NiO films has been shown to give H2. Compared to conventional solution-based procedures, the ALD approach offers significant advantages in scope and flexibility for the preparation of stable surface structures.« less

Multilayer block copolymer meshes by orthogonal self-assembly

PubMed Central

Tavakkoli K. G., Amir; Nicaise, Samuel M.; Gadelrab, Karim R.; Alexander-Katz, Alfredo; Ross, Caroline A.; Berggren, Karl K.

2016-01-01

Continued scaling-down of lithographic-pattern feature sizes has brought templated self-assembly of block copolymers (BCPs) into the forefront of nanofabrication research. Technologies now exist that facilitate significant control over otherwise unorganized assembly of BCP microdomains to form both long-range and locally complex monolayer patterns. In contrast, the extension of this control into multilayers or 3D structures of BCP microdomains remains limited, despite the possible technological applications in next-generation devices. Here, we develop and analyse an orthogonal self-assembly method in which multiple layers of distinct-molecular-weight BCPs naturally produce nanomesh structures of cylindrical microdomains without requiring layer-by-layer alignment or high-resolution lithographic templating. The mechanisms for orthogonal self-assembly are investigated with both experiment and simulation, and we determine that the control over height and chemical preference of templates are critical process parameters. The method is employed to produce nanomeshes with the shapes of circles and Y-intersections, and is extended to produce three layers of orthogonally oriented cylinders. PMID:26796218

Design Principles of Regulatory Networks: Searching for the Molecular Algorithms of the Cell

PubMed Central

Lim, Wendell A.; Lee, Connie M.; Tang, Chao

2013-01-01

A challenge in biology is to understand how complex molecular networks in the cell execute sophisticated regulatory functions. Here we explore the idea that there are common and general principles that link network structures to biological functions, principles that constrain the design solutions that evolution can converge upon for accomplishing a given cellular task. We describe approaches for classifying networks based on abstract architectures and functions, rather than on the specific molecular components of the networks. For any common regulatory task, can we define the space of all possible molecular solutions? Such inverse approaches might ultimately allow the assembly of a design table of core molecular algorithms that could serve as a guide for building synthetic networks and modulating disease networks. PMID:23352241

Carbene based photochemical molecular assemblies for solar driven hydrogen generation.

PubMed

Peuntinger, Katrin; Pilz, T David; Staehle, Robert; Schaub, Markus; Kaufhold, Simon; Petermann, Lydia; Wunderlin, Markus; Görls, Helmar; Heinemann, Frank W; Li, Jing; Drewello, Thomas; Vos, Johannes G; Guldi, Dirk M; Rau, Sven

2014-09-28

Novel photocatalysts based on ruthenium complexes with NHC (N-heterocyclic carbene)-type bridging ligands have been prepared and structurally and photophysically characterised. The identity of the NHC-unit of the bridging ligand was established unambiguously by means of X-ray structural analysis of a heterodinuclear ruthenium-silver complex. The photophysical data indicate ultrafast intersystem crossing into an emissive and a non-emissive triplet excited state after excitation of the ruthenium centre. Exceptionally high luminescence quantum yields of up to 39% and long lifetimes of up to 2 μs are some of the triplet excited state characteristics. Preliminary studies into the visible light driven photocatalytic hydrogen formation show no induction phase and constant turnover frequencies that are independent on the concentration of the photocatalyst. In conclusion this supports the notion of a stable assembly under photocatalytic conditions.

Real-time monitoring of enzyme-free strand displacement cascades by colorimetric assays

NASA Astrophysics Data System (ADS)

Duan, Ruixue; Wang, Boya; Hong, Fan; Zhang, Tianchi; Jia, Yongmei; Huang, Jiayu; Hakeem, Abdul; Liu, Nannan; Lou, Xiaoding; Xia, Fan

2015-03-01

The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications.The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications. Electronic supplementary information (ESI) available: Experimental procedures and analytical data are provided. See DOI: 10.1039/c5nr00697j

Ordered patterns and structures via interfacial self-assembly: superlattices, honeycomb structures and coffee rings.

PubMed

Ma, Hongmin; Hao, Jingcheng

2011-11-01

Self-assembly is now being intensively studied in chemistry, physics, biology, and materials engineering and has become an important "bottom-up" approach to create intriguing structures for different applications. Self-assembly is not only a practical approach for creating a variety of nanostructures, but also shows great superiority in building hierarchical structures with orders on different length scales. The early work in self-assembly focused on molecular self-assembly in bulk solution, including the resultant dye aggregates, liposomes, vesicles, liquid crystals, gels and so on. Interfacial self-assembly has been a great concern over the last two decades, largely because of the unique and ingenious roles of this method for constructing materials at interfaces, such as self-assembled monolayers, Langmuir-Blodgett films, and capsules. Nanocrystal superlattices, honeycomb films and coffee rings are intriguing structural materials with more complex features and can be prepared by interfacial self-assembly on different length scales. In this critical review, we outline the recent development in the preparation and application of colloidal nanocrystal superlattices, honeycomb-patterned macroporous structures by the breath figure method, and coffee-ring-like patterns (247 references). This journal is © The Royal Society of Chemistry 2011

Discrete and polymeric self-assembled dendrimers: Hydrogen bond-mediated assembly with high stability and high fidelity

PubMed Central

Corbin, Perry S.; Lawless, Laurence J.; Li, Zhanting; Ma, Yuguo; Witmer, Melissa J.; Zimmerman, Steven C.

2002-01-01

Hydrogen bond-mediated self-assembly is a powerful strategy for creating nanoscale structures. However, little is known about the fidelity of assembly processes that must occur when similar and potentially competing hydrogen-bonding motifs are present. Furthermore, there is a continuing need for new modules and strategies that can amplify the relatively weak strength of a hydrogen bond to give more stable assemblies. Herein we report quantitative complexation studies on a ureidodeazapterin-based module revealing an unprecedented stability for dimers of its self-complementary acceptoracceptor-donor-donor (AADD) array. Linking two such units together with a semirigid spacer that carries a first-, second-, or third-generation Fréchet-type dendron affords a ditopic structure programmed to self assemble. The specific structure that is formed depends both on the size of the dendron and the solvent, but all of the assemblies have exceptionally high stability. The largest discrete nanoscale assembly is a hexamer with a molecular mass of about 17.8 kDa. It is stabilized by 30 hydrogen bonds, including six AADD⋅DDAA contacts. The hexamer forms and is indefinitely stable in the presence of a hexamer containing six ADD⋅DAA hydrogen-bonding arrays. PMID:11917113

A novel form of β-strand assembly observed in Aβ33-42 adsorbed onto graphene

NASA Astrophysics Data System (ADS)

Wang, Xiaofeng; Weber, Jeffrey K.; Liu, Lei; Dong, Mingdong; Zhou, Ruhong; Li, Jingyuan

2015-09-01

Peptide assembly plays a seminal role in the fabrication of structural and functional architectures in cells. Characteristically, peptide assemblies are often dominated by β-sheet structures, wherein component molecules are connected by backbone hydrogen bonds in a parallel or an antiparallel fashion. While β-rich peptide scaffolds are implicated in an array of neurodegenerative diseases, the mechanisms by which toxic peptides assemble and mediate neuropathic effects are still poorly understood. In this work, we employ molecular dynamics simulations to study the adsorption and assembly of the fragment Aβ33-42 (taken from the Aβ-42 peptide widely associated with Alzheimer's disease) on a graphene surface. We observe that such Aβ33-42 fragments, which are largely hydrophobic in character, readily adsorb onto the graphitic surface and coalesce into a well-structured, β-strand-like assembly. Strikingly, the structure of such complex is quite unique: hydrophobic side-chains extend over the graphene surface and interact with adjacent peptides, yielding a well-defined mosaic of hydrophobic interaction patches. This ordered structure is markedly depleted of backbone hydrogen bonds. Hence, our simulation results reveal a distinct type of β-strand assembly, maintained by hydrophobic side-chain interactions. Our finding suggests the backbone hydrogen bond is no longer crucial to the peptide assembly. Further studies concerning whether such β-strand assembly can be realized in other peptide systems and in biologically-relevant contexts are certainly warranted.Peptide assembly plays a seminal role in the fabrication of structural and functional architectures in cells. Characteristically, peptide assemblies are often dominated by β-sheet structures, wherein component molecules are connected by backbone hydrogen bonds in a parallel or an antiparallel fashion. While β-rich peptide scaffolds are implicated in an array of neurodegenerative diseases, the mechanisms by which toxic peptides assemble and mediate neuropathic effects are still poorly understood. In this work, we employ molecular dynamics simulations to study the adsorption and assembly of the fragment Aβ33-42 (taken from the Aβ-42 peptide widely associated with Alzheimer's disease) on a graphene surface. We observe that such Aβ33-42 fragments, which are largely hydrophobic in character, readily adsorb onto the graphitic surface and coalesce into a well-structured, β-strand-like assembly. Strikingly, the structure of such complex is quite unique: hydrophobic side-chains extend over the graphene surface and interact with adjacent peptides, yielding a well-defined mosaic of hydrophobic interaction patches. This ordered structure is markedly depleted of backbone hydrogen bonds. Hence, our simulation results reveal a distinct type of β-strand assembly, maintained by hydrophobic side-chain interactions. Our finding suggests the backbone hydrogen bond is no longer crucial to the peptide assembly. Further studies concerning whether such β-strand assembly can be realized in other peptide systems and in biologically-relevant contexts are certainly warranted. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr00555h

Molecular Analysis of Core Kinetochore Composition and Assembly in Drosophila melanogaster

PubMed Central

Przewloka, Marcin R.; Archambault, Vincent; D'Avino, Pier Paolo; Lilley, Kathryn S.; Laue, Ernest D.; McAinsh, Andrew D.; Glover, David M.

2007-01-01

Background Kinetochores are large multiprotein complexes indispensable for proper chromosome segregation. Although Drosophila is a classical model organism for studies of chromosome segregation, little is known about the organization of its kinetochores. Methodology/Principal Findings We employed bioinformatics, proteomics and cell biology methods to identify and analyze the interaction network of Drosophila kinetochore proteins. We have shown that three Drosophila proteins highly diverged from human and yeast Ndc80, Nuf2 and Mis12 are indeed their orthologues. Affinity purification of these proteins from cultured Drosophila cells identified a further five interacting proteins with weak similarity to subunits of the SPC105/KNL-1, MIND/MIS12 and NDC80 kinetochore complexes together with known kinetochore associated proteins such as dynein/dynactin, spindle assembly checkpoint components and heterochromatin proteins. All eight kinetochore complex proteins were present at the kinetochore during mitosis and MIND/MIS12 complex proteins were also centromeric during interphase. Their down-regulation led to dramatic defects in chromosome congression/segregation frequently accompanied by mitotic spindle elongation. The systematic depletion of each individual protein allowed us to establish dependency relationships for their recruitment onto the kinetochore. This revealed the sequential recruitment of individual members of first, the MIND/MIS12 and then, NDC80 complex. Conclusions/Significance The Drosophila MIND/MIS12 and NDC80 complexes and the Spc105 protein, like their counterparts from other eukaryotic species, are essential for chromosome congression and segregation, but are highly diverged in sequence. Hierarchical dependence relationships of individual proteins regulate the assembly of Drosophila kinetochore complexes in a manner similar, but not identical, to other organisms. PMID:17534428

The origin of cellular life.

PubMed

Ingber, D E

2000-12-01

This essay presents a scenario of the origin of life that is based on analysis of biological architecture and mechanical design at the microstructural level. My thesis is that the same architectural and energetic constraints that shape cells today also guided the evolution of the first cells and that the molecular scaffolds that support solid-phase biochemistry in modern cells represent living microfossils of past life forms. This concept emerged from the discovery that cells mechanically stabilize themselves using tensegrity architecture and that these same building rules guide hierarchical self-assembly at all size scales (Sci. Amer 278:48-57;1998). When combined with other fundamental design principles (e.g., energy minimization, topological constraints, structural hierarchies, autocatalytic sets, solid-state biochemistry), tensegrity provides a physical basis to explain how atomic and molecular elements progressively self-assembled to create hierarchical structures with increasingly complex functions, including living cells that can self-reproduce.

The origin of cellular life

NASA Technical Reports Server (NTRS)

Ingber, D. E.

2000-01-01

This essay presents a scenario of the origin of life that is based on analysis of biological architecture and mechanical design at the microstructural level. My thesis is that the same architectural and energetic constraints that shape cells today also guided the evolution of the first cells and that the molecular scaffolds that support solid-phase biochemistry in modern cells represent living microfossils of past life forms. This concept emerged from the discovery that cells mechanically stabilize themselves using tensegrity architecture and that these same building rules guide hierarchical self-assembly at all size scales (Sci. Amer 278:48-57;1998). When combined with other fundamental design principles (e.g., energy minimization, topological constraints, structural hierarchies, autocatalytic sets, solid-state biochemistry), tensegrity provides a physical basis to explain how atomic and molecular elements progressively self-assembled to create hierarchical structures with increasingly complex functions, including living cells that can self-reproduce.

Characterization of the major cyanogen bromide fragment of alpha-A crystallin

NASA Technical Reports Server (NTRS)

Ifeanyi, F.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

1991-01-01

Alpha crystallin from the bovine lens has been digested with cyanogen bromide, and the major fragment (CB-1) has been purified using reverse phase HPLC. Characterization of this fragment by Edman degradation and antisera to synthetic peptides indicates that it originates from alpha-A crystallin, but lacks the N-terminal methionine and the last 35 amino acids from the C-terminus of the molecule. The purified CB-1 fragment binds as well as native alpha crystallin to lens membrane, but is unable to self-assemble into the correct size of high molecular weight oligomeric complexes characteristic of the intact alpha-A chain. Together, these results demonstrate that the alpha-A chain is comprised of at least two functional domains, one of which is involved in binding of alpha-A crystallin to lens membrane, and another which is necessary for correct self-assembly of the molecule into high molecular weight oligomers.

Bacteriophage lambda: early pioneer and still relevant

PubMed Central

Casjens, Sherwood R.; Hendrix, Roger W.

2015-01-01

Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid 1950's to mid 1980's was critically important in the attainment of our current understanding of the sophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virus assembly and of the molecular nature of lysogeny. The development of molecular cloning techniques, ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their efforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and its relatives have continued to give important new insights. In this review we give some relevant early history and describe recent developments in understanding the molecular biology of lambda's life cycle. PMID:25742714

Molecular docking of superantigens with class II major histocompatibility complex proteins.

PubMed

Olson, M A; Cuff, L

1997-01-01

The molecular recognition of two superantigens with class II major histocompatibility complex molecules was simulated by using protein-protein docking. Superantigens studied were staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1) in their crystallographic assemblies with HLA-DR1. Rigid-body docking was performed sampling configurational space of the interfacial surfaces by employing a strategy of partitioning the contact regions on HLA-DR1 into separate molecular recognition units. Scoring of docked conformations was based on an electrostatic continuum model evaluated with the finite-difference Poisson-Boltzmann method. Estimates of nonpolar contributions were derived from the buried molecular surface areas. We found for both superantigens that docking the HLA-DR1 surface complementary with the SEB and TSST-1 contact regions containing a homologous hydrophobic surface loop provided sufficient recognition for the reconstitution of native-like conformers exhibiting the highest-scoring free energies. For the SEB complex, the calculations were successful in reproducing the total association free energy. A comparison of the free-energy determinants of the conserved hydrophobic contact residue indicates functional similarity between the two proteins for this interface. Though both superantigens share a common global association mode, differences in binding topology distinguish the conformational specificities underlying recognition.

Assembly of the β-Barrel Outer Membrane Proteins in Gram-Negative Bacteria, Mitochondria, and Chloroplasts

PubMed Central

Misra, Rajeev

2012-01-01

In the last decade, there has been an explosion of publications on the assembly of β-barrel outer membrane proteins (OMPs), which carry out diverse cellular functions, including solute transport, protein secretion, and assembly of protein and lipid components of the outer membrane. Of the three outer membrane model systems—Gram-negative bacteria, mitochondria and chloroplasts—research on bacterial and mitochondrial systems has so far led the way in dissecting the β-barrel OMP assembly pathways. Many exciting discoveries have been made, including the identification of β-barrel OMP assembly machineries in bacteria and mitochondria, and potentially the core assembly component in chloroplasts. The atomic structures of all five components of the bacterial β-barrel assembly machinery (BAM) complex, except the β-barrel domain of the core BamA protein, have been solved. Structures reveal that these proteins contain domains/motifs known to facilitate protein-protein interactions, which are at the heart of the assembly pathways. While structural information has been valuable, most of our current understanding of the β-barrel OMP assembly pathways has come from genetic, molecular biology, and biochemical analyses. This paper provides a comparative account of the β-barrel OMP assembly pathways in Gram-negative bacteria, mitochondria, and chloroplasts. PMID:27335668

Clustering and negative feedback by endocytosis in planar cell polarity signaling is modulated by ubiquitinylation of prickle.

PubMed

Cho, Bomsoo; Pierre-Louis, Gandhy; Sagner, Andreas; Eaton, Suzanne; Axelrod, Jeffrey D

2015-05-01

The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.

High-performance mussel-inspired adhesives of reduced complexity

PubMed Central

Ahn, B. Kollbe; Das, Saurabh; Linstadt, Roscoe; Kaufman, Yair; Martinez-Rodriguez, Nadine R.; Mirshafian, Razieh; Kesselman, Ellina; Talmon, Yeshayahu; Lipshutz, Bruce H.; Israelachvili, Jacob N.; Waite, J. Herbert

2015-01-01

Despite the recent progress in and demand for wet adhesives, practical underwater adhesion remains limited or non-existent for diverse applications. Translation of mussel-inspired wet adhesion typically entails catechol functionalization of polymers and/or polyelectrolytes, and solution processing of many complex components and steps that require optimization and stabilization. Here we reduced the complexity of a wet adhesive primer to synthetic low-molecular-weight catecholic zwitterionic surfactants that show very strong adhesion (∼50 mJ m−2) and retain the ability to coacervate. This catecholic zwitterion adheres to diverse surfaces and self-assembles into a molecularly smooth, thin (<4 nm) and strong glue layer. The catecholic zwitterion holds particular promise as an adhesive for nanofabrication. This study significantly simplifies bio-inspired themes for wet adhesion by combining catechol with hydrophobic and electrostatic functional groups in a small molecule. PMID:26478273

Molecular assembly and subcellular distribution of ATP-sensitive potassium channel proteins in rat hearts.

PubMed

Kuniyasu, Akihiko; Kaneko, Kazuyoshi; Kawahara, Kohichi; Nakayama, Hitoshi

2003-09-25

Cardiac ATP-sensitive K(+) (K(ATP)) channels are proposed to contribute to cardio-protection and ischemic preconditioning. Although mRNAs for all subunits of K(ATP) channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti-peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin-solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2-SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2)(4)-(SUR2A)(4), suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac K(ATP) channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning.

Spatial Rule-Based Modeling: A Method and Its Application to the Human Mitotic Kinetochore

PubMed Central

Ibrahim, Bashar; Henze, Richard; Gruenert, Gerd; Egbert, Matthew; Huwald, Jan; Dittrich, Peter

2013-01-01

A common problem in the analysis of biological systems is the combinatorial explosion that emerges from the complexity of multi-protein assemblies. Conventional formalisms, like differential equations, Boolean networks and Bayesian networks, are unsuitable for dealing with the combinatorial explosion, because they are designed for a restricted state space with fixed dimensionality. To overcome this problem, the rule-based modeling language, BioNetGen, and the spatial extension, SRSim, have been developed. Here, we describe how to apply rule-based modeling to integrate experimental data from different sources into a single spatial simulation model and how to analyze the output of that model. The starting point for this approach can be a combination of molecular interaction data, reaction network data, proximities, binding and diffusion kinetics and molecular geometries at different levels of detail. We describe the technique and then use it to construct a model of the human mitotic inner and outer kinetochore, including the spindle assembly checkpoint signaling pathway. This allows us to demonstrate the utility of the procedure, show how a novel perspective for understanding such complex systems becomes accessible and elaborate on challenges that arise in the formulation, simulation and analysis of spatial rule-based models. PMID:24709796

Assembly and function of the botulinum neurotoxin progenitor complex.

PubMed

Gu, Shenyan; Jin, Rongsheng

2013-01-01

Botulinum neurotoxins (BoNTs) are among the most poisonous substances known to man, but paradoxically, BoNT-containing medicines and cosmetics have been used with great success in the clinic. Accidental BoNT poisoning mainly occurs through oral ingestion of food contaminated with Clostridium botulinum. BoNTs are naturally produced in the form of progenitor toxin complexes (PTCs), which are high molecular weight (up to ~900 kDa) multiprotein complexes composed of BoNT and several non-toxic neurotoxin-associated proteins (NAPs). NAPs protect the inherently fragile BoNTs against the hostile environment of the gastrointestinal (GI) tract and help BoNTs pass through the intestinal epithelial barrier before they are released into the general circulation. These events are essential for ingested BoNTs to gain access to motoneurons, where they inhibit neurotransmitter release and cause muscle paralysis. In this review, we discuss the structural basis for assembly of NAPs and BoNT into the PTC that protects BoNT and facilitate its delivery into the bloodstream.

Mitochondrial heat shock protein (Hsp) 70 and Hsp10 cooperate in the formation of Hsp60 complexes.

PubMed

Böttinger, Lena; Oeljeklaus, Silke; Guiard, Bernard; Rospert, Sabine; Warscheid, Bettina; Becker, Thomas

2015-05-01

Mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis, like import and folding of proteins. In these processes, the chaperone cooperates with cochaperones, the presequence translocase, and other chaperone systems. The chaperonin Hsp60, together with its cofactor Hsp10, catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures that provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study, we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to the unassembled Hsp60 precursor to promote its assembly into mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate the biogenesis of the heptameric Hsp60 rings. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A Theoretical and Experimental Study of DNA Self-assembly

NASA Astrophysics Data System (ADS)

Chandran, Harish

The control of matter and phenomena at the nanoscale is fast becoming one of the most important challenges of the 21st century with wide-ranging applications from energy and health care to computing and material science. Conventional top-down approaches to nanotechnology, having served us well for long, are reaching their inherent limitations. Meanwhile, bottom-up methods such as self-assembly are emerging as viable alternatives for nanoscale fabrication and manipulation. A particularly successful bottom up technique is DNA self-assembly where a set of carefully designed DNA strands form a nanoscale object as a consequence of specific, local interactions among the different components, without external direction. The final product of the self-assembly process might be a static nanostructure or a dynamic nanodevice that performs a specific function. Over the past two decades, DNA self-assembly has produced stunning nanoscale objects such as 2D and 3D lattices, polyhedra and addressable arbitrary shaped substrates, and a myriad of nanoscale devices such as molecular tweezers, computational circuits, biosensors and molecular assembly lines. In this dissertation we study multiple problems in the theory, simulations and experiments of DNA self-assembly. We extend the Turing-universal mathematical framework of self-assembly known as the Tile Assembly Model by incorporating randomization during the assembly process. This allows us to reduce the tile complexity of linear assemblies. We develop multiple techniques to build linear assemblies of expected length N using far fewer tile types than previously possible. We abstract the fundamental properties of DNA and develop a biochemical system, which we call meta-DNA, based entirely on strands of DNA as the only component molecule. We further develop various enzyme-free protocols to manipulate meta-DNA systems and provide strand level details along with abstract notations for these mechanisms. We simulate DNA circuits by providing detailed designs for local molecular computations that involve spatially contiguous molecules arranged on addressable substrates via enzyme-free DNA hybridization reaction cascades. We use the Visual DSD simulation software in conjunction with localized reaction rates obtained from biophysical modeling to create chemical reaction networks of localized hybridization circuits that are then model checked using the PRISM model checking software. We develop a DNA detection system employing the triggered self-assembly of a novel DNA dendritic nanostructure. Detection begins when a specific, single-stranded target DNA strand triggers a hybridization chain reaction between two distinct DNA hairpins. Each hairpin opens and hybridizes up to two copies of the other, and hence each layer of the growing dendritic nanostructure can in principle accommodate an exponentially increasing number of cognate molecules, generating a nanostructure with high molecular weight. We build linear activatable assemblies employing a novel protection/deprotection strategy to strictly enforce the direction of tiling assembly growth to ensure the robustness of the assembly process. Our system consists of two tiles that can form a linear co-polymer. These tiles, which are initially protected such that they do not react with each other, can be activated to form linear co-polymers via the use of a strand displacing enzyme.

