Formation Of Higher-order Structures Research Articles

Global remodeling of nucleosome positions in C. elegans

BackgroundEukaryotic chromatin architecture is affected by intrinsic histone-DNA sequence preferences, steric exclusion between nucleosome particles, formation of higher-order structures, and in vivo activity of chromatin remodeling enzymes.ResultsTo disentangle sequence-dependent nucleosome positioning from the other factors, we have created two high-throughput maps of nucleosomes assembled in vitro on genomic DNA from the nematode worm Caenorhabditis elegans. A comparison of in vitro nucleosome positions with those observed in a mixed-stage, mixed-tissue population of C. elegans cells reveals that in vivo sequence preferences are modified on the genomic scale. Indeed, G/C dinucleotides are predicted to be most favorable for nucleosome formation in vitro but not in vivo. Nucleosome sequence read coverage in vivo is distinctly lower in chromosome arms than in central regions; the observed changes in apparent nucleosome sequence specificity, likely due to genome-wide chromatin remodeler activity, contribute to the formation of these megabase-scale chromatin domains. We also observe that the majority of well-positioned in vivo nucleosomes do not occupy thermodynamically favorable sequences observed in vitro. Finally, we find that exons are intrinsically more amenable to nucleosome formation compared to introns. Nucleosome occupancy of introns and exons consistently increases with G/C content in vitro but not in vivo, in agreement with our observation that G/C dinucleotide enrichment does not strongly promote in vivo nucleosome formation.ConclusionsOur findings highlight the importance of both sequence specificity and active nucleosome repositioning in creating large-scale chromatin domains, and the antagonistic roles of intrinsic sequence preferences and chromatin remodelers in C. elegans.Sequence read data has been deposited into Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra; accession number SRA050182). Additional data, software and computational predictions are available on the Nucleosome Explorer website (http://nucleosome.rutgers.edu).

PcG-Mediated Higher-Order Chromatin Structures Modulate Replication Programs at the Drosophila BX-C

Polycomb group proteins (PcG) exert conserved epigenetic functions that convey maintenance of repressed transcriptional states, via post-translational histone modifications and high order structure formation. During S-phase, in order to preserve cell identity, in addition to DNA information, PcG-chromatin-mediated epigenetic signatures need to be duplicated requiring a tight coordination between PcG proteins and replication programs. However, the interconnection between replication timing control and PcG functions remains unknown. Using Drosophila embryonic cell lines, we find that, while presence of specific PcG complexes and underlying transcription state are not the sole determinants of cellular replication timing, PcG-mediated higher-order structures appear to dictate the timing of replication and maintenance of the silenced state. Using published datasets we show that PRC1, PRC2, and PhoRC complexes differently correlate with replication timing of their targets. In the fully repressed BX-C, loss of function experiments revealed a synergistic role for PcG proteins in the maintenance of replication programs through the mediation of higher-order structures. Accordingly, replication timing analysis performed on two Drosophila cell lines differing for BX-C gene expression states, PcG distribution, and chromatin domain conformation revealed a cell-type-specific replication program that mirrors lineage-specific BX-C higher-order structures. Our work suggests that PcG complexes, by regulating higher-order chromatin structure at their target sites, contribute to the definition and the maintenance of genomic structural domains where genes showing the same epigenetic state replicate at the same time.

Hierarchical self-assembly of Tjernberg peptide at nanoscale

Molecular self-assembly of biomolecules, especially proteins and peptides has elucidated much interest in recent years due to the reproducibility of structures and the high degree of control on the formation kinetics and morphology by varying the self-assembling conditions. These self-assembled structures have found widespread applications in tissue engineering, drug delivery and electronics. Two pentapeptides, namely Soto peptide (LPFFD) and the Tjernberg peptide (KLVFF), have been proposed to effectively curtail amyloid plaque deposits. Being amphipathic, these molecules can undergo self-assembly that can be influenced by a host of factors including concentration, pH, proteins, salt content, etc. However, this facet is scarcely explored till date, which might pave way for the novel treatment strategy for Alzheimer's disease. Our data show that the peptide spontaneously forms micelles and they are rich in beta sheet structures, which is confirmed using FT-IR, SAED and thioflavin binding studies. While, acidic pH seems to have no role in the formation of higher order structures, alkaline pH promotes longitudinal and lateral stacking interactions resulting in formation of higher order structures. We also found that the hydrophobicity and hydrophilicity of the substrate also play a major role in the formation of higher order structures. Moreover, the micelles formed by the KLVFF peptide under various environmental conditions exhibits excellent surface tension lowering effects which is in good agreement with the cell proliferation data that showed the protective ability of KLVFF peptide against Aβ1–42 cytotoxicity indicating its potential in Alzheimer's disease therapy as an aggregation blocking agent.

Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein's digestibility is not affected by such processing.