The Past, Present, and Future of Human Centromere Genomics

PubMed Central

Aldrup-MacDonald, Megan E.; Sullivan, Beth A.

2014-01-01

The centromere is the chromosomal locus essential for chromosome inheritance and genome stability. Human centromeres are located at repetitive alpha satellite DNA arrays that compose approximately 5% of the genome. Contiguous alpha satellite DNA sequence is absent from the assembled reference genome, limiting current understanding of centromere organization and function. Here, we review the progress in centromere genomics spanning the discovery of the sequence to its molecular characterization and the work done during the Human Genome Project era to elucidate alpha satellite structure and sequence variation. We discuss exciting recent advances in alpha satellite sequence assembly that have provided important insight into the abundance and complex organization of this sequence on human chromosomes. In light of these new findings, we offer perspectives for future studies of human centromere assembly and function. PMID:24683489

Molecular Packing of Amphiphilic Nanosheets Resolved by X-ray Scattering

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

Harutyunyan, Boris; Dannenhoffer, Adam; Kewalramani, Sumit

2016-12-29

Molecular packing in light harvesting 2D assemblies of photocatalytic materials is a critical factor for solar-to-fuel conversion efficiency. However, structure–function correlations have yet to be fully established. This is partly due to the difficulties in extracting the molecular arrangements from the complex 3D powder averaged diffraction patterns of 2D lattices, obtained via in situ wide-angle X-ray scattering. Here, we develop a scattering theory formalism and couple it with a simple geometrical model for the molecular shape of chromophore 9-methoxy-N-(sodium hexanoate)perylene-3,4-dicarboximide (MeO-PMI) used in our study. This generally applicable method fully reproduces the measured diffraction pattern including the asymmetric line shapesmore » for the Bragg reflections and yields the molecular packing arrangement within a 2D crystal structure with a remarkable degree of detail. We find an approximate edge-centered herringbone structure for the PMI fused aromatic rings and ordering of the carboxypentyl chains above and below the nanosheets. Such a packing arrangement differs from the more symmetric face-to-face orientation of the unsubstituted PMI rings. This structural difference is correlated to our measurement of the reduced catalytic performance of MeO-PMI nanosheets as compared to the mesoscopically similar unsubstituted PMI assemblies.« less

Insight into the architecture of the NuRD complex: structure of the RbAp48-MTA1 subcomplex.

PubMed

Alqarni, Saad S M; Murthy, Andal; Zhang, Wei; Przewloka, Marcin R; Silva, Ana P G; Watson, Aleksandra A; Lejon, Sara; Pei, Xue Y; Smits, Arne H; Kloet, Susan L; Wang, Hongxin; Shepherd, Nicholas E; Stokes, Philippa H; Blobel, Gerd A; Vermeulen, Michiel; Glover, David M; Mackay, Joel P; Laue, Ernest D

2014-08-08

The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

COX16 promotes COX2 metallation and assembly during respiratory complex IV biogenesis

PubMed Central

Aich, Abhishek; Wang, Cong; Chowdhury, Arpita; Ronsör, Christin; Pacheu-Grau, David; Richter-Dennerlein, Ricarda; Dennerlein, Sven

2018-01-01

Cytochrome c oxidase of the mitochondrial oxidative phosphorylation system reduces molecular oxygen with redox equivalent-derived electrons. The conserved mitochondrial-encoded COX1- and COX2-subunits are the heme- and copper-center containing core subunits that catalyze water formation. COX1 and COX2 initially follow independent biogenesis pathways creating assembly modules with subunit-specific, chaperone-like assembly factors that assist in redox centers formation. Here, we find that COX16, a protein required for cytochrome c oxidase assembly, interacts specifically with newly synthesized COX2 and its copper center-forming metallochaperones SCO1, SCO2, and COA6. The recruitment of SCO1 to the COX2-module is COX16- dependent and patient-mimicking mutations in SCO1 affect interaction with COX16. These findings implicate COX16 in CuA-site formation. Surprisingly, COX16 is also found in COX1-containing assembly intermediates and COX2 recruitment to COX1. We conclude that COX16 participates in merging the COX1 and COX2 assembly lines. PMID:29381136

A multiscale simulation technique for molecular electronics: design of a directed self-assembled molecular n-bit shift register memory device.

PubMed

Lambropoulos, Nicholas A; Reimers, Jeffrey R; Crossley, Maxwell J; Hush, Noel S; Silverbrook, Kia

2013-12-20

A general method useful in molecular electronics design is developed that integrates modelling on the nano-scale (using quantum-chemical software) and on the micro-scale (using finite-element methods). It is applied to the design of an n-bit shift register memory that could conceivably be built using accessible technologies. To achieve this, the entire complex structure of the device would be built to atomic precision using feedback-controlled lithography to provide atomic-level control of silicon devices, controlled wet-chemical synthesis of molecular insulating pillars above the silicon, and controlled wet-chemical self-assembly of modular molecular devices to these pillars that connect to external metal electrodes (leads). The shift register consists of n connected cells that read data from an input electrode, pass it sequentially between the cells under the control of two external clock electrodes, and deliver it finally to an output device. The proposed cells are trimeric oligoporphyrin units whose internal states are manipulated to provide functionality, covalently connected to other cells via dipeptide linkages. Signals from the clock electrodes are conveyed by oligoporphyrin molecular wires, and μ-oxo porphyrin insulating columns are used as the supporting pillars. The developed multiscale modelling technique is applied to determine the characteristics of this molecular device, with in particular utilization of the inverted region for molecular electron-transfer processes shown to facilitate latching and control using exceptionally low energy costs per logic operation compared to standard CMOS shift register technology.

A multiscale simulation technique for molecular electronics: design of a directed self-assembled molecular n-bit shift register memory device

NASA Astrophysics Data System (ADS)

Lambropoulos, Nicholas A.; Reimers, Jeffrey R.; Crossley, Maxwell J.; Hush, Noel S.; Silverbrook, Kia

2013-12-01

A general method useful in molecular electronics design is developed that integrates modelling on the nano-scale (using quantum-chemical software) and on the micro-scale (using finite-element methods). It is applied to the design of an n-bit shift register memory that could conceivably be built using accessible technologies. To achieve this, the entire complex structure of the device would be built to atomic precision using feedback-controlled lithography to provide atomic-level control of silicon devices, controlled wet-chemical synthesis of molecular insulating pillars above the silicon, and controlled wet-chemical self-assembly of modular molecular devices to these pillars that connect to external metal electrodes (leads). The shift register consists of n connected cells that read data from an input electrode, pass it sequentially between the cells under the control of two external clock electrodes, and deliver it finally to an output device. The proposed cells are trimeric oligoporphyrin units whose internal states are manipulated to provide functionality, covalently connected to other cells via dipeptide linkages. Signals from the clock electrodes are conveyed by oligoporphyrin molecular wires, and μ-oxo porphyrin insulating columns are used as the supporting pillars. The developed multiscale modelling technique is applied to determine the characteristics of this molecular device, with in particular utilization of the inverted region for molecular electron-transfer processes shown to facilitate latching and control using exceptionally low energy costs per logic operation compared to standard CMOS shift register technology.

Nanofibers formed through pi...pi stacking of the complexes of glucosyl-C2-salicyl-imine and phenylalanine: characterization by microscopy, modeling by molecular mechanics, and interaction by alpha-helical and beta-sheet proteins.

PubMed

Acharya, Amitabha; Ramanujam, Balaji; Mitra, Atanu; Rao, Chebrolu P

2010-07-27

This paper deals with the self-assembly of the 1:1 complex of two different amphiphiles, namely, a glucosyl-salicyl-imino conjugate (L) and phenylalanine (Phe), forming nanofibers over a period of time through pi...pi interactions. Significant enhancement observed in the fluorescence intensity of L at approximately 423 nm band and the significant decrease observed in the absorbance of the approximately 215 nm band are some characteristics of this self-assembly. Matrix-assisted laser desorption ionization/time of flight titration carried out at different time intervals supports the formation of higher aggregates. Atomic force microscopy (AFM), transmission electron microscopy, and scanning electron miscroscopy results showed the formation of nanofibers for the solutions of L with phenylalanine. In dynamic light scattering measurements, the distribution of the particles extends to a higher diameter range over time, indicating a slow kinetic process of assembly. Similar spectral and microscopy studies carried out with the control molecules support the role of the amino acid moiety over the simple -COOH moiety as well as the side chain phenyl moiety in association with the amino acid, in the formation of these fibers. All these observations support the presence of pi...pi interactions between the initially formed 1:1 complexes leading to the fiber formation. The aggregation of 1:1 complexes leading to fibers followed by the formation of bundles has been modeled by molecular mechanics studies. Thus the fiber formation with L is limited to phenylalanine and not to any other naturally occurring amino acid and hence a polymer composed of two different biocompatible amphiphiles. AFM studies carried out between the fiber forming mixture and proteins resulted in the observation that only BSA selectively adheres to the fiber among the three alpha-helical and two beta-sheet proteins studied and hence may be of use in some medical applications.

Insulin stimulates syntaxin4 SNARE complex assembly via a novel regulatory mechanism.

PubMed

Kioumourtzoglou, Dimitrios; Gould, Gwyn W; Bryant, Nia J

2014-04-01

Insulin stimulates glucose transport into fat and muscle cells by increasing the exocytic trafficking rate of the GLUT4 facilitative glucose transporter from intracellular stores to the plasma membrane. Delivery of GLUT4 to the plasma membrane is mediated by formation of functional SNARE complexes containing syntaxin4, SNAP23, and VAMP2. Here we have used an in situ proximity ligation assay to integrate these two observations by demonstrating for the first time that insulin stimulation causes an increase in syntaxin4-containing SNARE complex formation in adipocytes. Furthermore, we demonstrate that insulin brings about this increase in SNARE complex formation by mobilizing a pool of syntaxin4 held in an inactive state under basal conditions. Finally, we have identified phosphorylation of the regulatory protein Munc18c, a direct target of the insulin receptor, as a molecular switch to coordinate this process. Hence, this report provides molecular detail of how the cell alters membrane traffic in response to an external stimulus, in this case, insulin.

Defining the Architecture of the Core Machinery for the Assembly of Fe-S Clusters in Human Mitochondria.

PubMed

Gakh, Oleksandr; Ranatunga, Wasantha; Galeano, Belinda K; Smith, Douglas S; Thompson, James R; Isaya, Grazia

2017-01-01

Although Fe-S clusters may assemble spontaneously from elemental iron and sulfur in protein-free systems, the potential toxicity of free Fe 2+ , Fe 3+ , and S 2- ions in aerobic environments underscores the requirement for specialized proteins to oversee the safe assembly of Fe-S clusters in living cells. Prokaryotes first developed multiprotein systems for Fe-S cluster assembly, from which mitochondria later derived their own system and became the main Fe-S cluster suppliers for eukaryotic cells. Early studies in yeast and human mitochondria indicated that Fe-S cluster assembly in eukaryotes is centered around highly conserved Fe-S proteins (human ISCU) that serve as scaffolds upon which new Fe-S clusters are assembled from (i) elemental sulfur, provided by a pyridoxal phosphate-dependent cysteine desulfurase (human NFS1) and its stabilizing-binding partner (human ISD11), and (ii) elemental iron, provided by an iron-binding protein of the frataxin family (human FXN). Further studies revealed that all of these proteins could form stable complexes that could reach molecular masses of megadaltons. However, the protein-protein interaction surfaces, catalytic mechanisms, and overall architecture of these macromolecular machines remained undefined for quite some time. The delay was due to difficulties inherent in reconstituting these very large multiprotein complexes in vitro or isolating them from cells in sufficient quantities to enable biochemical and structural studies. Here, we describe approaches we developed to reconstitute the human Fe-S cluster assembly machinery in Escherichia coli and to define its remarkable architecture. © 2017 Elsevier Inc. All rights reserved.

Molecular Self-Assembly of Group 11 Pyrazolate Complexes as Phosphorescent Chemosensors for Detection of Benzene

NASA Astrophysics Data System (ADS)

Ghazalli, N. F.; Yuliati, L.; Lintang, H. O.

2018-01-01

We highlight the systematic study on vapochromic sensing of aromatic vapors such as benzene using phosphorescent trinuclear pyrazolate complexes (2) with supramolecular assembly of a weak intermolecular metal-metal interaction consisting of 4-(3,5-dimethoxybenzyl)-3,5-dimethyl pyrazole ligand (1) and group 11 metal ions (Cu(I), Ag(I), Au(I)). The resulting chemosensor 2(Cu) revealed positive response to benzene vapors in 5 mins by blue-shifting its emission band in 44 nm (from 616 to 572 nm) and emitted bright orange to green, where this change cannot be recovered even with external stimuli. Comparing to 2(Ag) with longer metal-metal distance (473 nm) with same sensing time and quenching in 37%, 2(Au) gave quenching in 81% from its original intensity at 612 nm with reusability in 82% without external stimuli and emitted less emissive of red-orange from its original color. The shifting phenomenon in 2(Cu) suggests diffusion of benzene vapors to inside molecules for formation of intermolecular interaction with Cu(I)-Cu(I) interaction while quenching phenomenon in 2(Au) suggests diffusion of benzene vapors to between the Au(I)-Au(I) interaction. These results indicate that suitable molecular structure of ligand and metal ion in pyrazolate complex is important for designing chemosensor in the detection of benzene vapors.

Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins?

PubMed Central

2016-01-01

RNA silencing is a eukaryote‐specific phenomenon in which microRNAs and small interfering RNAs degrade messenger RNAs containing a complementary sequence. To this end, these small RNAs need to be loaded onto an Argonaute protein (AGO protein) to form the effector complex referred to as RNA‐induced silencing complex (RISC). RISC assembly undergoes multiple and sequential steps with the aid of Hsc70/Hsp90 chaperone machinery. The molecular mechanisms for this assembly process remain unclear, despite their significance for the development of gene silencing techniques and RNA interference‐based therapeutics. This review dissects the currently available structures of AGO proteins and proposes models and hypotheses for RISC assembly, covering the conformation of unloaded AGO proteins, the chaperone‐assisted duplex loading, and the slicer‐dependent and slicer‐independent duplex separation. The differences in the properties of RISC between prokaryotes and eukaryotes will also be clarified. WIREs RNA 2016, 7:637–660. doi: 10.1002/wrna.1356 For further resources related to this article, please visit the WIREs website. PMID:27184117

Nano-Assemblies of Modified Cyclodextrins and Their Complexes with Guest Molecules: Incorporation in Nanostructured Membranes and Amphiphile Nanoarchitectonics Design

PubMed Central

Zerkoune, Leïla; Angelova, Angelina; Lesieur, Sylviane

2014-01-01

A variety of cyclodextrin-based molecular structures, with substitutions of either primary or secondary faces of the natural oligosaccharide macrocycles of α-, β-, or γ-cyclodextrins, have been designed towards innovative applications of self-assembled cyclodextrin nanomaterials. Amphiphilic cyclodextrins have been obtained by chemical or enzymatic modifications of their macrocycles using phospholipidyl, peptidolipidyl, cholesteryl, and oligo(ethylene oxide) anchors as well as variable numbers of grafted hydrophobic hydrocarbon or fluorinated chains. These novel compounds may self-assemble in an aqueous medium into different types of supramolecular nanoassemblies (vesicles, micelles, nanorods, nanospheres, and other kinds of nanoparticles and liquid crystalline structures). This review discusses the supramolecular nanoarchitectures, which can be formed by amphiphilic cyclodextrin derivatives in mixtures with other molecules (phospholipids, surfactants, and olygonucleotides). Biomedical applications are foreseen for nanoencapsulation of drug molecules in the hydrophobic interchain volumes and nanocavities of the amphiphilic cyclodextrins (serving as drug carriers or pharmaceutical excipients), anticancer phototherapy, gene delivery, as well as for protection of instable active ingredients through inclusion complexation in nanostructured media. PMID:28344245

Strength of Neisseria meningitidis binding to endothelial cells requires highly-ordered CD147/β2-adrenoceptor clusters assembled by alpha-actinin-4

PubMed Central

Maïssa, Nawal; Covarelli, Valentina; Janel, Sébastien; Durel, Beatrice; Simpson, Nandi; Bernard, Sandra C.; Pardo-Lopez, Liliana; Bouzinba-Ségard, Haniaa; Faure, Camille; Scott, Mark G.H.; Coureuil, Mathieu; Morand, Philippe C.; Lafont, Frank; Nassif, Xavier; Marullo, Stefano; Bourdoulous, Sandrine

2017-01-01

Neisseria meningitidis (meningococcus) is an invasive bacterial pathogen that colonizes human vessels, causing thrombotic lesions and meningitis. Establishment of tight interactions with endothelial cells is crucial for meningococci to resist haemodynamic forces. Two endothelial receptors, CD147 and the β2-adrenergic receptor (β2AR), are sequentially engaged by meningococci to adhere and promote signalling events leading to vascular colonization, but their spatiotemporal coordination is unknown. Here we report that CD147 and β2AR form constitutive hetero-oligomeric complexes. The scaffolding protein α-actinin-4 directly binds to the cytosolic tail of CD147 and governs the assembly of CD147–β2AR complexes in highly ordered clusters at bacterial adhesion sites. This multimolecular assembly process increases the binding strength of meningococci to endothelial cells under shear stress, and creates molecular platforms for the elongation of membrane protrusions surrounding adherent bacteria. Thus, the specific organization of cellular receptors has major impacts on host–pathogen interaction. PMID:28569760

Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins?

PubMed

Nakanishi, Kotaro

2016-09-01

RNA silencing is a eukaryote-specific phenomenon in which microRNAs and small interfering RNAs degrade messenger RNAs containing a complementary sequence. To this end, these small RNAs need to be loaded onto an Argonaute protein (AGO protein) to form the effector complex referred to as RNA-induced silencing complex (RISC). RISC assembly undergoes multiple and sequential steps with the aid of Hsc70/Hsp90 chaperone machinery. The molecular mechanisms for this assembly process remain unclear, despite their significance for the development of gene silencing techniques and RNA interference-based therapeutics. This review dissects the currently available structures of AGO proteins and proposes models and hypotheses for RISC assembly, covering the conformation of unloaded AGO proteins, the chaperone-assisted duplex loading, and the slicer-dependent and slicer-independent duplex separation. The differences in the properties of RISC between prokaryotes and eukaryotes will also be clarified. WIREs RNA 2016, 7:637-660. doi: 10.1002/wrna.1356 For further resources related to this article, please visit the WIREs website. © 2016 The Authors. WIREs RNA published by Wiley Periodicals, Inc.

Structural analysis and modeling reveals new mechanisms governing ESCRT-III spiral filament assembly

PubMed Central

Shen, Qing-Tao; Schuh, Amber L.; Zheng, Yuqing; Quinney, Kyle; Wang, Lei; Hanna, Michael; Mitchell, Julie C.; Otegui, Marisa S.; Ahlquist, Paul; Cui, Qiang

2014-01-01

The scission of biological membranes is facilitated by a variety of protein complexes that bind and manipulate lipid bilayers. ESCRT-III (endosomal sorting complex required for transport III) filaments mediate membrane scission during the ostensibly disparate processes of multivesicular endosome biogenesis, cytokinesis, and retroviral budding. However, mechanisms by which ESCRT-III subunits assemble into a polymer remain unknown. Using cryogenic electron microscopy (cryo-EM), we found that the full-length ESCRT-III subunit Vps32/CHMP4B spontaneously forms single-stranded spiral filaments. The resolution afforded by two-dimensional cryo-EM combined with molecular dynamics simulations revealed that individual Vps32/CHMP4B monomers within a filament are flexible and able to accommodate a range of bending angles. In contrast, the interface between monomers is stable and refractory to changes in conformation. We additionally found that the carboxyl terminus of Vps32/CHMP4B plays a key role in restricting the lateral association of filaments. Our findings highlight new mechanisms by which ESCRT-III filaments assemble to generate a unique polymer capable of membrane remodeling in multiple cellular contexts. PMID:25202029

The molecular biology of Bluetongue virus replication.

PubMed

Patel, Avnish; Roy, Polly

2014-03-01

The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Koepf, Matthieu; Bergkamp, Jesse J.; Teillout, Anne-Lucie

The association of different metals in stable, well-defined molecular assemblies remains a great challenge of supramolecular chemistry. In such constructs, the emergence of synergism, or cooperative effects between the different metal centers is particularly intriguing. These effects can lead to uncommon reactivity or remarkable physico-chemical properties that are not otherwise achievable. For example, the association of alkaline or alkaline-earth cations and transition metals is pivotal for the activity of several biomolecules and human-made catalysts that carry out fundamental redox transformations (water oxidation, nitrogen reduction, water–gas shift reaction, etc.). In many cases the precise nature of the interactions between the alkaline-earthmore » cations and the redox-active transition metals remains elusive due to the difficulty of building stable molecular heterometallic assemblies that associate transition metals and alkaline or alkaline-earth cations in a controlled way. In this work we present the rational design of porphyrin-based ligands possessing a second binding site for alkaline-earth cations above the porphyrin macrocycle primary complexation site. We demonstrate that by using a combination of crown ether and carboxylic acid substituents suitably positioned on the periphery of the porphyrin, bitopic ligands can be obtained. The binding of calcium, a typical alkaline-earth cation, by the newly prepared ligands has been studied in detail and we show that a moderately large binding constant can be achieved in protic media using ligands that possess some degree of structural flexibility. The formation of Zn–Ca assemblies discussed in this work is viewed as a stepping stone towards the assembly of well defined molecular transition metal-alkaline earth bimetallic centers using a versatile organic scaffold.« less

Assembly/disassembly of a complex icosahedral virus to incorporate heterologous nucleic acids

NASA Astrophysics Data System (ADS)

Pascual, Elena; Mata, Carlos P.; Carrascosa, José L.; Castón, José R.

2017-12-01

Hollow protein containers are widespread in nature, and include virus capsids as well as eukaryotic and bacterial complexes. Protein cages are studied extensively for applications in nanotechnology, nanomedicine and materials science. Their inner and outer surfaces can be modified chemically or genetically, and the internal cavity can be used to template, store and/or arrange molecular cargos. Virus capsids and virus-like particles (VLP, noninfectious particles) provide versatile platforms for nanoscale bioengineering. Study of capsid protein self-assembly into monodispersed particles, and of VLP structure and biophysics is necessary not only to understand natural processes, but also to infer how these platforms can be redesigned to furnish novel functional VLP. Here we address the assembly dynamics of infectious bursal disease virus (IBDV), a complex icosahedral virus. IBDV has a ~70 nm-diameter T  =  13 capsid with VP2 trimers as the only structural subunits. During capsid assembly, VP2 is synthesized as a precursor (pVP2) whose C terminus is cleaved. The pVP2 C terminus has an amphipathic helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, necessary for control of assembly, 466/456-residue pVP2 intermediates bearing this helix assemble into VLP only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for genetic insertion of proteins (cargo space ~78 000 nm3). We established an in vitro assembly/disassembly system of HT-VP2-466-based VLP for heterologous nucleic acid packaging and/or encapsulation of drugs and other molecules. HT-VP2-466 (empty) capsids were disassembled and reassembled by dialysis against low-salt/basic pH and high-salt/acid pH buffers, respectively, thus illustrating the reversibility in vitro of IBDV capsid assembly. HT-VP2-466 VLP also packed heterologous DNA by non-specific confinement during assembly. These and previous results establish the bases for biotechnological applications based on the IBDV capsid and its ability to incorporate exogenous proteins and nucleic acids.

Imparting the unique properties of DNA into complex material architectures and functions.

PubMed

Xu, Phyllis F; Noh, Hyunwoo; Lee, Ju Hun; Domaille, Dylan W; Nakatsuka, Matthew A; Goodwin, Andrew P; Cha, Jennifer N

2013-07-01

While the remarkable chemical and biological properties of DNA have been known for decades, these properties have only been imparted into materials with unprecedented function much more recently. The inimitable ability of DNA to form programmable, complex assemblies through stable, specific, and reversible molecular recognition has allowed the creation of new materials through DNA's ability to control a material's architecture and properties. In this review we discuss recent progress in how DNA has brought unmatched function to materials, focusing specifically on new advances in delivery agents, devices, and sensors.

Modular Homogeneous Chromophore–Catalyst Assemblies

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

Mulfort, Karen L.; Utschig, Lisa M.

2016-05-17

Photosynthetic reaction center (RC) proteins convert incident solar energy to chemical energy through a network of molecular cofactors which have been evolutionarily tuned to couple efficient light-harvesting, directional electron transfer, and long-lived charge separation with secondary reaction sequences. These molecular cofactors are embedded within a complex protein environment which precisely positions each cofactor in optimal geometries along efficient electron transfer pathways with localized protein environments facilitating sequential and accumulative charge transfer. By contrast, it is difficult to approach a similar level of structural complexity in synthetic architectures for solar energy conversion. However, by using appropriate self-assembly strategies, we anticipate thatmore » molecular modules, which are independently synthesized and optimized for either light-harvesting or redox catalysis, can be organized into spatial arrangements that functionally mimic natural photosynthesis. In this Account, we describe a modular approach to new structural designs for artificial photosynthesis which is largely inspired by photosynthetic RC proteins. We focus on recent work from our lab which uses molecular modules for light-harvesting or proton reduction catalysis in different coordination geometries and different platforms, spanning from discrete supramolecular assemblies to molecule–nanoparticle hybrids to protein-based biohybrids. Molecular modules are particularly amenable to high-resolution characterization of the ground and excited state of each module using a variety of physical techniques; such spectroscopic interrogation helps our understanding of primary artificial photosynthetic mechanisms. In particular, we discuss the use of transient optical spectroscopy, EPR, and X-ray scattering techniques to elucidate dynamic structural behavior and light-induced kinetics and the impact on photocatalytic mechanism. Two different coordination geometries of supramolecular photocatalyst based on the [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) light-harvesting module with cobaloxime-based catalyst module are compared, with progress in stabilizing photoinduced charge separation identified. These same modules embedded in the small electron transfer protein ferredoxin exhibit much longer charge-separation, enabled by stepwise electron transfer through the native [2Fe-2S] cofactor. We anticipate that the use of interchangeable, molecular modules which can interact in different coordination geometries or within entirely different structural platforms will provide important fundamental insights into the effect of environment on parameters such as electron transfer and charge separation, and ultimately drive more efficient designs for artificial photosynthesis.« less

An Assembly Funnel Makes Biomolecular Complex Assembly Efficient

PubMed Central

Zenk, John; Schulman, Rebecca

2014-01-01

Like protein folding and crystallization, the self-assembly of complexes is a fundamental form of biomolecular organization. While the number of methods for creating synthetic complexes is growing rapidly, most require empirical tuning of assembly conditions and/or produce low yields. We use coarse-grained simulations of the assembly kinetics of complexes to identify generic limitations on yields that arise because of the many simultaneous interactions allowed between the components and intermediates of a complex. Efficient assembly occurs when nucleation is fast and growth pathways are few, i.e. when there is an assembly “funnel”. For typical complexes, an assembly funnel occurs in a narrow window of conditions whose location is highly complex specific. However, by redesigning the components this window can be drastically broadened, so that complexes can form quickly across many conditions. The generality of this approach suggests assembly funnel design as a foundational strategy for robust biomolecular complex synthesis. PMID:25360818

Non-muscle (NM) myosin heavy chain phosphorylation regulates the formation of NM myosin filaments, adhesome assembly and smooth muscle contraction.

PubMed

Zhang, Wenwu; Gunst, Susan J

2017-07-01

Non-muscle (NM) and smooth muscle (SM) myosin II are both expressed in smooth muscle tissues, however the role of NM myosin in SM contraction is unknown. Contractile stimulation of tracheal smooth muscle tissues stimulates phosphorylation of the NM myosin heavy chain on Ser1943 and causes NM myosin filament assembly at the SM cell cortex. Expression of a non-phosphorylatable NM myosin mutant, NM myosin S1943A, in SM tissues inhibits ACh-induced NM myosin filament assembly and SM contraction, and also inhibits the assembly of membrane adhesome complexes during contractile stimulation. NM myosin regulatory light chain (RLC) phosphorylation but not SM myosin RLC phosphorylation is regulated by RhoA GTPase during ACh stimulation, and NM RLC phosphorylation is required for NM myosin filament assembly and SM contraction. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. The molecular function of non-muscle (NM) isoforms of myosin II in smooth muscle (SM) tissues and their possible role in contraction are largely unknown. We evaluated the function of NM myosin during contractile stimulation of canine tracheal SM tissues. Stimulation with ACh caused NM myosin filament assembly, as assessed by a Triton solubility assay and a proximity ligation assay aiming to measure interactions between NM myosin monomers. ACh stimulated the phosphorylation of NM myosin heavy chain on Ser1943 in tracheal SM tissues, which can regulate NM myosin IIA filament assembly in vitro. Expression of the non-phosphorylatable mutant NM myosin S1943A in SM tissues inhibited ACh-induced endogenous NM myosin Ser1943 phosphorylation, NM myosin filament formation, the assembly of membrane adhesome complexes and tension development. The NM myosin cross-bridge cycling inhibitor blebbistatin suppressed adhesome complex assembly and SM contraction without inhibiting NM myosin Ser1943 phosphorylation or NM myosin filament assembly. RhoA inactivation selectively inhibited phosphorylation of the NM myosin regulatory light chain (RLC), NM myosin filament assembly and contraction, although it did not inhibit SM RLC phosphorylation. We conclude that the assembly and activation of NM myosin II is regulated during contractile stimulation of airway SM tissues by RhoA-mediated NM myosin RLC phosphorylation and by NM myosin heavy chain Ser1943 phosphorylation. NM myosin II actomyosin cross-bridge cycling regulates the assembly of membrane adhesome complexes that mediate the cytoskeletal processes required for tension generation. NM myosin II plays a critical role in airway SM contraction that is independent and distinct from the function of SM myosin. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Ultrafast dynamics in multifunctional Ru(II)-loaded polymers for solar energy conversion.

PubMed

Morseth, Zachary A; Wang, Li; Puodziukynaite, Egle; Leem, Gyu; Gilligan, Alexander T; Meyer, Thomas J; Schanze, Kirk S; Reynolds, John R; Papanikolas, John M

2015-03-17

The use of sunlight to make chemical fuels (i.e., solar fuels) is an attractive approach in the quest to develop sustainable energy sources. Using nature as a guide, assemblies for artificial photosynthesis will need to perform multiple functions. They will need to be able to harvest light across a broad region of the solar spectrum, transport excited-state energy to charge-separation sites, and then transport and store redox equivalents for use in the catalytic reactions that produce chemical fuels. This multifunctional behavior will require the assimilation of multiple components into a single macromolecular system. A wide variety of different architectures including porphyrin arrays, peptides, dendrimers, and polymers have been explored, with each design posing unique challenges. Polymer assemblies are attractive due to their relative ease of production and facile synthetic modification. However, their disordered nature gives rise to stochastic dynamics not present in more ordered assemblies. The rational design of assemblies requires a detailed understanding of the energy and electron transfer events that follow light absorption, which can occur on time scales ranging from femtoseconds to hundreds of microseconds, necessitating the use of sophisticated techniques. We have used a combination of time-resolved absorption and emission spectroscopies with observation times that span 9 orders of magnitude to follow the excited-state evolution within polymer-based molecular assemblies. We complement experimental observations with molecular dynamics simulations to develop a microscopic view of these dynamics. This Account provides an overview of our work on polymers decorated with pendant Ru(II) chromophores, both in solution and on surfaces. We have examined site-to-site energy transport among the Ru(II) complexes, and in systems incorporating π-conjugated polymers, we have observed ultrafast formation of a long-lived charge-separated state. When attached to TiO2, these assemblies exhibit multifunctional behavior in which photon absorption is followed by energy transport to the surface and electron injection to produce an oxidized metal complex. The oxidizing equivalent is then transferred to the conjugated polymer, giving rise to a long-lived charge-separated state.

A nanometre-scale electronic switch consisting of a metal cluster and redox-addressable groups.

PubMed

Gittins, D I; Bethell, D; Schiffrin, D J; Nichols, R J

2000-11-02

So-called bottom-up fabrication methods aim to assemble and integrate molecular components exhibiting specific functions into electronic devices that are orders of magnitude smaller than can be fabricated by lithographic techniques. Fundamental to the success of the bottom-up approach is the ability to control electron transport across molecular components. Organic molecules containing redox centres-chemical species whose oxidation number, and hence electronic structure, can be changed reversibly-support resonant tunnelling and display promising functional behaviour when sandwiched as molecular layers between electrical contacts, but their integration into more complex assemblies remains challenging. For this reason, functionalized metal nanoparticles have attracted much interest: they exhibit single-electron characteristics (such as quantized capacitance charging) and can be organized through simple self-assembly methods into well ordered structures, with the nanoparticles at controlled locations. Here we report scanning tunnelling microscopy measurements showing that organic molecules containing redox centres can be used to attach metal nanoparticles to electrode surfaces and so control the electron transport between them. Our system consists of gold nanoclusters a few nanometres across and functionalized with polymethylene chains that carry a central, reversibly reducible bipyridinium moiety. We expect that the ability to electronically contact metal nanoparticles via redox-active molecules, and to alter profoundly their tunnelling properties by charge injection into these molecules, can form the basis for a range of nanoscale electronic switches.

Novel calix[4]pyrrole assembly: Punctilious recognition of F- and Cu+2 ions

NASA Astrophysics Data System (ADS)

Bhatt, Keyur D.; Shah, Hemangini; Modi, Krunal M.; Kongor, Anita; Panchal, Manthan; Jain, Vinod K.

2017-12-01

A new tetra hydroxyl methoxy substituted calix[4]pyrrole (HMCP) has been synthesized and found to form stable complex with F- ions and Cu+2 ions. The red-shift in absorption band of HMCP was observed due to the presence of both cation (Cu+2) and anion (F-). These results displayed that formation of the complex is mainly attributed to the charge-transfer interactions between HMCP with electron deficient pyrrole rings and the electron-rich guest ions. Molecular dynamics simulation predicts intermolecular H-bonds and van der Waals types of interaction for the complex formation of HMCP-Cu+2 and HMCP-F-.

The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes.

PubMed

Wang, Zheng; Miao, Guangyan; Xue, Xue; Guo, Xiangyang; Yuan, Chongzhen; Wang, Zhaoyu; Zhang, Gangming; Chen, Yingyu; Feng, Du; Hu, Junjie; Zhang, Hong

2016-09-01

Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

PSB27: A thylakoid protein enabling Arabidopsis to adapt to changing light intensity

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

Hou, Xin; Garcia, Veder J.; Buchanan, Bob B.

Project Title: Immunophilins in the assembly and maintenance of photosynthetic electron transport chain in Arabidopsis Applicant: The Regents of the University of California PI: Sheng Luan, University of California at Berkeley Photosynthetic light energy conversion entails coordinated function of complex molecular machines that capture and convert light energy into chemical forms through photosynthetic electron transport chain. Each molecular machine, such as photosystem II (PSII), may consist of dozens of protein subunits and small molecule cofactors. Despite advanced understanding of the structure and function of these complexes, little is known about “How individual proteins and cofactors assemble into a functional machinemore » and how do these molecular machines maintain their structure and function under a highly hazardous lumenal environment.” Our studies on immunophilins have unexpectedly contributed to the understanding of this question. Originally defined as cellular receptors for immunosuppressants, immunophilins have been discovered in a wide range of organisms from bacteria, fungi, plants, to animals. Immunophilins function in protein folding processes as chaperones and foldases. Arabidopsis genome encodes ca. 50 immunophilins. The most striking finding is that 16 immunophilin members are targeted to chloroplast thylakoid lumen, by far the largest group in the lumenal proteome. What is the function of immunophilins in the thylakoid lumen? Our studies have demonstrated critical roles for several immunophilins in the biogenesis and maintenance of photosynthetic complexes such as PSII. These studies have made a critical link between immunophilins and the assembly of photosynthetic machines and thus opened up a new area of research in photosynthesis. Our goal is to dissect the roles of immunophilins and their partners in the assembly and maintenance of the photosynthetic electron transport chain. The specific objectives for this funding period will be: 1. To dissect the mechanism of action for CYP38. Our studies suggest an “autoinhibitory” model for CYP38 action. We plan to test this model and determine the mechanistic details for CYP38 function by biochemical and genetic procedures. 2. To determine the mechanism of action of FKBP20-2/FKBP16-3. Studies on FBKP16-3 and FKBP20-2 suggest that they may work together as a redox-dependent duo in PSII assembly. We will test this hypothesis using biochemical and genetic tools. 3. To determine the function of lumenal FKBPs by multi-gene mutagenesis approach. Using new genetic knockout (KO) procedures especially CRISPR/CAS9 system, we plan to generate multi-gene KO models that will likely provide vital information on those FKBPs with functional redundancy. 4. Functional analysis of thylakoid lumen network. We will focus our effort on objectives 1-3. Objective 4 represents a long-term goal to establish a more comprehensive network of proteins in the thylakoid lumen and their function in the assembly and maintenance of photosynthetic complexes. I hypothesize that the 16 immunophilin-type chaperones and foldases in the thylakoid lumen constitute an “assembly line” for the various components in the photosynthetic electron transport chain. Understanding this assembly line will enable further engineering of more efficient photosynthetic machines to capture more light energy and enhance plant productivity. Furthermore, it is envisioned that such molecular “assembly line” may be rebuilt to produce artificial photosynthesis in vitro or in non-photosynthetic organisms. These are highly relevant to the mission of “Photosynthetic Systems” and “Physical Biosciences” programs to “enhance our understanding of energy capture, conversion, and/or storage.” During the funding period, we have performed a number of experiments under the proposed objectives and obtained several new results. We have found in Objective 1 that CYP38 is associated to the thylokoid membrane with a larger complex that might be PSII supercomplex. Under objective 2, we have found that FKBP16-2 interacted with PSB27 that was further pursuited and published a research article in PNAS (attached). Under Objective 3, we have identified several mutants of other FKBPs in the thyalkoid lumen that should be further studied if future funding is available. Under Objective 4, we have started to build a network of lumenal proteins that play a number of roles in photosynthesis. For example, the CYP37 and CYP28 are linked to chloroplast signaling to nucleus, critical for controlling plant response to high light and adaptation to climate change. Unfortunately these studies have been terminated due to funding shortage.« less

High molecular diversity of extraterrestrial organic matter in Murchison meteorite revealed 40 years after its fall

PubMed Central

Schmitt-Kopplin, Philippe; Gabelica, Zelimir; Gougeon, Régis D.; Fekete, Agnes; Kanawati, Basem; Harir, Mourad; Gebefuegi, Istvan; Eckel, Gerhard; Hertkorn, Norbert

2010-01-01

Numerous descriptions of organic molecules present in the Murchison meteorite have improved our understanding of the early interstellar chemistry that operated at or just before the birth of our solar system. However, all molecular analyses were so far targeted toward selected classes of compounds with a particular emphasis on biologically active components in the context of prebiotic chemistry. Here we demonstrate that a nontargeted ultrahigh-resolution molecular analysis of the solvent-accessible organic fraction of Murchison extracted under mild conditions allows one to extend its indigenous chemical diversity to tens of thousands of different molecular compositions and likely millions of diverse structures. This molecular complexity, which provides hints on heteroatoms chronological assembly, suggests that the extraterrestrial chemodiversity is high compared to terrestrial relevant biological- and biogeochemical-driven chemical space. PMID:20160129

High molecular diversity of extraterrestrial organic matter in Murchison meteorite revealed 40 years after its fall.

PubMed

Schmitt-Kopplin, Philippe; Gabelica, Zelimir; Gougeon, Régis D; Fekete, Agnes; Kanawati, Basem; Harir, Mourad; Gebefuegi, Istvan; Eckel, Gerhard; Hertkorn, Norbert

2010-02-16

Numerous descriptions of organic molecules present in the Murchison meteorite have improved our understanding of the early interstellar chemistry that operated at or just before the birth of our solar system. However, all molecular analyses were so far targeted toward selected classes of compounds with a particular emphasis on biologically active components in the context of prebiotic chemistry. Here we demonstrate that a nontargeted ultrahigh-resolution molecular analysis of the solvent-accessible organic fraction of Murchison extracted under mild conditions allows one to extend its indigenous chemical diversity to tens of thousands of different molecular compositions and likely millions of diverse structures. This molecular complexity, which provides hints on heteroatoms chronological assembly, suggests that the extraterrestrial chemodiversity is high compared to terrestrial relevant biological- and biogeochemical-driven chemical space.

Self-assembled supramolecular hetero-bimetallacycles for anticancer potency via intracellular release

PubMed Central

Mishra, Anurag; Lee, Seung Chang; Kaushik, Neha; Cook, Timothy R.; Choi, Eun Ha

2015-01-01

Two new tetracationic hetero-bimetallacycles, 4 and 5, have been constructed from an N,N′-bis(4-(pyridin-4-ylethynyl)phenyl)pyridine-2,6-dicarboxamide ligand, 1, and cis-blocked complexes [M(dppf)](OTf)2 (dppf = 1,1′-bis(diphenylphosphino)ferrocene; M = Pd (2), Pt (3)) in CH3NO2/CH2Cl2 (1:1) solvent. Both complexes were isolated with adequate yields as triflate salts and were then characterized using 1H, 13C, and 31P NMR spectroscopy, elemental analysis, UV-Vis spectroscopy, and high-resolution electrospray mass spectrometry (HR-ESMS). The molecular structure of 4 was determined by molecular mechanics force field calculations. The cytotoxic effect of both new complexes were analyzed against T98G (brain tumor), KB (head and neck cancer), SNU-80 (thyroid cancer), and HEK-293 non-malignant cell lines. The cytotoxicity of complexes 4 and 5 were found to be considerably more effective against cancer cells than reference drug cisplatin. Annexin-V/PI staining, caspase-3/-7 activity, reduction in mitochondrial membrane potential justify a significant level of apoptosis in complex treated cells. PMID:25209962

One pot synthesis of two Mn(II) perchlorate complexes with s-triazine NNN-pincer ligand; molecular structure, Hirshfeld analysis and DFT studies

NASA Astrophysics Data System (ADS)

Soliman, Saied M.; El-Faham, Ayman

2018-07-01

Self assembly of Mn(II) perchlorate and bis(pyrazolo)-s-triazine pincer ligand (L) in methanol-water mixture afforded the homoleptic [MnL2](ClO4)2 complex (1) as plate colorless crystals. Following the crystallization process till the near dryness of the solution, we noted few needle like crystals of the heteroleptic [MnL(H2O)3](ClO4)2·H2O complex (2). Their molecular and supramolecular structures were analyzed using single crystal structure combined with Hirshfeld analysis. The packing of complexes 1 and 2 is dominated by weak Csbnd H⋯O and strong Osbnd H⋯O hydrogen bonds, respectively, as well as anion-π stacking interactions. Using Hirshfeld analysis, the percentages of the O⋯H intermolecular contacts are 32.7% and 36.8% for 1 and 2, respectively. The Mnsbnd N distances correlated well with the atoms in molecules (AIM) topological parameters. The amount of electron density transferred from the ligand units to the manganese centre are nearly the same (0.9 e) in both complexes.

Artificial enzymes based on supramolecular scaffolds.

PubMed

Dong, Zeyuan; Luo, Quan; Liu, Junqiu

2012-12-07

Enzymes are nanometer-sized molecules with three-dimensional structures created by the folding and self-assembly of polymeric chain-like components through supramolecular interactions. They are capable of performing catalytic functions usually accompanied by a variety of conformational states. The conformational diversities and complexities of natural enzymes exerted in catalysis seriously restrict the detailed understanding of enzymatic mechanisms in molecular terms. A supramolecular viewpoint is undoubtedly helpful in understanding the principle of enzyme catalysis. The emergence of supramolecular artificial enzymes therefore provides an alternative way to approach the structural complexity and thus to unravel the mystery of enzyme catalysis. This critical review covers the recent development of artificial enzymes designed based on supramolecular scaffolds ranging from the synthetic macrocycles to self-assembled nanometer-sized objects. Such findings are anticipated to facilitate the design of supramolecular artificial enzymes as well as their potential uses in important fields, such as manufacturing and food industries, environmental biosensors, pharmaceutics and so on.

Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20.

PubMed

Maio, N; Rouault, T A

2016-10-01

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery.