Transiently populated intermediate functions as a branching point of the FF domain folding pathway

Studies of protein folding and the intermediates that are formed along the folding pathway provide valuable insights into the process by which an unfolded ensemble forms a functional native conformation. However, because intermediates on folding pathways can serve as initiation points of aggregation (implicated in a number of diseases), their characterization assumes an even greater importance. Establishing the role of such intermediates in folding, misfolding, and aggregation remains a major challenge due to their often low populations and short lifetimes. We recently used NMR relaxation dispersion methods and computational techniques to determine an atomic resolution structure of the folding intermediate of a small protein module--the FF domain--with an equilibrium population of 2-3% and a millisecond lifetime, 25 °C. Based on this structure a variant FF domain has been designed in which the native state is selectively destabilized by removing the carboxyl-terminal helix in the native structure to produce a highly populated structural mimic of the intermediate state. Here, we show via solution NMR studies of the designed mimic that the mimic forms distinct conformers corresponding to monomeric and dimeric (K(d) = 0.2 mM) forms of the protein. The conformers exchange on the seconds timescale with a monomer association rate of 1.1 · 10(4) M(-1) s(-1) and with a region responsible for dimerization localized to the amino-terminal residues of the FF domain. This study establishes the FF domain intermediate as a central player in both folding and misfolding pathways and illustrates how incomplete folding can lead to the formation of higher-order structures.

Pri-miR-17-92a transcript folds into a tertiary structure and autoregulates its processing

MicroRNAs control gene expression either by RNA transcript degradation or translational repression. Expressions of miRNAs are highly regulated in tissues, disruption of which leads to disease. How this regulation is achieved and maintained is still largely unknown. MiRNAs that reside on clustered or polycistronic transcripts represent a more complex case where individual miRNAs from a cluster are processed with different efficiencies despite being cotranscribed. To shed light on the regulatory mechanisms that might be operating in these cases, we considered the long polycistronic primary miRNA transcript pri-miR-17-92a that contains six miRNAs with diverse functions. The six miRNA domains on this cluster are differentially processed to produce varying amounts of resultant mature miRNAs in different tissues. How this is achieved is not known. We show, using various biochemical and biophysical methods coupled with mutational studies, that pri-miR-17-92a adopts a specific three-dimensional architecture that poses a kinetic barrier to its own processing. This tertiary structure could create suboptimal protein recognition sites on the pri-miRNA cluster due to higher-order structure formation.

Biotechnology and Bioengineering: Volume 109, Number 5, May 2012

Biotechnology and BioengineeringVolume 109, Issue 5 p. C1-C1 CoverFree Access Biotechnology and Bioengineering: Volume 109, Number 5, May 2012 First published: 15 March 2012 https://doi.org/10.1002/bit.24293AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Cover Picture: Viral structural protein aggregates during bioprocessing of virus-like particles (VLPs). Formation of higher order structures was investigated with high-resolution static light scattering technique, providing quantitative information on the competition between productive VLP assembly and undesirable aggregation. See related article by Middelberg et. al., Biotechnology and Bioengineering, Volume 107, Number 3 (October 15, 2010), pages 550–560. (Image courtesy of Dr. Anton Middelberg et. al.). Volume109, Issue5May 2012Pages C1-C1 RelatedInformation

Using DNA as a Fiducial Marker To Study SMC Complex Interactions with the Atomic Force Microscope

Atomic force microscopy can potentially provide information on protein volumes, shapes, and interactions but is susceptible to variable tip-induced artifacts. In this study, we present an atomic force microscopy approach that can measure volumes of nonglobular polypeptides such as structural maintenance of chromosomes (SMC) proteins, and use it to study the interactions that occur within and between SMC complexes. Together with the protein of interest, we coadsorb a DNA molecule and use it as a fiducial marker to account for tip-induced artifacts that affect both protein and DNA, allowing normalization of protein volumes from images taken on different days and with different tips. This approach significantly reduced the error associated with volume analysis, and allowed determination of the oligomeric states and architecture of the Bacillus subtilis SMC complex, formed by the SMC protein, and by the smaller ScpA and ScpB subunits. This work reveals that SMC and ScpB are dimers and that ScpA is a stable monomer. Moreover, whereas ScpA binds directly to SMC, ScpB only binds to SMC in the presence of ScpA. Notably, the presence of both ScpA and ScpB favored the formation of higher-order structures of SMC complexes, suggesting a role for these subunits in the organization of SMC oligomers.

Functions of chromatin remodeling factors in heterochromatin formation and maintenance

Heterochromatin is characteristically more compact than euchromatin in the eukaryotic genome. The establishment of heterochromatin is mediated by special histone modifications, recruitment and propagation of heterochromatin specific proteins, as well as formation of special primary and high order structures of chromatin. Chromatin remodeling factors are ATPases that can alter the conformation and/or positioning of nucleosomes along DNA in an ATP-dependent manner. There is increasing evidence implicating chromatin remodeling activities in heterochromatin in various organisms ranging from yeasts to humans. Chromatin remodeling factors play roles in the establishment, maintenance and epigenetic inheritance of heterochromatin, but the underlying molecular mechanisms have just begun to be investigated.