[Modulation of Kv4 channels by KChIPs clamping].

PubMed

Cui, Yuan-Yuan; Wang, Ke-Wei

2009-01-01

The rapidly inactivating (A-type) potassium channels regulate membrane excitability that defines the fundamental mechanism of neuronal functions such as pain signaling. Cytosolic Kv channel-interacting proteins KChIPs co-assemble with Kv4 (Shal) alpha subunits to form a native complex. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Based on recent structural efforts, here we attempt to emphasize the interaction between KChIPs and Kv4 channel complex in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner. Greater insights into molecular mechanism between KChIPs and Kv4 interaction may provide therapeutic potentials by structure-based design of chemical compounds aimed at disrupting the protein-protein interaction for treatment of membrane excitability-related disorders.

New Directions in X-Ray Light Sources

ScienceCinema

Falcone, Roger

2017-12-09

July 15, 2008 Berkeley Lab lecture: Molecular movies of chemical reactions and material phase transformations need a strobe of x-rays, the penetrating light that reveals how atoms and molecules assemble in chemical and biological systems and complex materials. Roger Falcone, Director of the Advanced Light Source,will discuss a new generation of x ray sources that will enable a new science of atomic dynamics on ultrafast timescales.

High molecular weight DNA assembly in vivo for synthetic biology applications.

PubMed

Juhas, Mario; Ajioka, James W

2017-05-01

DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.

Proteomic analysis in non-denaturing condition of the secretome reveals the presence of multienzyme complexes in Penicillium purpurogenum.

PubMed

Gonzalez-Vogel, Alvaro; Eyzaguirre, Jaime; Oleas, Gabriela; Callegari, Eduardo; Navarrete, Mario

2011-01-01

Proteins secreted by filamentous fungi play key roles in different aspects of their biology. The fungus Penicillium purpurogenum, used as a model organism, is able to degrade hemicelluloses and pectins by secreting a variety of enzymes to the culture medium. This work shows that these enzymes interact with each other to form high molecular weight, catalytically active complexes. By using a proteomics approach, we were able to identify several protein complexes in the secretome of this fungus. The expression and assembly of these complexes depend on the carbon source used and display molecular masses ranging from 300 to 700 kDa. These complexes are composed of a variety of enzymes, including arabinofuranosidases, acetyl xylan esterases, feruloyl esterases, β-glucosidases and xylanases. The protein-protein interactions in these multienzyme complexes were confirmed by coimmunoprecipitation assays. One of the complexes was purified from sugar beet pulp cultures and the subunits identified by tandem mass spectrometry. A better understanding of the biological significance of these kinds of interactions will help in the comprehension of the degradation mechanisms used by fungi and may be of special interest to the biotechnology industry.

Complete subunit structure of the Clostridium botulinum type D toxin complex via intermediate assembly with nontoxic components.

PubMed

Mutoh, Shingo; Kouguchi, Hirokazu; Sagane, Yoshimasa; Suzuki, Tomonori; Hasegawa, Kimiko; Watanabe, Toshihiro; Ohyama, Tohru

2003-09-23

Clostridium botulinum serotype D strains usually produce two types of stable toxin complex (TC), namely, the 300 kDa M (M-TC) and the 660 kDa L (L-TC) toxin complexes. We previously proposed assembly pathways for both TCs [Kouguchi, H., et al. (2002) J. Biol. Chem. 277, 2650-2656]: M-TC is composed by association of neurotoxin (NT) and nontoxic nonhemagglutinin (NTNHA); conjugation of M-TC with three auxiliary types of hemagglutinin subcomponents (HA-33, HA-17, and HA-70) leads to the formation of L-TC. In this study, we found three TC species, 410, 540, and 610 kDa TC species, in the culture supernatant of type D strain 4947. The 540 and 610 kDa TC species displayed banding patterns on SDS-PAGE similar to that of L-TC but with less staining intensity of the HA-33 and HA-17 bands than those of L-TC, indicating that these are intermediate species in the pathway to L-TC assembly. In contrast, the 410 kDa TC species consisted of M-TC and two molecules of HA-70. All of the TC species, except L-TC, demonstrated no hemagglutination activity. When the intermediate TC species were mixed with an isolated HA-33/17 complex, every TC species converted to 650 kDa L-TC with full hemagglutination activity and had the same molecular composition of L-TC. On the basis of titration analysis with the HA-33/17 complex, the stoichiometry of the HA-33/17 complex molecules in the L-TC, 610 kDa, and 540 kDa TC species was estimated as 4, 3, and 2, respectively. In conclusion, the complete subunit composition of mature L-TC is deduced to be a dodecamer assembled by a single NT, a single NTNHA, two HA-70, four HA-33, and four HA-17 molecules.

Photoresponsive Molecular Memory Films Composed of Sequentially Assembled Heterolayers Containing Ruthenium Complexes.

PubMed

Nagashima, Takumi; Ozawa, Hiroaki; Suzuki, Takashi; Nakabayashi, Takuya; Kanaizuka, Katsuhiko; Haga, Masa-Aki

2016-01-26

Photoresponsive molecular memory films were fabricated by a layer-by-layer (LbL) assembling of two dinuclear Ru complexes with tetrapodal phosphonate anchors, containing either 2,3,5,6-tetra(2-pyridyl)pyrazine or 1,2,4,5-tetra(2-pyridyl)benzene as a bridging ligand (Ru-NP and Ru-CP, respectively), using zirconium phosphonate to link the layers. Various types of multilayer homo- and heterostructures were constructed. In the multilayer heterofilms such as ITO||(Ru-NP)m |(Ru-CP)n , the difference in redox potentials between Ru-NP and Ru-CP layers was approximately 0.7 V, which induced a potential gradient determined by the sequence of the layers. In the ITO||(Ru-NP)m |(Ru-CP)n multilayer heterofilms, the direct electron transfer (ET) from the outer Ru-CP layers to the ITO were observed to be blocked for m>2, and charge trapping in the outer Ru-CP layers became evident from the appearance of an intervalence charge transfer (IVCT) band at 1140 nm from the formation of the mixed-valent state of Ru-CP units, resulting from the reductive ET mediation of the inner Ru-NP layers. Therefore, the charging/discharging ("1"and "0") states in the outer Ru-CP layers could be addressed and interconverted by applying potential pulses between -0.5 and +0.7 V. The two states could be read out by the direction of the photocurrent (anodic or cathodic). The molecular heterolayer films thus represent a typical example of a photoresponsive memory device; that is, the writing process may be achieved by the applied potential (-0.5 or +0.7 V), while the readout process is achieved by measuring the direction of the photocurrent (anodic or cathodic). Sequence-sensitive multilayer heterofilms, using redox-active complexes as building blocks, thus demonstrate great potential for the design of molecular functional devices. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Configuration-specific electronic structure of strongly interacting interfaces: TiOPc on Cu(110)

NASA Astrophysics Data System (ADS)

Maughan, Bret; Zahl, Percy; Sutter, Peter; Monti, Oliver L. A.

2017-12-01

We use low-temperature scanning tunneling microscopy in combination with angle-resolved ultraviolet and two-photon photoemission spectroscopy to investigate the interfacial electronic structure of titanyl phthalocyanine (TiOPc) on Cu(110). We show that the presence of two unique molecular adsorption configurations is crucial for a molecular-level analysis of the hybridized interfacial electronic structure. Specifically, thermally induced self-assembly exposes marked adsorbate-configuration-specific contributions to the interfacial electronic structure. The results of this work demonstrate an avenue towards understanding and controlling interfacial electronic structure in chemisorbed films even for the case of complex film structure.

On the Free Energy That Drove Primordial Anabolism

PubMed Central

Kaufmann, Michael

2009-01-01

A key problem in understanding the origin of life is to explain the mechanism(s) that led to the spontaneous assembly of molecular building blocks that ultimately resulted in the appearance of macromolecular structures as they are known in modern biochemistry today. An indispensable thermodynamic prerequisite for such a primordial anabolism is the mechanistic coupling to processes that supplied the free energy required. Here I review different sources of free energy and discuss the potential of each form having been involved in the very first anabolic reactions that were fundamental to increase molecular complexity and thus were essential for life. PMID:19468343

Coarse-Grained Molecular Simulation of the Hierarchical Self-Assembly of π-Conjugated Optoelectronic Peptides

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

Mansbach, Rachael A.; Ferguson, Andrew L.

Self-assembled aggregates of peptides containing aromatic groups possess optoelectronic properties that make them attractive targets for the fabrication of biocompatible electronics. Molecular-level understanding of how the microscopic peptide chemistry influences the properties of the aggregates is vital for rational peptide design. We construct a coarse-grained model of Asp-Phe-Ala-Gly-OPV3-Gly-Ala-Phe-Asp (DFAG-OPV3-GAFD) peptides containing OPV3 (distyrylbenzene) π-conjugated cores explicitly parameterized against all-atom calculations and perform molecular dynamics simulations of the self-assembly of hundreds of molecules over hundreds of nanoseconds. We observe a hierarchical assembly mechanism wherein ~2-8 peptides assemble into stacks with aligned aromatic cores that subsequently form elliptical aggregates and ultimately amore » branched network with a fractal dimensionality of ~1.5. The assembly dynamics are well described by a Smoluchowski coagulation process for which we extract rate constants from the molecular simulations to both furnish insight into the microscopic assembly kinetics and extrapolate our aggregation predictions to time and length scales beyond the reach of molecular simulation. Lastly, this study presents new molecular-level understanding of the morphology and dynamics of the spontaneous self-assembly of DFAG-OPV3-GAFD peptides and establishes a systematic protocol to develop coarse-grained models of optoelectronic peptides for the exploration and design of π-conjugated peptides with tunable optoelectronic properties.« less

Coarse-Grained Molecular Simulation of the Hierarchical Self-Assembly of π-Conjugated Optoelectronic Peptides

DOE PAGES

Mansbach, Rachael A.; Ferguson, Andrew L.

2017-02-10

Self-assembled aggregates of peptides containing aromatic groups possess optoelectronic properties that make them attractive targets for the fabrication of biocompatible electronics. Molecular-level understanding of how the microscopic peptide chemistry influences the properties of the aggregates is vital for rational peptide design. We construct a coarse-grained model of Asp-Phe-Ala-Gly-OPV3-Gly-Ala-Phe-Asp (DFAG-OPV3-GAFD) peptides containing OPV3 (distyrylbenzene) π-conjugated cores explicitly parameterized against all-atom calculations and perform molecular dynamics simulations of the self-assembly of hundreds of molecules over hundreds of nanoseconds. We observe a hierarchical assembly mechanism wherein ~2-8 peptides assemble into stacks with aligned aromatic cores that subsequently form elliptical aggregates and ultimately amore » branched network with a fractal dimensionality of ~1.5. The assembly dynamics are well described by a Smoluchowski coagulation process for which we extract rate constants from the molecular simulations to both furnish insight into the microscopic assembly kinetics and extrapolate our aggregation predictions to time and length scales beyond the reach of molecular simulation. Lastly, this study presents new molecular-level understanding of the morphology and dynamics of the spontaneous self-assembly of DFAG-OPV3-GAFD peptides and establishes a systematic protocol to develop coarse-grained models of optoelectronic peptides for the exploration and design of π-conjugated peptides with tunable optoelectronic properties.« less

The mechanism of a nuclear pore assembly: a molecular biophysics view.

PubMed

Kuvichkin, Vasily V

2011-06-01

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA-PC liposomes-Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) < 100 nm in diameter, a "big" liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The "big" membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.

Stepwise Synthesis of Giant Unilamellar Vesicles on a Microfluidic Assembly Line

PubMed Central

2011-01-01

Among the molecular milieu of the cell, the membrane bilayer stands out as a complex and elusive synthetic target. We report a microfluidic assembly line that produces uniform cellular compartments from droplet, lipid, and oil/water interface starting materials. Droplets form in a lipid-containing oil flow and travel to a junction where the confluence of oil and extracellular aqueous media establishes a flow-patterned interface that is both stable and reproducible. A triangular post mediates phase transfer bilayer assembly by deflecting droplets from oil, through the interface, and into the extracellular aqueous phase to yield a continuous stream of unilamellar phospholipid vesicles with uniform and tunable size. The size of the droplet precursor dictates vesicle size, encapsulation of small-molecule cargo is highly efficient, and the single bilayer promotes functional insertion of a bacterial transmembrane pore. PMID:21309555

Synthesis, Delivery and Regulation of Eukaryotic Heme and Fe-S Cluster Cofactors

PubMed Central

Barupala, Dulmini P.; Dzul, Stephen P.; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L.

2016-01-01

In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. PMID:26785297

Artificial muscle-like function from hierarchical supramolecular assembly of photoresponsive molecular motors

NASA Astrophysics Data System (ADS)

Chen, Jiawen; Leung, Franco King-Chi; Stuart, Marc C. A.; Kajitani, Takashi; Fukushima, Takanori; van der Giessen, Erik; Feringa, Ben L.

2018-02-01

A striking feature of living systems is their ability to produce motility by amplification of collective molecular motion from the nanoscale up to macroscopic dimensions. Some of nature's protein motors, such as myosin in muscle tissue, consist of a hierarchical supramolecular assembly of very large proteins, in which mechanical stress induces a coordinated movement. However, artificial molecular muscles have often relied on covalent polymer-based actuators. Here, we describe the macroscopic contractile muscle-like motion of a supramolecular system (comprising 95% water) formed by the hierarchical self-assembly of a photoresponsive amphiphilic molecular motor. The molecular motor first assembles into nanofibres, which further assemble into aligned bundles that make up centimetre-long strings. Irradiation induces rotary motion of the molecular motors, and propagation and accumulation of this motion lead to contraction of the fibres towards the light source. This system supports large-amplitude motion, fast response, precise control over shape, as well as weight-lifting experiments in water and air.

PLEKHA7 Recruits PDZD11 to Adherens Junctions to Stabilize Nectins.

PubMed

Guerrera, Diego; Shah, Jimit; Vasileva, Ekaterina; Sluysmans, Sophie; Méan, Isabelle; Jond, Lionel; Poser, Ina; Mann, Matthias; Hyman, Anthony A; Citi, Sandra

2016-05-20

PLEKHA7 is a junctional protein implicated in stabilization of the cadherin protein complex, hypertension, cardiac contractility, glaucoma, microRNA processing, and susceptibility to bacterial toxins. To gain insight into the molecular basis for the functions of PLEKHA7, we looked for new PLEKHA7 interactors. Here, we report the identification of PDZ domain-containing protein 11 (PDZD11) as a new interactor of PLEKHA7 by yeast two-hybrid screening and by mass spectrometry analysis of PLEKHA7 immunoprecipitates. We show that PDZD11 (17 kDa) is expressed in epithelial and endothelial cells, where it forms a complex with PLEKHA7, as determined by co-immunoprecipitation analysis. The N-terminal Trp-Trp (WW) domain of PLEKHA7 interacts directly with the N-terminal 44 amino acids of PDZD11, as shown by GST-pulldown assays. Immunofluorescence analysis shows that PDZD11 is localized at adherens junctions in a PLEKHA7-dependent manner, because its junctional localization is abolished by knock-out of PLEKHA7, and is rescued by re-expression of exogenous PLEKHA7. The junctional recruitment of nectin-1 and nectin-3 and their protein levels are decreased via proteasome-mediated degradation in epithelial cells where either PDZD11 or PLEKHA7 have been knocked-out. PDZD11 forms a complex with nectin-1 and nectin-3, and its PDZ domain interacts directly with the PDZ-binding motif of nectin-1. PDZD11 is required for the efficient assembly of apical junctions of epithelial cells at early time points in the calcium-switch model. These results show that the PLEKHA7-PDZD11 complex stabilizes nectins to promote efficient early junction assembly and uncover a new molecular mechanism through which PLEKHA7 recruits PDZ-binding membrane proteins to epithelial adherens junctions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

In vitro assembly of catalase.

PubMed

Baureder, Michael; Barane, Elisabeth; Hederstedt, Lars

2014-10-10

Most aerobic organisms contain catalase, which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components, but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Optimized Assembly of a Multifunctional RNA-Protein Nanostructure in a Cell-Free Gene Expression System.

PubMed

Schwarz-Schilling, Matthaeus; Dupin, Aurore; Chizzolini, Fabio; Krishnan, Swati; Mansy, Sheref S; Simmel, Friedrich C

2018-04-11

Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.

Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies.

PubMed

Fong, Monica; Berrin, Jean-Guy; Paës, Gabriel

2016-01-01

Enzymes degrading plant biomass polymers are widely used in biotechnological applications. Their efficiency can be limited by non-specific interactions occurring with some chemical motifs. In particular, the lignin component is known to bind enzymes irreversibly. In order to determine interactions of enzymes with their substrates, experiments are usually performed on isolated simple polymers which are not representative of plant cell wall complexity. But when using natural plant substrates, the role of individual chemical and structural features affecting enzyme-binding properties is also difficult to decipher. We have designed and used lignified model assemblies of plant cell walls as templates to characterize binding properties of multi-modular cellulases. These three-dimensional assemblies are modulated in their composition using the three principal polymers found in secondary plant cell walls (cellulose, hemicellulose, and lignin). Binding properties of enzymes are obtained from the measurement of their mobility that depends on their interactions with the polymers and chemical motifs of the assemblies. The affinity of the multi-modular GH45 cellulase was characterized using a statistical analysis to determine the role played by each assembly polymer. Presence of hemicellulose had much less impact on affinity than cellulose and model lignin. Depending on the number of CBMs appended to the cellulase catalytic core, binding properties toward cellulose and lignin were highly contrasted. Model assemblies bring new insights into the molecular determinants that are responsible for interactions between enzymes and substrate without the need of complex analysis. Consequently, we believe that model bioinspired assemblies will provide relevant information for the design and optimization of enzyme cocktails in the context of biorefineries.

Programmable colloidal molecules from sequential capillarity-assisted particle assembly

PubMed Central

Ni, Songbo; Leemann, Jessica; Buttinoni, Ivo; Isa, Lucio; Wolf, Heiko

2016-01-01

The assembly of artificial nanostructured and microstructured materials which display structures and functionalities that mimic nature’s complexity requires building blocks with specific and directional interactions, analogous to those displayed at the molecular level. Despite remarkable progress in synthesizing “patchy” particles encoding anisotropic interactions, most current methods are restricted to integrating up to two compositional patches on a single “molecule” and to objects with simple shapes. Currently, decoupling functionality and shape to achieve full compositional and geometrical programmability remains an elusive task. We use sequential capillarity-assisted particle assembly which uniquely fulfills the demands described above. This is a new method based on simple, yet essential, adaptations to the well-known capillary assembly of particles over topographical templates. Tuning the depth of the assembly sites (traps) and the surface tension of moving droplets of colloidal suspensions enables controlled stepwise filling of traps to “synthesize” colloidal molecules. After deposition and mechanical linkage, the colloidal molecules can be dispersed in a solvent. The template’s shape solely controls the molecule’s geometry, whereas the filling sequence independently determines its composition. No specific surface chemistry is required, and multifunctional molecules with organic and inorganic moieties can be fabricated. We demonstrate the “synthesis” of a library of structures, ranging from dumbbells and triangles to units resembling bar codes, block copolymers, surfactants, and three-dimensional chiral objects. The full programmability of our approach opens up new directions not only for assembling and studying complex materials with single-particle-level control but also for fabricating new microscale devices for sensing, patterning, and delivery applications. PMID:27051882

Corepressors: custom tailoring and alterations while you wait

PubMed Central

Goodson, Michael; Jonas, Brian A.; Privalsky, Martin A.

2005-01-01

A diverse cadre of metazoan transcription factors mediate repression by recruiting protein complexes containing the SMRT (silencing mediator of retinoid and thyroid hormone receptor) or N-CoR (nuclear receptor corepressor) corepressors. SMRT and N-CoR nucleate the assembly of still larger corepressor complexes that perform the specific molecular incantations necessary to confer transcriptional repression. Although SMRT and N-CoR are paralogs and possess similar molecular architectures and mechanistic strategies, they nonetheless exhibit distinct molecular and biological properties. It is now clear that the functions of both SMRT and N-CoR are further diversified through alternative mRNA splicing, yielding a series of corepressor protein variants that participate in distinctive transcription factor partnerships and display distinguishable repression properties. This review will discuss what is known about the structure and actions of SMRT, N-CoR, and their splicing variants, and how alternative splicing may allow the functions of these corepressors to be adapted and tailored to different cells and to different developmental stages. PMID:16604171

Chiral signs of TPPS co-assemblies with chiral gelators: role of molecular and supramolecular chirality.

PubMed

Wang, Qiuling; Zhang, Li; Yang, Dong; Li, Tiesheng; Liu, Minghua

2016-10-13

A dianionic tetrakis(4-sulfonatophenyl)porphyrin (TPPS) self-assembled into J-aggregates when it co-assembled with a chiral cationic amphiphile via supramolecular gelation. The chiral signs of TPPS J aggregates followed the supramolecular chirality of amphiphilic assemblies rather than the molecular chirality of the amphiphile.

Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope

PubMed Central

Otsuka, Shotaro; Bui, Khanh Huy; Schorb, Martin; Hossain, M Julius; Politi, Antonio Z; Koch, Birgit; Eltsov, Mikhail; Beck, Martin; Ellenberg, Jan

2016-01-01

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM. DOI: http://dx.doi.org/10.7554/eLife.19071.001 PMID:27630123

Force feedback controls motor activity and mechanical properties of self-assembling branched actin networks

PubMed Central

Bieling, Peter; Li, Tai-De; Weichsel, Julian; McGorty, Ryan; Jreij, Pamela; Huang, Bo; Fletcher, Daniel A.; Mullins, R. Dyche

2016-01-01

Branched actin networks–created by the Arp2/3 complex, capping protein, and a nucleation promoting factor– generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and atomic force microscopy to quantify their molecular composition and material properties under various forces. Remarkably, mechanical loading of these self-assembling materials increases their density, power, and efficiency. Microscopically, increased density reflects increased filament number and altered geometry, but no change in average length. Macroscopically, increased density enhances network stiffness and resistance to mechanical failure beyond those of isotropic actin networks. These effects endow branched actin networks with memory of their mechanical history that shapes their material properties and motor activity. This work reveals intrinsic force feedback mechanisms by which mechanical resistance makes self-assembling actin networks stiffer, stronger, and more powerful. PMID:26771487

Diffusion and self-assembly of C60 molecules on monolayer graphyne sheets

PubMed Central

Ozmaian, Masoumeh; Fathizadeh, Arman; Jalalvand, Morteza; Ejtehadi, Mohammad Reza; Allaei, S. Mehdi Vaez

2016-01-01

The motion of a fullerene (C60) on 5 different types of graphyne is studied by all-atom molecular dynamics simulations and compared with former studies on the motion of C60 on graphene. The motion shows a diffusive behavior which consists of either a continuous motion or discrete movements between trapping sites depending on the type of the graphyne sheet. For graphyne-4 and graphyne-5, fullerenes could detach from the surface of the graphyne sheet at room temperature which was not reported for similar cases on graphene sheets. Collective motion of a group of fullerenes interacting with a graphyne studied and it is shown that fullerenes exhibit stable assemblies. Depending on the type of graphyne, these assemblies can have either single or double layers. The mobility of the assembled structures is also dependent on the type of the graphyne sheet. The observed properties of the motion suggests novel applications for the complexes of fullerene and monolayer graphynes. PMID:26912386

Electronic Transport through Self Assembled Thiol Molecules: Effect of Monolayer Order, Dynamics and Temperature

NASA Technical Reports Server (NTRS)

Dholakia, Geetha; Fan, Wendy; Meyyappan, M.

2005-01-01

We present the charge transport and tunneling conductance of self assembled organic thiol molecules and discuss the influence of order and dynamics in the monolayer on the transport behavior and the effect of temperature. Conjugated thiol molecular wires and organometals such as terpyridine metal complexes provide a new platform for molecular electronic devices and we study their self assembly on Au(111) substrates by the scanning tunneling microscope. Determining the organization of the molecule and the ability to control the nature of its interface with the substrate is important for reliable performance of the molecular electronic devices. By concurrent scanning tunneling microscopy and spectroscopy studies on SAMs formed from oligo (phenelyne ethynelyne) monolayers with and without molecular order, we show that packing and order determine the response of a self assembled monolayer (SAM) to competing interactions. Molecular resolution STM imaging in vacuum shows that the OPES adopt an imcommensurate SAM structure on Au(111) with a rectangular unit cell. Tunneling spectroscopic measurements were performed on the SAM as a function of junction resistance. STS results show that the I-Vs are non linear and asymmetric due to the inherent asymmetry in the molecular structure, with larger currents at negative sample biases. The asymmetry increases with increasing junction resistance due to the asymmetry in the coupling to the leads. This is brought out clearly in the differential conductance, which also shows a gap at the Fermi level. We also studied the effect of order and dynamics in the monolayer on the charge transport and found that competing forces between the electric field, intermolecular interactions, tip-molecule physisorption and substrate-molecule chemisorption impact the transport measurements and its reliability and that the presence of molecular order is very important for reproducible transport measurements. Thus while developing new electronic platforms based on molecules, it is important to have a good control of the molecule-substrate interface, for the devices to perform reliably. While such a control would minimize fluctuations and dynamics in the ensemble, the real challenge is to develop device architectures that are tolerant to fluctuations, since they cannot be totally eliminated in these low dimensional soft systems. Results of temperature dependent STS measurements will also be discussed.

DNA binding of supramolecular mixed-metal complexes

NASA Astrophysics Data System (ADS)

Swavey, Shawn; Williams, Rodd L.; Fang, Zhenglai; Milkevitch, Matthew; Brewer, Karen J.

2001-10-01

The high binding affinity of cisplatin toward DNA has led to its popularity as an anticancer agent. Due to cumulative drug resistance and toxic side effects, researchers are exploring related metallodrugs. Our approach involves the use of supramolecular complexes. These mixed-metal complexes incorporate a reactive platinum moiety bridged by a polyazine ligand to a light absorbing metal-based chromophore. The presence of the light absorber allows excitation of these systems, opening up the possibility of photoactivation. The use of a supramolecular design allows components of the assembly to be varied to enhance device function and light absorbing properties. Aspects of our molecular design process and results on the DNA binding properties for a number of these mixed-metal complexes will be discussed.

Molecular dynamics simulations of structural transformation of perfluorooctane sulfonate (PFOS) at water/rutile interfaces.

PubMed

He, Guangzhi; Zhang, Meiyi; Zhou, Qin; Pan, Gang

2015-09-01

Concentration and salinity conditions are the dominant environmental factors affecting the behavior of perfluorinated compounds (PFCs) on the surfaces of a variety of solid matrices (suspended particles, sediments, and natural minerals). However, the mechanism has not yet been examined at molecular scales. Here, the structural transformation of perfluorooctane sulfonate (PFOS) at water/rutile interfaces induced by changes of the concentration level of PFOS and salt condition was investigated using molecular dynamics (MD) simulations. At low and intermediate concentrations all PFOS molecules directly interacted with the rutile (110) surface mainly by the sulfonate headgroups through electrostatic attraction, yielding a typical monolayer structure. As the concentration of PFOS increased, the molecules aggregated in a complex multi-layered structure, where an irregular assembling configuration was adsorbed on the monolayer structure by the van der Waals interactions between the perfluoroalkyl chains. When adding CaCl2 to the system, the multi-layered structure changed to a monolayer again, indicating that the addition of CaCl2 enhanced the critical concentration value to yield PFOS multilayer assemblies. The divalent Ca(2+) substituted for monovalent K(+) as the bridging counterion in PFOS adsorption. MD simulation may trigger wide applications in study of perfluorinated compounds (PFCs) from atomic/molecular scale. Copyright © 2015 Elsevier Ltd. All rights reserved.

Exploring contribution of intermolecular interactions in supramolecular layered assembly of naphthyridine co-crystals: Insights from Hirshfeld surface analysis of their crystalline states

NASA Astrophysics Data System (ADS)

Seth, Saikat Kumar; Das, Nirmal Kumar; Aich, Krishnendu; Sen, Debabrata; Fun, Hoong-Kun; Goswami, Shyamaprasad

2013-09-01

Co-crystals of 1a and 1b have been prepared by slow evaporation of the solutions of mixtures of 2,7-dimethyl-1,8-naphthyridine (1), urea (a) and thiourea (b). The structures of the complexes are determined by the single crystal X-ray diffraction and a detailed investigation of the crystal packing and classification of intermolecular interactions is presented by means of Hirshfeld surface analysis which is of considerable current interest in crystal engineering. The X-ray study reveals that the co-crystal formers are envisioned to produce N-H⋯N hydrogen bond as well as N-H⋯O/N-H⋯S pair-wise hydrogen bonds and also the weaker aromatic π⋯π interactions which cooperatively take part in the crystal packing. The recurring feature of the self-assembly in the compounds is the appearance of the molecular ribbon through multiple hydrogen bonding which are further stacked into molecular layers by π⋯π stacking interactions. Hirshfeld surface analysis for visually analyzing intermolecular interactions in crystal structures employing molecular surface contours and 2D Fingerprint plots have been used to examine molecular shapes. Crystal structure analysis supported with the Hirshfeld surface and fingerprint plots enabled the identification of the significant intermolecular interactions.

Self-assembly concepts for multicompartment nanostructures

NASA Astrophysics Data System (ADS)

Gröschel, André H.; Müller, Axel H. E.

2015-07-01

Compartmentalization is ubiquitous to many biological and artificial systems, be it for the separate storage of incompatible matter or to isolate transport processes. Advancements in the synthesis of sequential block copolymers offer a variety of tools to replicate natural design principles with tailor-made soft matter for the precise spatial separation of functionalities on multiple length scales. Here, we review recent trends in the self-assembly of amphiphilic block copolymers to multicompartment nanostructures (MCNs) under (semi-)dilute conditions, with special emphasis on ABC triblock terpolymers. The intrinsic immiscibility of connected blocks induces short-range repulsion into discrete nano-domains stabilized by a third, soluble block or molecular additive. Polymer blocks can be synthesized from an arsenal of functional monomers directing self-assembly through packing frustration or response to various fields. The mobility in solution further allows the manipulation of self-assembly processes into specific directions by clever choice of environmental conditions. This review focuses on practical concepts that direct self-assembly into predictable nanostructures, while narrowing particle dispersity with respect to size, shape and internal morphology. The growing understanding of underlying self-assembly mechanisms expands the number of experimental concepts providing the means to target and manipulate progressively complex superstructures.

Peptides at the Interface: Self-Assembly of Amphiphilic Designer Peptides and Their Membrane Interaction Propensity

PubMed Central

2016-01-01

Self-assembling amphiphilic designer peptides have been successfully applied as nanomaterials in biomedical applications. Understanding molecular interactions at the peptide–membrane interface is crucial, since interactions at this site often determine (in)compatibility. The present study aims to elucidate how model membrane systems of different complexity (in particular single-component phospholipid bilayers and lipoproteins) respond to the presence of amphiphilic designer peptides. We focused on two short anionic peptides, V4WD2 and A6YD, which are structurally similar but showed a different self-assembly behavior. A6YD self-assembled into high aspect ratio nanofibers at low peptide concentrations, as evidenced by synchrotron small-angle X-ray scattering and electron microscopy. These supramolecular assemblies coexisted with membranes without remarkable interference. In contrast, V4WD2 formed only loosely associated assemblies over a large concentration regime, and the peptide promoted concentration-dependent disorder on the membrane arrangement. Perturbation effects were observed on both membrane systems although most likely induced by different modes of action. These results suggest that membrane activity critically depends on the peptide’s inherent ability to form highly cohesive supramolecular structures. PMID:27741400

Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

PubMed Central

Lakatos, Lóránt; Csorba, Tibor; Pantaleo, Vitantonio; Chapman, Elisabeth J; Carrington, James C; Liu, Yu-Ping; Dolja, Valerian V; Calvino, Lourdes Fernández; López-Moya, Juan José; Burgyán, József

2006-01-01

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. PMID:16724105

Combining QD-FRET and microfluidics to monitor DNA nanocomplex self-assembly in real-time.

PubMed

Ho, Yi-Ping; Chen, Hunter H; Leong, Kam W; Wang, Tza-Huei

2009-08-26

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships.

Modular synthesis of amphiphilic Janus glycodendrimers and their self-assembly into glycodendrimersomes and other complex architectures with bioactivity to biomedically relevant lectins.

PubMed

Percec, Virgil; Leowanawat, Pawaret; Sun, Hao-Jan; Kulikov, Oleg; Nusbaum, Christopher D; Tran, Tam M; Bertin, Annabelle; Wilson, Daniela A; Peterca, Mihai; Zhang, Shaodong; Kamat, Neha P; Vargo, Kevin; Moock, Diana; Johnston, Eric D; Hammer, Daniel A; Pochan, Darrin J; Chen, Yingchao; Chabre, Yoann M; Shiao, Tze C; Bergeron-Brlek, Milan; André, Sabine; Roy, René; Gabius, Hans-J; Heiney, Paul A

2013-06-19

The modular synthesis of 7 libraries containing 51 self-assembling amphiphilic Janus dendrimers with the monosaccharides D-mannose and D-galactose and the disaccharide D-lactose in their hydrophilic part is reported. These unprecedented sugar-containing dendrimers are named amphiphilic Janus glycodendrimers. Their self-assembly by simple injection of THF or ethanol solution into water or buffer and by hydration was analyzed by a combination of methods including dynamic light scattering, confocal microscopy, cryogenic transmission electron microscopy, Fourier transform analysis, and micropipet-aspiration experiments to assess mechanical properties. These libraries revealed a diversity of hard and soft assemblies, including unilamellar spherical, polygonal, and tubular vesicles denoted glycodendrimersomes, aggregates of Janus glycodendrimers and rodlike micelles named glycodendrimer aggregates and glycodendrimermicelles, cubosomes denoted glycodendrimercubosomes, and solid lamellae. These assemblies are stable over time in water and in buffer, exhibit narrow molecular-weight distribution, and display dimensions that are programmable by the concentration of the solution from which they are injected. This study elaborated the molecular principles leading to single-type soft glycodendrimersomes assembled from amphiphilic Janus glycodendrimers. The multivalency of glycodendrimersomes with different sizes and their ligand bioactivity were demonstrated by selective agglutination with a diversity of sugar-binding protein receptors such as the plant lectins concanavalin A and the highly toxic mistletoe Viscum album L. agglutinin, the bacterial lectin PA-IL from Pseudomonas aeruginosa, and, of special biomedical relevance, human adhesion/growth-regulatory galectin-3 and galectin-4. These results demonstrated the candidacy of glycodendrimersomes as new mimics of biological membranes with programmable glycan ligand presentations, as supramolecular lectin blockers, vaccines, and targeted delivery devices.

Aerospace Power Scholarly Research Program. Delivery Order 0011: Modeling Lithium-Ion Conducting Channel

DTIC Science & Technology

2005-12-01

Brinkmann, D. NMR, DSC, and conductivity study of a poly (ethylene oxide) complex electrolyte: PEO(LiClO4)x, Sol. St. Ionics 1986, 18-19, 295. (27...Electrochemical Characterizations of Dilithium Octacyanophthalocyanine Langmuir - Blodgett films, Langmuir 2002, 18, 2223. 20 ...phthalocyanine (Li2Pc) has been used in this development since it can undergo molecular self-assembly to form the ionic ally conducting channel. The

Solution-Phase Dynamic Assembly of Permanently Interlocked Aryleneethynylene Cages through Alkyne Metathesis

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

Wang, Qi; Yu, Chao; Long, Hai

2015-05-08

Highly stable permanently interlocked aryleneethynylene molecular cages were synthesized from simple triyne monomers using dynamic alkyne metathesis. The interlocked complexes are predominantly formed in the reaction solution in the absence of any recognition motif and were isolated in a pure form using column chromatography. This study is the first example of the thermodynamically controlled solution-phase synthesis of interlocked organic cages with high stability.

Post-Self-Assembly Cross-Linking to Integrate Molecular Nanofibers with Copolymers in Oscillatory Hydrogels

DTIC Science & Technology

2013-05-09

The BZ reaction provides a model system to mimic a variety of complex processes, such as biological morphogenesis, in monodisperse microemulsions .15...surfaces, ion-exchange resins, membranes, and microemulsions . For example, in addition to minimizing the hydrodynamic effects and formation of bubbles...Reaction-Diffusion Microemulsions Reveals Three-Dimensional Tu- ring Patterns. Science (Washington, DC, U.S.) 2011, 331, 1309−1312. (16) Agladze, K. I

Investigation of the heparin-thrombin interaction by dynamic force spectroscopy.

PubMed

Wang, Congzhou; Jin, Yingzi; Desai, Umesh R; Yadavalli, Vamsi K

2015-06-01

The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process. In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin-thrombin interaction from an energy viewpoint at the molecular scale. Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters. An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces. These results contribute to understanding the role of specific and nonspecific forces that drive heparin-thrombin interactions under applied force or flow conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of simulation approach for two-dimensional chiral molecular self-assembly driven by hydrogen bond at the liquid/solid interface

NASA Astrophysics Data System (ADS)

Qin, Yuan; Yao, Man; Hao, Ce; Wan, Lijun; Wang, Yunhe; Chen, Ting; Wang, Dong; Wang, Xudong; Chen, Yonggang

2017-09-01

Two-dimensional (2D) chiral self-assembly system of 5-(benzyloxy)-isophthalic acid derivative/(S)-(+)-2-octanol/highly oriented pyrolytic graphite was studied. A combined density functional theory/molecular mechanics/molecular dynamics (DFT/MM/MD) approach for system of 2D chiral molecular self-assembly driven by hydrogen bond at the liquid/solid interface was thus proposed. Structural models of the chiral assembly were built on the basis of scanning tunneling microscopy (STM) images and simplified for DFT geometry optimization. Merck Molecular Force Field (MMFF) was singled out as the suitable force field by comparing the optimized configurations of MM and DFT. MM and MD simulations for hexagonal unit model which better represented the 2D assemble network were then preformed with MMFF. The adhesion energy, evolution of self-assembly process and characteristic parameters of hydrogen bond were obtained and analyzed. According to the above simulation, the stabilities of the clockwise and counterclockwise enantiomorphous networks were evaluated. The calculational results were supported by STM observations and the feasibility of the simulation method was confirmed by two other systems in the presence of chiral co-absorbers (R)-(-)-2-octanol and achiral co-absorbers 1-octanol. This theoretical simulation method assesses the stability trend of 2D enantiomorphous assemblies with atomic scale and can be applied to the similar hydrogen bond driven 2D chirality of molecular self-assembly system.

Self-assembly behaviours of peptide-drug conjugates: influence of multiple factors on aggregate morphology and potential self-assembly mechanism

NASA Astrophysics Data System (ADS)

Fan, Qin; Ji, Yujie; Wang, Jingjing; Wu, Li; Li, Weidong; Chen, Rui; Chen, Zhipeng

2018-04-01

Peptide-drug conjugates (PDCs) as self-assembly prodrugs have the unique and specific features to build one-component nanomedicines. Supramolecular structure based on PDCs could form various morphologies ranging from nanotube, nanofibre, nanobelt to hydrogel. However, the assembly process of PDCs is too complex to predict or control. Herein, we investigated the effects of extrinsic factors on assembly morphology and the possible formation of nanostructures based on PDCs. To this end, we designed a PDC consisting of hydrophobic drug (S)-ketoprofen (Ket) and valine-glutamic acid dimeric repeats peptide (L-VEVE) to study their assembly behaviour. Our results showed that the critical assembly concentration of Ket-L-VEVE was 0.32 mM in water to form various nanostructures which experienced from micelle, nanorod, nanofibre to nanoribbon. The morphology was influenced by multiple factors including molecular design, assembly time, pH and hydrogen bond inhibitor. On the basis of experimental results, we speculated the possible assembly mechanism of Ket-L-VEVE. The π-π stacking interaction between Ket molecules could serve as an anchor, and hydrogen bonded-induced β-sheets and hydrophilic/hydrophobic balance between L-VEVE peptide play structure-directing role in forming filament-like or nanoribbon morphology. This work provides a new sight to rationally design and precisely control the nanostructure of PDCs based on aromatic fragment.

Self-assembly behaviours of peptide-drug conjugates: influence of multiple factors on aggregate morphology and potential self-assembly mechanism.

PubMed

Fan, Qin; Ji, Yujie; Wang, Jingjing; Wu, Li; Li, Weidong; Chen, Rui; Chen, Zhipeng

2018-04-01

Peptide-drug conjugates (PDCs) as self-assembly prodrugs have the unique and specific features to build one-component nanomedicines. Supramolecular structure based on PDCs could form various morphologies ranging from nanotube, nanofibre, nanobelt to hydrogel. However, the assembly process of PDCs is too complex to predict or control. Herein, we investigated the effects of extrinsic factors on assembly morphology and the possible formation of nanostructures based on PDCs. To this end, we designed a PDC consisting of hydrophobic drug ( S )-ketoprofen (Ket) and valine-glutamic acid dimeric repeats peptide (L-VEVE) to study their assembly behaviour. Our results showed that the critical assembly concentration of Ket-L-VEVE was 0.32 mM in water to form various nanostructures which experienced from micelle, nanorod, nanofibre to nanoribbon. The morphology was influenced by multiple factors including molecular design, assembly time, pH and hydrogen bond inhibitor. On the basis of experimental results, we speculated the possible assembly mechanism of Ket-L-VEVE. The π-π stacking interaction between Ket molecules could serve as an anchor, and hydrogen bonded-induced β-sheets and hydrophilic/hydrophobic balance between L-VEVE peptide play structure-directing role in forming filament-like or nanoribbon morphology. This work provides a new sight to rationally design and precisely control the nanostructure of PDCs based on aromatic fragment.

Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

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

Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi

Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing,more » Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.« less

Interaction of 4.1G and cGMP-gated channels in rod photoreceptor outer segments.

PubMed

Cheng, Christiana L; Molday, Robert S

2013-12-15

In photoreceptors, the assembly of signaling molecules into macromolecular complexes is important for phototransduction and maintaining the structural integrity of rod outer segments (ROSs). However, the molecular composition and formation of these complexes are poorly understood. Using immunoprecipitation and mass spectrometry, 4.1G was identified as a new interacting partner for the cyclic-nucleotide gated (CNG) channels in ROSs. 4.1G is a widely expressed multifunctional protein that plays a role in the assembly and stability of membrane protein complexes. Multiple splice variants of 4.1G were cloned from bovine retina. A smaller splice variant of 4.1G selectively interacted with CNG channels not associated with peripherin-2-CNG channel complex. A combination of truncation studies and domain-binding assays demonstrated that CNG channels selectively interacted with 4.1G through their FERM and CTD domains. Using immunofluorescence, labeling of 4.1G was seen to be punctate and partially colocalized with CNG channels in the ROS. Our studies indicate that 4.1G interacts with a subset of CNG channels in the ROS and implicate this protein-protein interaction in organizing the spatial arrangement of CNG channels in the plasma membrane of outer segments.

The Search Engine for Multi-Proteoform Complexes: An Online Tool for the Identification and Stoichiometry Determination of Protein Complexes.

PubMed

Skinner, Owen S; Schachner, Luis F; Kelleher, Neil L

2016-12-08

Recent advances in top-down mass spectrometry using native electrospray now enable the analysis of intact protein complexes with relatively small sample amounts in an untargeted mode. Here, we describe how to characterize both homo- and heteropolymeric complexes with high molecular specificity using input data produced by tandem mass spectrometry of whole protein assemblies. The tool described is a "search engine for multi-proteoform complexes," (SEMPC) and is available for free online. The output is a list of candidate multi-proteoform complexes and scoring metrics, which are used to define a distinct set of one or more unique protein subunits, their overall stoichiometry in the intact complex, and their pre- and post-translational modifications. Thus, we present an approach for the identification and characterization of intact protein complexes from native mass spectrometry data. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Self-assembled nanogaps for molecular electronics.

PubMed

Tang, Qingxin; Tong, Yanhong; Jain, Titoo; Hassenkam, Tue; Wan, Qing; Moth-Poulsen, Kasper; Bjørnholm, Thomas

2009-06-17

A nanogap for molecular devices was realized using solution-based self-assembly. Gold nanorods were assembled to gold nanoparticle-coated conducting SnO2:Sb nanowires via thiol end-capped oligo(phenylenevinylene)s (OPVs). The molecular gap was easily created by the rigid molecule itself during self-assembly and the gap length was determined by the molecule length. The gold nanorods and gold nanoparticles, respectively covalently bonded at the two ends of the molecule, had very small dimensions, e.g. a width of approximately 20 nm, and hence were expected to minimize the screening effect. The ultra-long conducting SnO2:Sb nanowires provided the bridge to connect one of the electrodes of the molecular device (gold nanoparticle) to the external circuit. The tip of the atomic force microscope (AFM) was contacted onto the other electrode (gold nanorod) for the electrical measurement of the OPV device. The conductance measurement confirmed that the self-assembly of the molecules and the subsequent self-assembly of the gold nanorods was a feasible method for the fabrication of the nanogap of the molecular devices.

Structure of the Polycomb Group protein PCGF1 (NSPC1) in complex with BCOR reveals basis for binding selectivity of PCGF homologs

PubMed Central

Junco, Sarah E.; Wang, Renjing; Gaipa, John C.; Taylor, Alexander B.; Schirf, Virgil; Gearhart, Micah D.; Bardwell, Vivian J.; Demeler, Borries; Hart, P. John; Kim, Chongwoo A.

2014-01-01

Summary Polycomb Group RING finger homologs (PCGF1, 2, 3, 4, 5 and 6) are critical components in the assembly of distinct Polycomb Repression Complex 1 (PRC1) related complexes. Here we identify a protein interaction domain in BCL6 co-repressor, BCOR, which binds the ubiquitin-like RAWUL domain of PCGF1 (NSPC1) and PCGF3 but not of PCGF2 (MEL18) or PCGF4 (BMI1). Because of the selective binding, we have named this domain PCGF Ub-like fold Discriminator (PUFD). The structure of BCOR PUFD bound to PCGF1 reveals 1. that PUFD binds to the same surfaces as observed for a different Polycomb Group RAWUL domain and 2. the ability of PUFD to discriminate among RAWULs stems from the identity of specific residues within these interaction surfaces. These data are the first to show the molecular basis for determining the binding preference for a PCGF homolog, which ultimately helps determine the identity of the larger PRC1-like assembly. PMID:23523425

Host-Guest Assembly of a Molecular Reporter with Chiral Cyanohydrins for Assignment of Absolute Stereochemistry.

PubMed

Gholami, Hadi; Anyika, Mercy; Zhang, Jun; Vasileiou, Chrysoula; Borhan, Babak

2016-06-27

The absolute stereochemistry of cyanohydrins, derived from ketones and aldehydes, is obtained routinely, in a microscale and derivatization-free manner, upon their complexation with Zn-MAPOL, a zincated porphyrin host with a binding pocket comprised of a biphenol core. The host-guest complex leads to observable exciton-coupled circular dichroism (ECCD), the sign of which is easily correlated to the absolute stereochemistry of the bound cyanohydrin. A working model, based on the ECCD signal of cyanohydrins with known configuration, is proposed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Free-energy landscape of protein oligomerization from atomistic simulations

PubMed Central

Barducci, Alessandro; Bonomi, Massimiliano; Prakash, Meher K.; Parrinello, Michele

2013-01-01

In the realm of protein–protein interactions, the assembly process of homooligomers plays a fundamental role because the majority of proteins fall into this category. A comprehensive understanding of this multistep process requires the characterization of the driving molecular interactions and the transient intermediate species. The latter are often short-lived and thus remain elusive to most experimental investigations. Molecular simulations provide a unique tool to shed light onto these complex processes complementing experimental data. Here we combine advanced sampling techniques, such as metadynamics and parallel tempering, to characterize the oligomerization landscape of fibritin foldon domain. This system is an evolutionarily optimized trimerization motif that represents an ideal model for experimental and computational mechanistic studies. Our results are fully consistent with previous experimental nuclear magnetic resonance and kinetic data, but they provide a unique insight into fibritin foldon assembly. In particular, our simulations unveil the role of nonspecific interactions and suggest that an interplay between thermodynamic bias toward native structure and residual conformational disorder may provide a kinetic advantage. PMID:24248370

Free-energy landscape of protein oligomerization from atomistic simulations.

PubMed

Barducci, Alessandro; Bonomi, Massimiliano; Prakash, Meher K; Parrinello, Michele

2013-12-03

In the realm of protein-protein interactions, the assembly process of homooligomers plays a fundamental role because the majority of proteins fall into this category. A comprehensive understanding of this multistep process requires the characterization of the driving molecular interactions and the transient intermediate species. The latter are often short-lived and thus remain elusive to most experimental investigations. Molecular simulations provide a unique tool to shed light onto these complex processes complementing experimental data. Here we combine advanced sampling techniques, such as metadynamics and parallel tempering, to characterize the oligomerization landscape of fibritin foldon domain. This system is an evolutionarily optimized trimerization motif that represents an ideal model for experimental and computational mechanistic studies. Our results are fully consistent with previous experimental nuclear magnetic resonance and kinetic data, but they provide a unique insight into fibritin foldon assembly. In particular, our simulations unveil the role of nonspecific interactions and suggest that an interplay between thermodynamic bias toward native structure and residual conformational disorder may provide a kinetic advantage.

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.

De novo assembly and next-generation sequencing to analyse full-length gene variants from codon-barcoded libraries.

PubMed

Cho, Namjin; Hwang, Byungjin; Yoon, Jung-ki; Park, Sangun; Lee, Joongoo; Seo, Han Na; Lee, Jeewon; Huh, Sunghoon; Chung, Jinsoo; Bang, Duhee

2015-09-21

Interpreting epistatic interactions is crucial for understanding evolutionary dynamics of complex genetic systems and unveiling structure and function of genetic pathways. Although high resolution mapping of en masse variant libraries renders molecular biologists to address genotype-phenotype relationships, long-read sequencing technology remains indispensable to assess functional relationship between mutations that lie far apart. Here, we introduce JigsawSeq for multiplexed sequence identification of pooled gene variant libraries by combining a codon-based molecular barcoding strategy and de novo assembly of short-read data. We first validate JigsawSeq on small sub-pools and observed high precision and recall at various experimental settings. With extensive simulations, we then apply JigsawSeq to large-scale gene variant libraries to show that our method can be reliably scaled using next-generation sequencing. JigsawSeq may serve as a rapid screening tool for functional genomics and offer the opportunity to explore evolutionary trajectories of protein variants.

Efficient 3D kinetic Monte Carlo method for modeling of molecular structure and dynamics.

PubMed

Panshenskov, Mikhail; Solov'yov, Ilia A; Solov'yov, Andrey V

2014-06-30

Self-assembly of molecular systems is an important and general problem that intertwines physics, chemistry, biology, and material sciences. Through understanding of the physical principles of self-organization, it often becomes feasible to control the process and to obtain complex structures with tailored properties, for example, bacteria colonies of cells or nanodevices with desired properties. Theoretical studies and simulations provide an important tool for unraveling the principles of self-organization and, therefore, have recently gained an increasing interest. The present article features an extension of a popular code MBN EXPLORER (MesoBioNano Explorer) aiming to provide a universal approach to study self-assembly phenomena in biology and nanoscience. In particular, this extension involves a highly parallelized module of MBN EXPLORER that allows simulating stochastic processes using the kinetic Monte Carlo approach in a three-dimensional space. We describe the computational side of the developed code, discuss its efficiency, and apply it for studying an exemplary system. Copyright © 2014 Wiley Periodicals, Inc.

Role of the protective antigen octamer in the molecular mechanism of anthrax lethal toxin stabilization in plasma

PubMed Central

Kintzer, Alexander F.; Sterling, Harry J.; Tang, Iok I.; Abdul-Gader, Ali; Miles, Andrew J.; Wallace, B. A.; Williams, Evan R.; Krantz, Bryan A.

2010-01-01

Anthrax is caused by strains of Bacillus anthracis that produce two key virulence factors, anthrax toxin (Atx) and a poly-γ-D-glutamic acid capsule. Atx is comprised of three-proteins: protective antigen (PA) and two enzymes, lethal factor (LF) and edema factor (EF). To disrupt cell function, these components must assemble into holotoxin complexes, which contain either a ring-shaped homooctameric or homoheptameric PA oligomer bound to multiple copies of either LF and/or EF, producing lethal toxin (LT), edema toxin, or mixtures thereof. Once a host cell endocytoses these complexes, PA converts into a membrane-inserted channel that translocates LF and EF into the cytosol. LT may assemble on host cell surfaces or extracellularly in plasma. We show that under physiological conditions in bovine plasma that LT complexes containing heptameric PA aggregate and inactivate more readily than LT complexes containing octameric PA. LT complexes containing octameric PA possess enhanced stability, channel forming activity, and macrophage cytotoxicity relative to those containing heptameric PA. Under physiological conditions, multiple biophysical probes reveal that heptameric PA can prematurely adopt the channel conformation, but octameric PA complexes remain in their soluble prechannel configuration allowing them to resist aggregation and inactivation. We conclude that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that may circulate freely in the blood. PMID:20433851

Use of π-π forces to steer the assembly of a NTA complex of Cu(II) into hydrogen bonded supramolecular layers (H 3NTA = nitrilotriacetic acid)

NASA Astrophysics Data System (ADS)

Dey, Biswajit; Choudhury, Somnath Ray; Suresh, Eringathodi; Jana, Atish Dipankar; Mukhopadhyay, Subrata

2009-03-01

We propose a crystal engineering principle where we show that it might be possible to direct the organization of molecular complexes into hydrogen bonded supramolecular layers through the use of suitable co-ligands possessing both the hydrogen-bonding as well as π-π stacking capability. This principle has been tested for the organization of [Cu(NTA) 2] units (H 3NTA = nitrilotriacetic acid, N(CH 2CO 2H) 3) in the molecular complex with formula (2-A-PH) 4[Cu(NTA) 2]·6H 2O ( 1), where 2-A-PH is protonated 2-amino-4-picoline. In 1, the 2-amino-4-picoline co-ligands have been utilized to direct the organization of [Cu(NTA) 2] units into hydrogen bonded layers. The linear stacking of π-π bonded protonated 2-amino-4-picoline molecules can be thought as the influencing agent for the organization of [Cu(NTA) 2] units into hydrogen bonded layers.

saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription.

PubMed

Portnoy, Victoria; Lin, Szu Hua Sharon; Li, Kathy H; Burlingame, Alma; Hu, Zheng-Hui; Li, Hao; Li, Long-Cheng

2016-03-01

Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

Structural characterisation of medically relevant protein assemblies by integrating mass spectrometry with computational modelling.

PubMed

Politis, Argyris; Schmidt, Carla

2018-03-20

Structural mass spectrometry with its various techniques is a powerful tool for the structural elucidation of medically relevant protein assemblies. It delivers information on the composition, stoichiometries, interactions and topologies of these assemblies. Most importantly it can deal with heterogeneous mixtures and assemblies which makes it universal among the conventional structural techniques. In this review we summarise recent advances and challenges in structural mass spectrometric techniques. We describe how the combination of the different mass spectrometry-based methods with computational strategies enable structural models at molecular levels of resolution. These models hold significant potential for helping us in characterizing the function of protein assemblies related to human health and disease. In this review we summarise the techniques of structural mass spectrometry often applied when studying protein-ligand complexes. We exemplify these techniques through recent examples from literature that helped in the understanding of medically relevant protein assemblies. We further provide a detailed introduction into various computational approaches that can be integrated with these mass spectrometric techniques. Last but not least we discuss case studies that integrated mass spectrometry and computational modelling approaches and yielded models of medically important protein assembly states such as fibrils and amyloids. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

A Solomon link through an interwoven molecular grid.

PubMed

Beves, Jonathon E; Danon, Jonathan J; Leigh, David A; Lemonnier, Jean-François; Vitorica-Yrezabal, Iñigo J

2015-06-22

A molecular Solomon link was synthesized through the assembly of an interwoven molecular grid consisting of four bis(benzimidazolepyridyl)benzthiazolo[5,4-d]thiazole ligands and four zinc(II), iron(II), or cobalt(II) cations, followed by ring-closing olefin metathesis. NMR spectroscopy, mass spectrometry, and X-ray crystallography confirmed the doubly interlocked topology, and subsequent demetalation afforded the wholly organic Solomon link. The synthesis, in which each metal ion defines the crossing point of two ligand strands, suggests that interwoven molecular grids should be useful scaffolds for the rational construction of other topologically complex structures. © 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 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Impact of disease-causing mutations on inter-domain interactions in cMyBP-C: a steered molecular dynamics study.

PubMed

Krishnamoorthy, Navaneethakrishnan; Gajendrarao, Poornima; Olivotto, Iacopo; Yacoub, Magdi

2017-07-01

The molecular interactions of the sarcomeric proteins are essential in the regulation of various cardiac functions. Mutations in the gene MYBPC3 coding for cardiac myosin-binding protein-C (cMyBP-C), a multi-domain protein, are the most common cause of hypertrophic cardiomyopathy (HCM). The N-terminal complex, C1-motif-C2 is a central region in cMyBP-C for the regulation of cardiac muscle contraction. However, the mechanism of binding/unbinding of this complex during health and disease is unknown. Here, we study possible mechanisms of unbinding using steered molecular dynamics simulations for the complex in the wild type, in single mutations (E258K in C1, E441K in C2), as well as in a double mutation (E258K in C1 + E441K in C2), which are associated with severe HCM. The observed molecular events and the calculation of force utilized for the unbinding suggest the following: (i) double mutation can encourage the formation of rigid complex that required large amount of force and long-time to unbind, (ii) C1 appears to start to unbind ahead of C2 regardless of the mutation, and (iii) unbinding of C2 requires larger amount of force than C1. This molecular insight suggests that key HCM-causing mutations might significantly modify the native affinity required for the assembly of the domains in cMyBP-C, which is essential for normal cardiac function.

Electron transfer and conformational change in complexes of trimethylamine dehydrogenase and electron transferring flavoprotein.

PubMed

Jones, Matthew; Talfournier, Francois; Bobrov, Anton; Grossmann, J Günter; Vekshin, Nikolai; Sutcliffe, Michael J; Scrutton, Nigel S

2002-03-08

The trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH.ETF) electron transfer complex has been studied by fluorescence and absorption spectroscopies. These studies indicate that a series of conformational changes occur during the assembly of the TMADH.ETF electron transfer complex and that the kinetics of assembly observed with mutant TMADH (Y442F/L/G) or ETF (alpha R237A) complexes are much slower than are the corresponding rates of electron transfer in these complexes. This suggests that electron transfer does not occur in the thermodynamically most favorable state (which takes too long to form), but that one or more metastable states (which are formed more rapidly) are competent in transferring electrons from TMADH to ETF. Additionally, fluorescence spectroscopy studies of the TMADH.ETF complex indicate that ETF undergoes a stable conformational change (termed structural imprinting) when it interacts transiently with TMADH to form a second, distinct, structural form. The mutant complexes compromise imprinting of ETF, indicating a dependence on the native interactions present in the wild-type complex. The imprinted form of semiquinone ETF exhibits an enhanced rate of electron transfer to the artificial electron acceptor, ferricenium. Overall molecular conformations as probed by small-angle x-ray scattering studies are indistinguishable for imprinted and non-imprinted ETF, suggesting that changes in structure likely involve confined reorganizations within the vicinity of the FAD. Our results indicate a series of conformational events occur during the assembly of the TMADH.ETF electron transfer complex, and that the properties of electron transfer proteins can be affected lastingly by transient interaction with their physiological redox partners. This may have significant implications for our understanding of biological electron transfer reactions in vivo, because ETF encounters TMADH at all times in the cell. Our studies suggest that caution needs to be exercised in extrapolating the properties of in vitro interprotein electron transfer reactions to those occurring in vivo.

Native Mass Spectrometry: What is in the Name?

NASA Astrophysics Data System (ADS)

Leney, Aneika C.; Heck, Albert J. R.

2017-01-01

Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein-protein and protein-ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as "native MS," has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure-function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by "native MS," when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions.

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

PubMed

Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel; Søgaard-Andersen, Lotte; Mignot, Tâm

2015-07-20

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature. © 2015 Treuner-Lange et al.

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions

PubMed Central

Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M.; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel

2015-01-01

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate–bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA–MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein–cytoskeleton interactions are a universally conserved feature. PMID:26169353

Two Heteromeric Kinesin Complexes in Chemosensory Neurons and Sensory Cilia of Caenorhabditis elegans

PubMed Central

Signor, Dawn; Wedaman, Karen P.; Rose, Lesilee S.; Scholey, Jonathan M.

1999-01-01

Chemosensation in the nervous system of the nematode Caenorhabditis elegans depends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia. PMID:9950681

Tetrahedral Arrangements of Perylene Bisimide Columns via Supramolecular Orientational Memory.

PubMed

Sahoo, Dipankar; Peterca, Mihai; Aqad, Emad; Partridge, Benjamin E; Heiney, Paul A; Graf, Robert; Spiess, Hans W; Zeng, Xiangbing; Percec, Virgil

2017-01-24

Chiral, shape, and liquid crystalline memory effects are well-known to produce commercial macroscopic materials with important applications as springs, sensors, displays, and memory devices. A supramolecular orientational memory effect that provides complex nanoscale arrangements was only recently reported. This supramolecular orientational memory was demonstrated to preserve the molecular orientation and packing within supramolecular units of a self-assembling cyclotriveratrylene crown at the nanoscale upon transition between its columnar hexagonal and Pm3̅n cubic periodic arrays. Here we report the discovery of supramolecular orientational memory in a dendronized perylene bisimide (G2-PBI) that self-assembles into tetrameric crowns and subsequently self-organizes into supramolecular columns and spheres. This supramolecular orientation memory upon transition between columnar hexagonal and body-centered cubic (BCC) mesophases preserves the 3-fold cubic [111] orientations rather than the 4-fold [100] axes, generating an unusual tetrahedral arrangement of supramolecular columns. These results indicate that the supramolecular orientational memory concept may be general for periodic arrays of self-assembling dendrons and dendrimers as well as for other periodic and quasiperiodic nanoscale organizations comprising supramolecular spheres, generated from other organized complex soft matter including block copolymers and surfactants.

Bipartite design of a self-fibrillating protein copolymer with nanopatterned peptide display capabilities.

PubMed

Bruning, Marc; Kreplak, Laurent; Leopoldseder, Sonja; Müller, Shirley A; Ringler, Philippe; Duchesne, Laurence; Fernig, David G; Engel, Andreas; Ucurum-Fotiadis, Zöhre; Mayans, Olga

2010-11-10

The development of biomatrices for technological and biomedical applications employs self-assembled scaffolds built from short peptidic motifs. However, biopolymers composed of protein domains would offer more varied molecular frames to introduce finer and more complex functionalities in bioreactive scaffolds using bottom-up approaches. Yet, the rules governing the three-dimensional organization of protein architectures in nature are complex and poorly understood. As a result, the synthetic fabrication of ordered protein association into polymers poses major challenges to bioengineering. We have now fabricated a self-assembling protein nanofiber with predictable morphologies and amenable to bottom-up customization, where features supporting function and assembly are spatially segregated. The design was inspired by the cross-linking of titin filaments by telethonin in the muscle sarcomere. The resulting fiber is a two-protein system that has nanopatterned peptide display capabilities as shown by the recruitment of functionalized gold nanoparticles at regular intervals of ∼ 5 nm, yielding a semiregular linear array over micrometers. This polymer promises the uncomplicated display of biologically active motifs to selectively bind and organize matter in the fine nanoscale. Further, its conceptual design has high potential for controlled plurifunctionalization.

Differential Regulation of Elastic Fiber Formation by Fibulin-4 and -5*

PubMed Central

Choudhury, Rawshan; McGovern, Amanda; Ridley, Caroline; Cain, Stuart A.; Baldwin, Andrew; Wang, Ming-Chuan; Guo, Chun; Mironov, Aleksandr; Drymoussi, Zoe; Trump, Dorothy; Shuttleworth, Adrian; Baldock, Clair; Kielty, Cay M.

2009-01-01

Fibulin-4 and -5 are extracellular glycoproteins with essential non-compensatory roles in elastic fiber assembly. We have determined how they interact with tropoelastin, lysyl oxidase, and fibrillin-1, thereby revealing how they differentially regulate assembly. Strong binding between fibulin-4 and lysyl oxidase enhanced the interaction of fibulin-4 with tropoelastin, forming ternary complexes that may direct elastin cross-linking. In contrast, fibulin-5 did not bind lysyl oxidase strongly but bound tropoelastin in terminal and central regions and could concurrently bind fibulin-4. Both fibulins differentially bound N-terminal fibrillin-1, which strongly inhibited their binding to lysyl oxidase and tropoelastin. Knockdown experiments revealed that fibulin-5 controlled elastin deposition on microfibrils, although fibulin-4 can also bind fibrillin-1. These experiments provide a molecular account of the distinct roles of fibulin-4 and -5 in elastic fiber assembly and how they act in concert to chaperone cross-linked elastin onto microfibrils. PMID:19570982

A component-based software environment for visualizing large macromolecular assemblies.

PubMed

Sanner, Michel F

2005-03-01

The interactive visualization of large biological assemblies poses a number of challenging problems, including the development of multiresolution representations and new interaction methods for navigating and analyzing these complex systems. An additional challenge is the development of flexible software environments that will facilitate the integration and interoperation of computational models and techniques from a wide variety of scientific disciplines. In this paper, we present a component-based software development strategy centered on the high-level, object-oriented, interpretive programming language: Python. We present several software components, discuss their integration, and describe some of their features that are relevant to the visualization of large molecular assemblies. Several examples are given to illustrate the interoperation of these software components and the integration of structural data from a variety of experimental sources. These examples illustrate how combining visual programming with component-based software development facilitates the rapid prototyping of novel visualization tools.

Molecular engineering of chiral colloidal liquid crystals using DNA origami

NASA Astrophysics Data System (ADS)

Siavashpouri, Mahsa; Wachauf, Christian H.; Zakhary, Mark J.; Praetorius, Florian; Dietz, Hendrik; Dogic, Zvonimir

2017-08-01

Establishing precise control over the shape and the interactions of the microscopic building blocks is essential for design of macroscopic soft materials with novel structural, optical and mechanical properties. Here, we demonstrate robust assembly of DNA origami filaments into cholesteric liquid crystals, one-dimensional supramolecular twisted ribbons and two-dimensional colloidal membranes. The exquisite control afforded by the DNA origami technology establishes a quantitative relationship between the microscopic filament structure and the macroscopic cholesteric pitch. Furthermore, it also enables robust assembly of one-dimensional twisted ribbons, which behave as effective supramolecular polymers whose structure and elastic properties can be precisely tuned by controlling the geometry of the elemental building blocks. Our results demonstrate the potential synergy between DNA origami technology and colloidal science, in which the former allows for rapid and robust synthesis of complex particles, and the latter can be used to assemble such particles into bulk materials.

Molecular engineering of chiral colloidal liquid crystals using DNA origami.

PubMed

Siavashpouri, Mahsa; Wachauf, Christian H; Zakhary, Mark J; Praetorius, Florian; Dietz, Hendrik; Dogic, Zvonimir

2017-08-01

Establishing precise control over the shape and the interactions of the microscopic building blocks is essential for design of macroscopic soft materials with novel structural, optical and mechanical properties. Here, we demonstrate robust assembly of DNA origami filaments into cholesteric liquid crystals, one-dimensional supramolecular twisted ribbons and two-dimensional colloidal membranes. The exquisite control afforded by the DNA origami technology establishes a quantitative relationship between the microscopic filament structure and the macroscopic cholesteric pitch. Furthermore, it also enables robust assembly of one-dimensional twisted ribbons, which behave as effective supramolecular polymers whose structure and elastic properties can be precisely tuned by controlling the geometry of the elemental building blocks. Our results demonstrate the potential synergy between DNA origami technology and colloidal science, in which the former allows for rapid and robust synthesis of complex particles, and the latter can be used to assemble such particles into bulk materials.

Weaving colloidal webs around droplets: spontaneous assembly of extended colloidal networks encasing microfluidic droplet ensembles.

PubMed

Zheng, Lu; Ho, Leon Yoon; Khan, Saif A

2016-10-26

The ability to form transient, self-assembling solid networks that 'cocoon' emulsion droplets on-demand allows new possibilities in the rapidly expanding area of microfluidic droplet-based materials science. In this communication, we demonstrate the spontaneous formation of extended colloidal networks that encase large microfluidic droplet ensembles, thus completely arresting droplet motion and effectively isolating each droplet from others in the ensemble. To do this, we employ molecular inclusion complexes of β-cyclodextrin, which spontaneously form and assemble into colloidal solids at the droplet interface and beyond, via the outward diffusion of a guest molecule (dichloromethane) from the droplets. We illustrate the advantage of such transient network-based droplet stabilization in the area of pharmaceutical crystallization, where we are able to fabricate monodisperse spherical crystalline microgranules of 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile (ROY), a model hydrophobic drug, with a dramatic enhancement of particle properties compared to conventional methods.

Self-organisation of dodeca-dendronized fullerene into supramolecular discs and helical columns containing a nanowire-like core† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc00449g Click here for additional data file.

PubMed Central

Guerra, Sebastiano; Iehl, Julien; Holler, Michel; Peterca, Mihai; Wilson, Daniela A.; Partridge, Benjamin E.; Zhang, Shaodong

2015-01-01

Twelve chiral and achiral self-assembling dendrons have been grafted onto a [60]fullerene hexa-adduct core by copper-catalyzed alkyne azide “click” cycloaddition. The structure adopted by these compounds was determined by the self-assembling peripheral dendrons. These twelve dendrons mediate the self-organisation of the dendronized [60]fullerene into a disc-shaped structure containing the [60]fullerene in the centre. The fullerene-containing discs self-organise into helical supramolecular columns with a fullerene nanowire-like core, forming a 2D columnar hexagonal periodic array. These unprecedented supramolecular structures and their assemblies are expected to provide new developments in chiral complex molecular systems and their application to organic electronics and solar cells. PMID:29142695

Synthesis, delivery and regulation of eukaryotic heme and Fe-S cluster cofactors.

PubMed

Barupala, Dulmini P; Dzul, Stephen P; Riggs-Gelasco, Pamela Jo; Stemmler, Timothy L

2016-02-15

In humans, the bulk of iron in the body (over 75%) is directed towards heme- or Fe-S cluster cofactor synthesis, and the complex, highly regulated pathways in place to accomplish biosynthesis have evolved to safely assemble and load these cofactors into apoprotein partners. In eukaryotes, heme biosynthesis is both initiated and finalized within the mitochondria, while cellular Fe-S cluster assembly is controlled by correlated pathways both within the mitochondria and within the cytosol. Iron plays a vital role in a wide array of metabolic processes and defects in iron cofactor assembly leads to human diseases. This review describes progress towards our molecular-level understanding of cellular heme and Fe-S cluster biosynthesis, focusing on the regulation and mechanistic details that are essential for understanding human disorders related to the breakdown in these essential pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Cryo-EM structure of a late pre-40S ribosomal subunit from Saccharomyces cerevisiae

PubMed Central

Schmidt, Christian; Berninghausen, Otto; Becker, Thomas

2017-01-01

Mechanistic understanding of eukaryotic ribosome formation requires a detailed structural knowledge of the numerous assembly intermediates, generated along a complex pathway. Here, we present the structure of a late pre-40S particle at 3.6 Å resolution, revealing in molecular detail how assembly factors regulate the timely folding of pre-18S rRNA. The structure shows that, rather than sterically blocking 40S translational active sites, the associated assembly factors Tsr1, Enp1, Rio2 and Pno1 collectively preclude their final maturation, thereby preventing untimely tRNA and mRNA binding and error prone translation. Moreover, the structure explains how Pno1 coordinates the 3’end cleavage of the 18S rRNA by Nob1 and how the late factor’s removal in the cytoplasm ensures the structural integrity of the maturing 40S subunit. PMID:29155690

Cancer cell death induced by the intracellular self-assembly of an enzyme-responsive supramolecular gelator.

PubMed

Tanaka, Akiko; Fukuoka, Yuki; Morimoto, Yuka; Honjo, Takafumi; Koda, Daisuke; Goto, Masahiro; Maruyama, Tatsuo

2015-01-21

We report cancer cell death initiated by the intracellular molecular self-assembly of a peptide lipid, which was derived from a gelator precursor. The gelator precursor was designed to form nanofibers via molecular self-assembly, after cleavage by a cancer-related enzyme (matrix metalloproteinase-7, MMP-7), leading to hydrogelation. The gelator precursor exhibited remarkable cytotoxicity to five different cancer cell lines, while the precursor exhibited low cytotoxicity to normal cells. Cancer cells secrete excessive amounts of MMP-7, which converted the precursor into a supramolecular gelator prior to its uptake by the cells. Once inside the cells, the supramolecular gelator formed a gel via molecular self-assembly, exerting vital stress on the cancer cells. The present study thus describes a new drug where molecular self-assembly acts as the mechanism of cytotoxicity.

Assembly, molecular organization, and membrane-binding properties of development-specific septins

PubMed Central

Garcia, Galo; Finnigan, Gregory C.; Heasley, Lydia R.; Sterling, Sarah M.; Aggarwal, Adeeti; Pearson, Chad G.

2016-01-01

Septin complexes display remarkable plasticity in subunit composition, yet how a new subunit assembled into higher-order structures confers different functions is not fully understood. Here, this question is addressed in budding yeast, where during meiosis Spr3 and Spr28 replace the mitotic septin subunits Cdc12 and Cdc11 (and Shs1), respectively. In vitro, the sole stable complex that contains both meiosis-specific septins is a linear Spr28–Spr3–Cdc3–Cdc10–Cdc10–Cdc3–Spr3–Spr28 hetero-octamer. Only coexpressed Spr3 and Spr28 colocalize with Cdc3 and Cdc10 in mitotic cells, indicating that incorporation requires a Spr28-Spr3 protomer. Unlike their mitotic counterparts, Spr28-Spr3–capped rods are unable to form higher-order structures in solution but assemble to form long paired filaments on lipid monolayers containing phosphatidylinositol-4,5-bisphosphate, mimicking presence of this phosphoinositide in the prospore membrane. Spr28 and Spr3 fail to rescue the lethality of a cdc11Δ cdc12Δ mutant, and Cdc11 and Cdc12 fail to restore sporulation proficiency to spr3Δ/spr3Δ spr28Δ/spr28Δ diploids. Thus, specific meiotic and mitotic subunits endow septin complexes with functionally distinct properties. PMID:26929450

Living matter—nexus of physics and biology in the 21st century

PubMed Central

Gardel, Margaret L.

2012-01-01

Cells are made up of complex assemblies of cytoskeletal proteins that facilitate force transmission from the molecular to cellular scale to regulate cell shape and force generation. The “living matter” formed by the cytoskeleton facilitates versatile and robust behaviors of cells, including their migration, adhesion, division, and morphology, that ultimately determine tissue architecture and mechanics. Elucidating the underlying physical principles of such living matter provides great opportunities in both biology and physics. For physicists, the cytoskeleton provides an exceptional toolbox to study materials far from equilibrium. For biologists, these studies will provide new understanding of how molecular-scale processes determine cell morphological changes. PMID:23112229

The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin-Coregulated Pilus.

PubMed

Ng, Dixon; Harn, Tony; Altindal, Tuba; Kolappan, Subramania; Marles, Jarrad M; Lala, Rajan; Spielman, Ingrid; Gao, Yang; Hauke, Caitlyn A; Kovacikova, Gabriela; Verjee, Zia; Taylor, Ronald K; Biais, Nicolas; Craig, Lisa

2016-12-01

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system.

Tailored Surfaces/Assemblies for Molecular Plasmonics and Plasmonic Molecular Electronics.

PubMed

Lacroix, Jean-Christophe; Martin, Pascal; Lacaze, Pierre-Camille

2017-06-12

Molecular plasmonics uses and explores molecule-plasmon interactions on metal nanostructures for spectroscopic, nanophotonic, and nanoelectronic devices. This review focuses on tailored surfaces/assemblies for molecular plasmonics and describes active molecular plasmonic devices in which functional molecules and polymers change their structural, electrical, and/or optical properties in response to external stimuli and that can dynamically tune the plasmonic properties. We also explore an emerging research field combining molecular plasmonics and molecular electronics.

In situ synthesis of di-n-butyl l-tartrate-boric acid complex chiral selector and its application in chiral microemulsion electrokinetic chromatography.

PubMed

Hu, Shaoqiang; Chen, Yonglei; Zhu, Huadong; Zhu, Jinhua; Yan, Na; Chen, Xingguo

2009-11-06

A novel procedure for in situ assembling a complex chiral selector, di-n-butyl l-tartrate-boric acid complex, by the reaction of di-n-butyl l-tartrate with boric acid in a running buffer was reported and its application in the enantioseparation of beta-blockers and structural related compounds by chiral microemulsion electrokinetic chromatography (MEEKC) has been demonstrated. In order to achieve a good enantioseparation, the effect of dibutyl l-tartrate and sodium tetraborate concentration, surfactant identity and concentration, cosurfactant, buffer pH and composition, organic modifiers, as well as applied voltage and capillary length were investigated. Ten pairs of enantiomers that could not be separated with only dibutyl l-tartrate, obtained good chiral separation using the complex chiral selector; among them, seven pairs could be baseline resolved under optimized experimental conditions. The fixation of chiral centers by the formation of five-membered rings, and being oppositely charged with basic analytes were thought to be the key factors giving the complex chiral selector a superior chiral recognition capability. The effect of the molecular structure of analytes on enantioseparation was discussed in terms of molecular interaction.

Development of self-assembled molecular structures on polymeric surfaces and their applications as ultrasonically responsive barrier coatings for on-demand, pulsatile drug delivery

NASA Astrophysics Data System (ADS)

Kwok, Connie Sau-Kuen

Nature in the form of DNA, proteins, and cells has the remarkable ability to interact with its environment by processing biological information through specific molecular recognition at the interface. As such, materials that are capable of triggering an appropriate biological response need to be engineered at the biomaterial surface. Chemically and structurally well-defined self-assembled monolayers (SAMs), biomimetics of the lipid bilayer in cell membranes, have been created and studied mostly on rigid metallic surfaces. This dissertation is motivated by the lack of methods to generate a molecularly designed surface for biomedical polymers and thus provides an enabling technology to engineer a polymeric surface precisely at a molecular and cellular level. To take this innovation one step further, we demonstrated that such self-assembled molecular structure coated on drug-containing polymeric devices could act as a stimulus-responsive barrier for controlled drug delivery. A simple, one-step procedure for generating ordered, crystalline methylene chains on polymeric surfaces via urethane linkages was successfully developed. The self-assemblies and molecular structures of these crystalline methylene chains are comparable to the SAM model surfaces, as evidenced by various surface characterization techniques (XPS, TOF-SIMS, and FTIR-ATR). For the first time, these self-assembled molecular structures are shown to function collectively as an ultrasound-responsive barrier membrane for pulsatile drug delivery, including delivery of low-molecular-weight ciprofloxacin and high-molecular-weight insulin. Encouraging results, based on the insulin-activated deoxyglucose uptakes in adipocytes, indicate that the released insulin remained biologically active. Both chemical and acoustic analyses suggest that the ultrasound-assisted release mechanism is primarily induced by transient cavitation, which causes temporary disruption of the self-assembled overlayer, and thus allows temporal release of the encapsulated drugs. In addition to acoustic energy, self-assembled surfaces experience order-disorder transition and have a transition temperature higher than body temperature if longer alkyl chains (C18) are used. The C18-assembled surface barrier membrane exhibits a relatively superior impermeable coating than the shorter C12 chains. The versatility of derivatizing the terminal groups of the self-assembled molecular structures is illustrated by attaching poly (ethyleneoxide) oligomers to the alkyl chains to minimize nonspecific protein adsorption. This study lays an important foundation for future work in conjugating other biomolecules to develop surface-based diagnostics and biomaterials. With much success, this original research work of forming self-assembled crystalline structures on synthetic materials still allows for numerous opportunities for new applications and possibly even more new discoveries.

Guided molecular self-assembly: a review of recent efforts

NASA Astrophysics Data System (ADS)

Huie, Jiyun C.

2003-04-01

This paper serves as an introductory review of significant and novel successes achieved in the fields of nanotechnology, particularly in the formation of nanostructures using guided molecular self-assembly methods. Self-assembly is a spontaneous process by which molecules and nanophase entities may materialize into organized aggregates or networks. Through various interactive mechanisms of self-assembly, such as electrostatics, chemistry, surface properties, and via other mediating agents, the technique proves indispensable to recent functional materials and device realizations. The discussion will extend to spontaneous and Langmuir-Blodgett formation of self-assembled monolayers on various substrates, and a number of different categories of self-assembly techniques based on the type of interaction exploited. Combinatorial techniques, known as soft lithography, of micro-contact printing and dip-pen nanolithography, which can be effectively used to up-size nanostructured molecular assemblies to submicrometer and micrometer scale patterns, will also be mentioned.

The Assembly Pathway of Mitochondrial Respiratory Chain Complex I.

PubMed

Guerrero-Castillo, Sergio; Baertling, Fabian; Kownatzki, Daniel; Wessels, Hans J; Arnold, Susanne; Brandt, Ulrich; Nijtmans, Leo

2017-01-10

Mitochondrial complex I is the largest integral membrane enzyme of the respiratory chain and consists of 44 different subunits encoded in the mitochondrial and nuclear genome. Its biosynthesis is a highly complicated and multifaceted process involving at least 14 additional assembly factors. How these subunits assemble into a functional complex I and where the assembly factors come into play is largely unknown. Here, we applied a dynamic complexome profiling approach to elucidate the assembly of human mitochondrial complex I and its further incorporation into respiratory chain supercomplexes. We delineate the stepwise incorporation of all but one subunit into a series of distinct assembly intermediates and their association with known and putative assembly factors, which had not been implicated in this process before. The resulting detailed and comprehensive model of complex I assembly is fully consistent with recent structural data and the remarkable modular architecture of this multiprotein complex. Copyright © 2017 Elsevier Inc. All rights reserved.

Understanding the Function of Tuberous Sclerosis Complex Genes in Neural Development: Roles in Synapse Assembly and Axon Guidance

DTIC Science & Technology

2012-02-01

The goal of our project was to use the fruitfly Drosophila melanogaster , to identify molecular mechanisms affecting nervous...includes tuberous sclerosis 1 and 2 (TSC1 and TSC2). This pathway is fully represented in the fruitfly Drosophila melanogaster and we took advantage...provided in the Appendix. 8 KEY RESEARCH ACCOMPLISHMENTS:  The goal of our project was to use the fruitfly Drosophila melanogaster ,

Programmable self-assembly of three-dimensional nanostructures from 104 unique components

PubMed Central

Ong, Luvena L.; Hanikel, Nikita; Yaghi, Omar K.; Grun, Casey; Strauss, Maximilian T.; Bron, Patrick; Lai-Kee-Him, Josephine; Schueder, Florian; Wang, Bei; Wang, Pengfei; Kishi, Jocelyn Y.; Myhrvold, Cameron A.; Zhu, Allen; Jungmann, Ralf

2017-01-01

Nucleic acids (DNA and RNA) are widely used to construct nanoscale structures with ever increasing complexity1–14 for possible applications in fields as diverse as structural biology, biophysics, synthetic biology and photonics. The nanostructures are formed through one-pot self-assembly, with early examples typically containing on the order of 10 unique DNA strands. The introduction of DNA origami4, which uses many staple strands to fold one long scaffold strand into a desired structure, gave access to kilo- to mega-dalton nanostructures containing about 102 unique DNA strands6,7,10,13 . Aiming for even larger DNA origami structures is in principle possible15,16, but faces the challenge of having to manufacture and route an increasingly long scaffold strand. An alternative and in principle more readily scalable approach uses DNA brick assembly8,9, which doesn’t need a scaffold and instead uses hundreds of short DNA brick strands that self-assemble according to specific inter-brick interactions. First-generation bricks used to create 3D structures are 32-nt long with four 8-nt binding domains that directed 102 distinct bricks into well-formed assemblies, but attempts to create larger structures encountered practical challenges and had limited success.9 Here we show that a new generation of DNA bricks with longer binding domains makes it possible to self-assemble 0.1 – 1 giga-dalton three-dimensional nanostructures from 104 unique components, including a 0.5 giga-dalton cuboid containing 30,000 unique bricks and a 1 giga-dalton rotationally symmetric tetramer. We also assemble a cuboid containing 10,000 bricks and 20,000 uniquely addressable ‘nano-voxels’ that serves as a molecular canvas for three-dimensional sculpting, with introduction of sophisticated user-prescribed 3D cavities yielding structures such as letters, a complex helicoid and a teddy bear. We anticipate that, with further optimization, even larger assemblies might be accessible and prove useful as scaffolds or for positioning functional components. PMID:29219968

Kinetics of DNA Tile Dimerization

PubMed Central

2015-01-01

Investigating how individual molecular components interact with one another within DNA nanoarchitectures, both in terms of their spatial and temporal interactions, is fundamentally important for a better understanding of their physical behaviors. This will provide researchers with valuable insight for designing more complex higher-order structures that can be assembled more efficiently. In this report, we examined several spatial factors that affect the kinetics of bivalent, double-helical (DH) tile dimerization, including the orientation and number of sticky ends (SEs), the flexibility of the double helical domains, and the size of the tiles. The rate constants we obtained confirm our hypothesis that increased nucleation opportunities and well-aligned SEs accelerate tile–tile dimerization. Increased flexibility in the tiles causes slower dimerization rates, an effect that can be reversed by introducing restrictions to the tile flexibility. The higher dimerization rates of more rigid tiles results from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. We believe that the results presented here will assist in improved implementation of DNA tile based algorithmic self-assembly, DNA based molecular robotics, and other specific nucleic acid systems, and will provide guidance to design and assembly processes to improve overall yield and efficiency. PMID:24794259

Kinetics of DNA tile dimerization.

PubMed

Jiang, Shuoxing; Yan, Hao; Liu, Yan

2014-06-24

Investigating how individual molecular components interact with one another within DNA nanoarchitectures, both in terms of their spatial and temporal interactions, is fundamentally important for a better understanding of their physical behaviors. This will provide researchers with valuable insight for designing more complex higher-order structures that can be assembled more efficiently. In this report, we examined several spatial factors that affect the kinetics of bivalent, double-helical (DH) tile dimerization, including the orientation and number of sticky ends (SEs), the flexibility of the double helical domains, and the size of the tiles. The rate constants we obtained confirm our hypothesis that increased nucleation opportunities and well-aligned SEs accelerate tile-tile dimerization. Increased flexibility in the tiles causes slower dimerization rates, an effect that can be reversed by introducing restrictions to the tile flexibility. The higher dimerization rates of more rigid tiles results from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. We believe that the results presented here will assist in improved implementation of DNA tile based algorithmic self-assembly, DNA based molecular robotics, and other specific nucleic acid systems, and will provide guidance to design and assembly processes to improve overall yield and efficiency.

Naturally engineered glycolipid biosurfactants leading to distinctive self-assembled structures.

PubMed

Imura, Tomohiro; Ohta, Noboru; Inoue, Katsuaki; Yagi, Naoto; Negishi, Hideyuki; Yanagishita, Hiroshi; Kitamoto, Dai

2006-03-08

Self-assembling properties of "natural" glycolipid biosurfactants, mannosyl-erythritol lipids A and B (MEL-A, MEL-B), which are abundantly produced from yeast strains, were investigated by using the fluorescence-probe method, dynamic light-scattering (DLS) analysis, freeze-fracture transmission electron microscopy (FF-TEM), and synchrotron small/wide-angle X-ray scattering (SAXS/WAXS) analysis, among other methods. Both MEL-A and MEL-B exhibit excellent self-assembly properties at extremely low concentrations; they self-assemble into large unilamellar vesicles (LUV) just above their critical-aggregation concentration (CAC). The CAC(I) value was found to be 4.0x10(-6) M for MEL-A and 6.0x10(-6) M for MEL-B. Moreover, the self-assembled structure of MEL-A above a CAC(II) value of 2.0x10(-5) M was found to drastically change into sponge structures (L3) composed of a network of randomly connected bilayers that are usually obtained from a complicated multicomponent "synthetic" surfactant system. Interestingly, the average water-channel diameter of the sponge structure was 100 nm. This is relatively large compared with those obtained from "synthetic" surfactant systems. In addition, MEL-B, which has a hydroxyl group at the C-4' position on mannose instead of an acetyl group, gives only one CAC; the self-assembled structure of MEL-B seems to gradually move from LUV to multilamellar vesicles (MLV) with lattice constants of 4.4 nm, depending on the concentration. Furthermore, the lyotropic-liquid-crystal-phase observation at high concentrations demonstrates the formation of an inverted hexagonal phase (H2) for MEL-A, together with a lamella phase (L(alpha)) for MEL-B, indicating a difference between MEL-A and MEL-B molecules in the spontaneous curvature of the assemblies. These results clearly show that the difference in spontaneous curvature caused by the single acetyl group on the head group probably decides the direction of self-assembly of glycolipid biosurfactants. The unique and complex molecular structures with several chiral centers that are molecularly engineered by microorganisms must have led to the sophisticated self-assembling properties of the glycolipid biosurfactants.

The yeast prefoldin-like URI-orthologue Bud27 associates with the RSC nucleosome remodeler and modulates transcription

PubMed Central

Mirón-García, María Carmen; Garrido-Godino, Ana Isabel; Martínez-Fernández, Verónica; Fernández-Pevida, Antonio; Cuevas-Bermúdez, Abel; Martín-Expósito, Manuel; Chávez, Sebastián; de la Cruz, Jesús; Navarro, Francisco

2014-01-01

Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATP-independent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity. PMID:25081216

Dual personality of Mad1: regulation of nuclear import by a spindle assembly checkpoint protein.

PubMed

Cairo, Lucas V; Ptak, Christopher; Wozniak, Richard W

2013-01-01

Nuclear transport is a dynamic process that can be modulated in response to changes in cellular physiology. We recently reported that the transport activity of yeast nuclear pore complexes (NPCs) is altered in response to kinetochore-microtubule (KT-MT) interaction defects. Specifically, KT detachment from MTs activates a signaling pathway that prevents the nuclear import of cargos by the nuclear transport factor Kap121p. This loss of Kap121p-mediated import is thought to influence the nuclear environment, including the phosphorylation state of nuclear proteins. A key regulator of this process is the spindle assembly checkpoint protein Mad1p. In response to unattached KTs, Mad1p dynamically cycles between NPCs and KTs. This cycling appears to induce NPC molecular rearrangements that prevent the nuclear import of Kap121p-cargo complexes. Here, we discuss the underlying mechanisms and the physiological relevance of Mad1p cycling and the inhibition of Kap121p-mediated nuclear import, focusing on outstanding questions within the pathway.

Self-Assembling Amphiphilic Molecules: A Possible Relationship Between Interstellar Chemistry and Meteoritic Organics

NASA Technical Reports Server (NTRS)

Sandford, Scott A.; Dworkin, Jason P.; Deamer, David W.; Allamandola, Louis J.; DeVincenzi, Donald (Technical Monitor)

2001-01-01

Interstellar gas and dust comprise the primary material from which the solar system formed. Evidence that some of this material was organic in nature and survived incorporation into the protosolar nebula is provided by the presence of deuterium-enriched organics in meteorites and interplanetary dust particles. Once the inner planets had sufficiently cooled, late accretionary infall of meteoroids and cosmic dust must have seeded them with some of these complex organic compounds. Delivery of such extraterrestrial compounds may have contributed to the organic inventory necessary for the origin of life. Interstellar ices, the building blocks of comets, tie up a large fraction of the biogenic elements available in molecular clouds. In our efforts to understand their synthesis, chemical composition, and physical properties, we report here that a complex mixture of molecules is produced by ultraviolet (UV) photolysis of realistic, interstellar ice analogs, and that some of the components have properties relevant to the origin of life, including the ability to self-assemble into vesicular structures.

Mammalian Fe-S proteins: definition of a consensus motif recognized by the co-chaperone HSC20

PubMed Central

Maio, N.; Rouault, T. A.

2017-01-01

Iron-sulfur (Fe-S) clusters are inorganic cofactors that are fundamental to several biological processes in all three kingdoms of life. In most organisms, Fe-S clusters are initially assembled on a scaffold protein, ISCU, and subsequently transferred to target proteins or to intermediate carriers by a dedicated chaperone/co-chaperone system. The delivery of assembled Fe-S clusters to recipient proteins is a crucial step in the biogenesis of Fe-S proteins, and, in mammals, it relies on the activity of a multiprotein transfer complex that contains the chaperone HSPA9, the co-chaperone HSC20 and the scaffold ISCU. How the transfer complex efficiently engages recipient Fe-S target proteins involves specific protein interactions that are not fully understood. This mini review focuses on recent insights into the molecular mechanism of amino acid motif recognition and discrimination by the co-chaperone HSC20, which guides Fe-S cluster delivery. PMID:27714045

TFIID TAF6-TAF9 Complex Formation Involves the HEAT Repeat-containing C-terminal Domain of TAF6 and Is Modulated by TAF5 Protein*

PubMed Central

Scheer, Elisabeth; Delbac, Frédéric; Tora, Laszlo; Moras, Dino; Romier, Christophe

2012-01-01

The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes. PMID:22696218

Structure of the Tropomyosin Overlap Complex from Chicken Smooth Muscle: Insight into the Diversity of N-Terminal Recognition

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

Frye, Jeremiah; Klenchin, Vadim A.; Rayment, Ivan

Tropomyosin is a stereotypical {alpha}-helical coiled coil that polymerizes to form a filamentous macromolecular assembly that lies on the surface of F-actin. The interaction between the C-terminal and N-terminal segments on adjacent molecules is known as the overlap region. We report here two X-ray structures of the chicken smooth muscle tropomyosin overlap complex. A novel approach was used to stabilize the C-terminal and N-terminal fragments. Globular domains from both the human DNA ligase binding protein XRCC4 and bacteriophage {phi}29 scaffolding protein Gp7 were fused to 37 and 28 C-terminal amino acid residues of tropomyosin, respectively, whereas the 29 N-terminal aminomore » acids of tropomyosin were fused to the C-terminal helix bundle of microtubule binding protein EB1. The structures of both the XRCC4 and Gp7 fusion proteins complexed with the N-terminal EB1 fusion contain a very similar helix bundle in the overlap region that encompasses {approx}15 residues. The C-terminal coiled coil opens to allow formation of the helix bundle, which is stabilized by hydrophobic interactions. These structures are similar to that observed in the NMR structure of the rat skeletal overlap complex [Greenfield, N. J., et al. (2006) J. Mol. Biol. 364, 80-96]. The interactions between the N- and C-terminal coiled coils of smooth muscle tropomyosin show significant curvature, which differs somewhat between the two structures and implies flexibility in the overlap complex, at least in solution. This is likely an important attribute that allows tropomyosin to assemble around the actin filaments. These structures provide a molecular explanation for the role of N-acetylation in the assembly of native tropomyosin.« less

Self-Assembled Polystyrene Beads for Templated Covalent Functionalization of Graphitic Substrates Using Diazonium Chemistry.

PubMed

Van Gorp, Hans; Walke, Peter; Bragança, Ana M; Greenwood, John; Ivasenko, Oleksandr; Hirsch, Brandon E; De Feyter, Steven

2018-04-11

A network of self-assembled polystyrene beads was employed as a lithographic mask during covalent functionalization reactions on graphitic surfaces to create nanocorrals for confined molecular self-assembly studies. The beads were initially assembled into hexagonal arrays at the air-liquid interface and then transferred to the substrate surface. Subsequent electrochemical grafting reactions involving aryl diazonium molecules created covalently bound molecular units that were localized in the void space between the nanospheres. Removal of the bead template exposed hexagonally arranged circular nanocorrals separated by regions of chemisorbed molecules. Small molecule self-assembly was then investigated inside the resultant nanocorrals using scanning tunneling microscopy to highlight localized confinement effects. Overall, this work illustrates the utility of self-assembly principles to transcend length scale gaps in the development of hierarchically patterned molecular materials.

A molecular engineering toolbox for the structural biologist

PubMed Central

Debelouchina, Galia T.; Muir, Tom W.

2018-01-01

Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease. PMID:29233219

A molecular engineering toolbox for the structural biologist.

PubMed

Debelouchina, Galia T; Muir, Tom W

2017-01-01

Exciting new technological developments have pushed the boundaries of structural biology, and have enabled studies of biological macromolecules and assemblies that would have been unthinkable not long ago. Yet, the enhanced capabilities of structural biologists to pry into the complex molecular world have also placed new demands on the abilities of protein engineers to reproduce this complexity into the test tube. With this challenge in mind, we review the contents of the modern molecular engineering toolbox that allow the manipulation of proteins in a site-specific and chemically well-defined fashion. Thus, we cover concepts related to the modification of cysteines and other natural amino acids, native chemical ligation, intein and sortase-based approaches, amber suppression, as well as chemical and enzymatic bio-conjugation strategies. We also describe how these tools can be used to aid methodology development in X-ray crystallography, nuclear magnetic resonance, cryo-electron microscopy and in the studies of dynamic interactions. It is our hope that this monograph will inspire structural biologists and protein engineers alike to apply these tools to novel systems, and to enhance and broaden their scope to meet the outstanding challenges in understanding the molecular basis of cellular processes and disease.

Assembly and microscopic characterization of DNA origami structures.

PubMed

Scheible, Max; Jungmann, Ralf; Simmel, Friedrich C

2012-01-01

DNA origami is a revolutionary method for the assembly of molecular nanostructures from DNA with precisely defined dimensions and with an unprecedented yield. This can be utilized to arrange nanoscale components such as proteins or nanoparticles into pre-defined patterns. For applications it will now be of interest to arrange such components into functional complexes and study their geometry-dependent interactions. While commonly DNA nanostructures are characterized by atomic force microscopy or electron microscopy, these techniques often lack the time-resolution to study dynamic processes. It is therefore of considerable interest to also apply fluorescence microscopic techniques to DNA nanostructures. Of particular importance here is the utilization of novel super-resolved microscopy methods that enable imaging beyond the classical diffraction limit.

Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology

PubMed Central

2011-01-01

Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution. PMID:21338175

M-DNA: a self-assembling molecular wire for nanoelectronics and biosensing.

PubMed

Wettig, Shawn D; Li, Chen-Zhong; Long, Yi-Tao; Kraatz, Heinz-Bernhard; Lee, Jeremy S

2003-01-01

M-DNA is a complex between divalent metal ions such as Zn2+ and duplex DNA which forms at pH 8.5. Unlike B-DNA, M-DNA does not bind ethidium so that M-DNA formation can be monitored conveniently by an ethidium fluorescence assay. M-DNA was shown to be a better conductor than B-DNA by fluorometric measurements of electron transport in donor-acceptor labelled duplexes; by direct conductivity measurements of M-DNA bound between gold electrodes and by cyclic voltammetric studies on ferrocene labelled duplexes attached to gold microelectrodes. As is the case with B-DNA, M-DNA can self-assemble into a variety of structures and is anticipated to find widespread use in nanoelectronics and biosensing.

Probing the Energetics of Dynactin Filament Assembly and the Binding of Cargo Adaptor Proteins Using Molecular Dynamics Simulation and Electrostatics-Based Structural Modeling.

PubMed

Zheng, Wenjun

2017-01-10

Dynactin, a large multiprotein complex, binds with the cytoplasmic dynein-1 motor and various adaptor proteins to allow recruitment and transportation of cellular cargoes toward the minus end of microtubules. The structure of the dynactin complex is built around an actin-like minifilament with a defined length, which has been visualized in a high-resolution structure of the dynactin filament determined by cryo-electron microscopy (cryo-EM). To understand the energetic basis of dynactin filament assembly, we used molecular dynamics simulation to probe the intersubunit interactions among the actin-like proteins, various capping proteins, and four extended regions of the dynactin shoulder. Our simulations revealed stronger intersubunit interactions at the barbed and pointed ends of the filament and involving the extended regions (compared with the interactions within the filament), which may energetically drive filament termination by the capping proteins and recruitment of the actin-like proteins by the extended regions, two key features of the dynactin filament assembly process. Next, we modeled the unknown binding configuration among dynactin, dynein tails, and a number of coiled-coil adaptor proteins (including several Bicaudal-D and related proteins and three HOOK proteins), and predicted a key set of charged residues involved in their electrostatic interactions. Our modeling is consistent with previous findings of conserved regions, functional sites, and disease mutations in the adaptor proteins and will provide a structural framework for future functional and mutational studies of these adaptor proteins. In sum, this study yielded rich structural and energetic information about dynactin and associated adaptor proteins that cannot be directly obtained from the cryo-EM structures with limited resolutions.

Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin.

PubMed

Scheele, Urte; Alves, Jurgen; Frank, Ronald; Duwel, Michael; Kalthoff, Christoph; Ungewickell, Ernst

2003-07-11

Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.

From porphyrins to pyrphyrins: adsorption study and metalation of a molecular catalyst on Au(111)

NASA Astrophysics Data System (ADS)

Mette, Gerson; Sutter, Denys; Gurdal, Yeliz; Schnidrig, Stephan; Probst, Benjamin; Iannuzzi, Marcella; Hutter, Jürg; Alberto, Roger; Osterwalder, Jürg

2016-04-01

The molecular ligand pyrphyrin, a tetradentate bipyridine based macrocycle, represents an interesting but widely unexplored class of molecules. It resembles the well-known porphyrin, but consists of pyridyl subunits instead of pyrroles. Metal complexes based on pyrphyrin ligands have recently shown promise as water reduction catalysts in homogeneous photochemical water splitting reactions. In this study, the adsorption and metalation of pyrphyrin on a single crystalline Au(111) surface is investigated in an ultrahigh vacuum by means of scanning tunneling microscopy, low-energy electron diffraction, X-ray photoelectron spectroscopy and density functional theory. Pyrphyrin coverages of approximately one monolayer and less are obtained by sublimation of the molecules on the substrate kept at room temperature. The molecules self-assemble in two distinct phases of long-range molecular ordering depending on the surface coverage. The deposition of cobalt metal and subsequent annealing lead to the formation of Co-ligated pyrphyrin molecules accompanied by a pronounced change of the molecular self-assembly. Electronic structure calculations taking the herringbone reconstruction of Au(111) into account show that the molecules are physisorbed, but preferred adsorption sites are identified where Co and the N atoms of the two terminal cyano groups are optimally coordinated to the surface Au atoms. An intermediate state of the metalation reaction is observed and the reaction steps for the Co metalation of pyrphyrin molecules on Au(111) are established in a joint experimental and computational effort.The molecular ligand pyrphyrin, a tetradentate bipyridine based macrocycle, represents an interesting but widely unexplored class of molecules. It resembles the well-known porphyrin, but consists of pyridyl subunits instead of pyrroles. Metal complexes based on pyrphyrin ligands have recently shown promise as water reduction catalysts in homogeneous photochemical water splitting reactions. In this study, the adsorption and metalation of pyrphyrin on a single crystalline Au(111) surface is investigated in an ultrahigh vacuum by means of scanning tunneling microscopy, low-energy electron diffraction, X-ray photoelectron spectroscopy and density functional theory. Pyrphyrin coverages of approximately one monolayer and less are obtained by sublimation of the molecules on the substrate kept at room temperature. The molecules self-assemble in two distinct phases of long-range molecular ordering depending on the surface coverage. The deposition of cobalt metal and subsequent annealing lead to the formation of Co-ligated pyrphyrin molecules accompanied by a pronounced change of the molecular self-assembly. Electronic structure calculations taking the herringbone reconstruction of Au(111) into account show that the molecules are physisorbed, but preferred adsorption sites are identified where Co and the N atoms of the two terminal cyano groups are optimally coordinated to the surface Au atoms. An intermediate state of the metalation reaction is observed and the reaction steps for the Co metalation of pyrphyrin molecules on Au(111) are established in a joint experimental and computational effort. Electronic supplementary information (ESI) available: More details and results of the XPS experiments and the DFT calculation including also the coordinates of the calculated configurations. See DOI: 10.1039/C5NR08953K

Optoelectronic functional materials based on alkylated-π molecules: self-assembled architectures and nonassembled liquids.

PubMed

Li, Hongguang; Choi, Jiyoung; Nakanishi, Takashi

2013-05-07

The engineering of single molecules into higher-order hierarchical assemblies is a current research focus in molecular materials chemistry. Molecules containing π-conjugated units are an important class of building blocks because their self-assembly is not only of fundamental interest, but also the key to fabricating functional systems for organic electronic and photovoltaic applications. Functionalizing the π-cores with "alkyl chains" is a common strategy in the molecular design that can give the system desirable properties, such as good solubility in organic solvents for solution processing. Moreover, the alkylated-π system can regulate the self-assembly behavior by fine-tuning the intermolecular forces. The optimally assembled structures can then exhibit advanced functions. However, while some general rules have been revealed, a comprehensive understanding of the function played by the attached alkyl chains is still lacking, and current methodology is system-specific in many cases. Better clarification of this issue requires contributions from carefully designed libraries of alkylated-π molecular systems in both self-assembly and nonassembly materialization strategies. Here, based on recent efforts toward this goal, we show the power of the alkyl chains in controlling the self-assembly of soft molecular materials and their resulting optoelectronic properties. The design of alkylated-C60 is selected from our recent research achievements, as the most attractive example of such alkylated-π systems. Some other closely related systems composed of alkyl chains and π-units are also reviewed to indicate the universality of the methodology. Finally, as a contrast to the self-assembled molecular materials, nonassembled, solvent-free, novel functional liquid materials are discussed. In doing so, a new journey toward the ultimate organic "soft" materials is introduced, based on alkylated-π molecular design.

Molecular switches and motors on surfaces.

PubMed

Pathem, Bala Krishna; Claridge, Shelley A; Zheng, Yue Bing; Weiss, Paul S

2013-01-01

Molecular switches and motors respond structurally, electronically, optically, and/or mechanically to external stimuli, testing and potentially enabling extreme miniaturization of optoelectronic devices, nanoelectromechanical systems, and medical devices. The assembly of motors and switches on surfaces makes it possible both to measure the properties of individual molecules as they relate to their environment and to couple function between assembled molecules. In this review, we discuss recent progress in assembling molecular switches and motors on surfaces, measuring static and dynamic structures, understanding switching mechanisms, and constructing functional molecular materials and devices. As demonstrative examples, we choose a representative molecule from three commonly studied classes including molecular switches, photochromic molecules, and mechanically interlocked molecules. We conclude by offering perspectives on the future of molecular switches and motors on surfaces.

Assembly and Transfer of Iron–Sulfur Clusters in the Plastid

PubMed Central

Lu, Yan

2018-01-01

Iron-Sulfur (Fe-S) clusters and proteins are essential to many growth and developmental processes. In plants, they exist in the plastids, mitochondria, cytosol, and nucleus. Six types of Fe-S clusters are found in the plastid: classic 2Fe-2S, NEET-type 2Fe-2S, Rieske-type 2Fe-2S, 3Fe-4S, 4Fe-4S, and siroheme 4Fe-4S. Classic, NEET-type, and Rieske-type 2Fe-2S clusters have the same 2Fe-2S core; similarly, common and siroheme 4Fe-4S clusters have the same 4Fe-4S core. Plastidial Fe-S clusters are assembled by the sulfur mobilization (SUF) pathway, which contains cysteine desulfurase (EC 2.8.1.7), sulfur transferase (EC 2.8.1.3), Fe-S scaffold complex, and Fe-S carrier proteins. The plastidial cysteine desulfurase-sulfur transferase-Fe-S-scaffold complex system is responsible for de novo assembly of all plastidial Fe-S clusters. However, different types of Fe-S clusters are transferred to recipient proteins via respective Fe-S carrier proteins. This review focuses on recent discoveries on the molecular functions of different assembly and transfer factors involved in the plastidial SUF pathway. It also discusses potential points for regulation of the SUF pathway, relationships among the plastidial, mitochondrial, and cytosolic Fe-S assembly and transfer pathways, as well as several open questions about the carrier proteins for Rieske-type 2Fe-2S, NEET-type 2Fe-2S, and 3F-4S clusters. PMID:29662496

Self-assembly of knots and links

NASA Astrophysics Data System (ADS)

Orlandini, Enzo; Polles, Guido; Marenduzzo, Davide; Micheletti, Cristian

2017-03-01

Guiding the self-assembly of identical building blocks towards complex three-dimensional structures with a set of desired properties is a major goal in material science, chemistry and physics. A particularly challenging problem, especially explored in synthetic chemistry, is that of self-assembling closed structures with a target topology starting by simple geometrical templates. Here we overview and revisit recent advancements, based on stochastic simulations, where the geometry of rigid helical templates with functionalised sticky ends has been designed for self-assembling efficiently and reproducibly into a wide range of three-dimensional closed structures. Notably, these include non trivial topologies of links and knots, including the 819 knot that we had predicted to be highly encodable and that has only recently been obtained experimentally. By appropriately tuning the parameters that define the template shape, we show that, for fixed concentration of templates, the assembly process can be directed towards the formation of specific knotted and linked structures such as the trefoils, pentafoil knots, Hopf and Solomon links. More exotic and unexpected knots and links are also found. Our results should be relevant to the design of new protocols that can both increase and broaden the population of synthetise molecular knots and catenanes.

Computational studies of sequence-specific driving forces in peptide self-assembly

NASA Astrophysics Data System (ADS)

Jeon, Joohyun

Peptides are biopolymers made from various sequences of twenty different types of amino acids, connected by peptide bonds. There are practically an infinite number of possible sequences and tremendous possible combinations of peptide-peptide interactions. Recently, an increasing number of studies have shown a stark variety of peptide self-assembled nanomaterials whose detailed structures depend on their sequences and environmental factors; these have end uses in medical and bio-electronic applications, for example. To understand the underlying physics of complex peptide self-assembly processes and to delineate sequence specific effects, in this study, I use various simulation tools spanning all-atom molecular dynamics to simple lattice models and quantify the balance of interactions in the peptide self-assembly processes. In contrast to the existing view that peptides' aggregation propensities are proportional to the net sequence hydrophobicity and inversely proportional to the net charge, I show the more nuanced effects of electrostatic interactions, including the cooperative effects between hydrophobic and electrostatic interactions. Notably, I suggest rather unexpected, yet important roles of entropies in the small scale oligomerization processes. Overall, this study broadens our understanding of the role of thermodynamic driving forces in peptide self-assembly.

Directed assembly of colloidal particles for micro/nano photonics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zheng, Yuebing

2017-02-01

Bottom-up fabrication of complex structures with chemically synthesized colloidal particles as building blocks pave an efficient and cost-effective way towards micro/nano photonics with unprecedented functionality and tunability. Novel properties can arise from quantum effects of colloidal particles, as well as inter-particle interactions and spatial arrangement in particle assemblies. Herein, I discuss our recent developments and applications of three types of techniques for directed assembly of colloidal particles: moiré nanosphere lithography (MNSL), bubble-pen lithography (BPL), and optothermal tweezers (OTTs). Specifically, MNSL provides an efficient approach towards creating moiré metasurface with tunable and multiband optical responses from visible to mid-infrared regime. Au moiré metasurfaces have been applied for surface-enhanced infrared spectroscopy, optical capture and patterning of bacteria, and photothermal denaturation of proteins. BPL is developed to pattern a variety of colloidal particles on plasmonic substrates and two-dimensional atomic-layer materials in an arbitrary manner. The laser-directed microbubble captures and immobilizes nanoparticles through coordinated actions of Marangoni convection, surface tension, gas pressure, and substrate adhesion. OTTs are developed to create dynamic nanoparticle assemblies at low optical power. Such nanoparticle assemblies have been used for surface-enhanced Raman spectroscopy for molecular analysis in their native environments.

Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes.

PubMed

Lu, Jonathan; Trnka, Michael J; Roh, Soung-Hun; Robinson, Philip J J; Shiau, Carrie; Fujimori, Danica Galonic; Chiu, Wah; Burlingame, Alma L; Guan, Shenheng

2015-12-01

Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise. Graphical Abstract ᅟ.

ePMV embeds molecular modeling into professional animation software environments.

PubMed

Johnson, Graham T; Autin, Ludovic; Goodsell, David S; Sanner, Michel F; Olson, Arthur J

2011-03-09

Increasingly complex research has made it more difficult to prepare data for publication, education, and outreach. Many scientists must also wade through black-box code to interface computational algorithms from diverse sources to supplement their bench work. To reduce these barriers we have developed an open-source plug-in, embedded Python Molecular Viewer (ePMV), that runs molecular modeling software directly inside of professional 3D animation applications (hosts) to provide simultaneous access to the capabilities of these newly connected systems. Uniting host and scientific algorithms into a single interface allows users from varied backgrounds to assemble professional quality visuals and to perform computational experiments with relative ease. By enabling easy exchange of algorithms, ePMV can facilitate interdisciplinary research, smooth communication between broadly diverse specialties, and provide a common platform to frame and visualize the increasingly detailed intersection(s) of cellular and molecular biology. Copyright © 2011 Elsevier Ltd. All rights reserved.

ePMV Embeds Molecular Modeling into Professional Animation Software Environments

PubMed Central

Johnson, Graham T.; Autin, Ludovic; Goodsell, David S.; Sanner, Michel F.; Olson, Arthur J.

2011-01-01

SUMMARY Increasingly complex research has made it more difficult to prepare data for publication, education, and outreach. Many scientists must also wade through black-box code to interface computational algorithms from diverse sources to supplement their bench work. To reduce these barriers, we have developed an open-source plug-in, embedded Python Molecular Viewer (ePMV), that runs molecular modeling software directly inside of professional 3D animation applications (hosts) to provide simultaneous access to the capabilities of these newly connected systems. Uniting host and scientific algorithms into a single interface allows users from varied backgrounds to assemble professional quality visuals and to perform computational experiments with relative ease. By enabling easy exchange of algorithms, ePMV can facilitate interdisciplinary research, smooth communication between broadly diverse specialties and provide a common platform to frame and visualize the increasingly detailed intersection(s) of cellular and molecular biology. PMID:21397181