Formation of higher-order structures of chiral poly(ethynylpyridine)s depending on size, temperature, and saccharide recognition

Amphiphilic 2,6-pyridylene ethynylene "meta-ethynylpyridine" polymers having chiral oligo(oxyethylene) side chains were developed as hosts for saccharide recognition. The polymers were prepared via a Sonogashira reaction and fractionated by gel permeation chromatography (GPC). They showed circular dichroism (CD) activity due to their higher-order chiral helical structures, and their CD and UV-vis spectra changed depending on not only saccharide recognition but also molecular size, temperature, and metal cation recognition.

Formation of Higher-Order Foot-and-Mouth Disease Virus 3D pol Complexes Is Dependent on Elongation Activity

The replication of many viruses involves the formation of higher-order structures or replication "factories." We show that the key replication enzyme of foot-and-mouth disease virus (FMDV), the RNA-dependent RNA polymerase, forms fibrils in vitro. Although there are similarities with previously characterized poliovirus polymerase fibrils, FMDV fibrils are narrower, are composed of both protein and RNA, and, importantly, are seen only when all components of an elongation assay are present. Furthermore, an inhibitory RNA aptamer prevents fibril formation.

A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering

Formation of higher-order structure of nucleic acids (hairpins or loops, for example) may impact not only gene regulation, but also molecular biology techniques and approaches critical for design and production of vectors needed for genetic engineering approaches. In the course of designing vectors aimed to modify the murine Foxd3 locus through homologous recombination in embryonic stem cells, we discovered a 370 nucleotide segment of DNA resistant to polymerase read-through. In addition to sequencing and PCR disruptions, we were unable to use BAC recombineering strategies to exchange sequences within the Foxd3 locus. This segment corresponds to a putative DNA hairpin region just upstream of the 5' untranslated region of Foxd3. This region is also highly conserved across vertebrate species, suggesting possible functional significance. Our findings provide a cautionary note for researchers experiencing technical challenges with BAC recombineering or other molecular biology methods requiring recombination or polymerase activity.

The Role of MukE in Assembling a Functional MukBEF Complex

The MukB–MukE–MukF protein complex is essential for chromosome condensation and segregation in Escherichia coli. The central component of this complex, the MukB protein, is related functionally and structurally to the ubiquitous SMC ( structural maintenance of chromosomes) proteins. In a manner similar to SMC, MukB requires the association of two accessory proteins (MukE and MukF) for its function. MukF is a constitutive dimer that bridges the interaction between MukB and MukE. While MukB can condense DNA on its own, it requires MukF and MukE to ensure proper chromosome segregation. Here, we present a novel structure of the E. coli MukE–MukF complex, in which the intricate crystal packing interactions reveal an alternative MukE dimerization interface spanning both N- and C-terminal winged-helix domains of the protein. The structure also unveils additional cross-linking interactions between adjacent MukE–MukF complexes mediated by MukE. A variant of MukE encompassing point mutations on one of these surfaces does not affect assembly of the MukB–MukE–MukF complex and yet cannot restore the temperature sensitivity of the mukE∷ kan strain, suggesting that this surface may mediate critical protein–protein interactions between MukB–MukE–MukF complexes. Since the dimerization interface of MukE overlaps with the region of the protein that interacts with MukB in the MukB–MukE–MukF complex, we suggest that competing MukB–MukE and MukE–MukE interactions may regulate the formation of higher-order structures of bacterial condensin.

Chromatin Organization by Repetitive Elements (CORE): A Genomic Principle for the Higher-Order Structure of Chromosomes

Eukaryotic genomes contain a large amount of DNA repeats (also known as repetitive DNA, repetitive elements, and repetitive sequences). Here, I propose a role of repetitive DNA in the formation of higher-order structures of chromosomes. The central idea of this theory is that chromatin regions with repetitive sequences pair with regions harboring homologous repeats and that such somatic repeat pairing (RP) assembles repetitive DNA chromatin into compact chromosomal domains that specify chromatin folding in a site-directed manner. According to this theory, DNA repeats are not randomly distributed in the genome. Instead, they form a core framework that coordinates the architecture of chromosomes. In contrast to the viewpoint that DNA repeats are genomic ‘junk’, this theory advocates that repetitive sequences are chromatin organizer modules that determine chromatin-chromatin contact points within chromosomes. This novel concept, if correct, would suggest that DNA repeats in the linear genome encode a blueprint for higher-order chromosomal organization.

Probing cationic selectivity of cardiac calsequestrin and its CPVT mutants

CASQ (calsequestrin) is a Ca2+-buffering protein localized in the muscle SR (sarcoplasmic reticulum); however, it is unknown whether Ca2+ binding to CASQ2 is due to its location inside the SR rich in Ca2+ or due to its preference for Ca2+ over other ions. Therefore a major aim of the present study was to determine how CASQ2 selects Ca2+ over other metal ions by studying monomer folding and subsequent aggregation upon exposure to alkali (monovalent), alkaline earth (divalent) and transition (polyvalent) metals. We additionally investigated how CPVT (catecholaminergic polymorphic ventricular tachycardia) mutations affect CASQ2 structure and its molecular behaviour when exposed to different metal ions. Our results show that alkali and alkaline earth metals can initiate similar molecular compaction (folding), but only Ca2+ can promote CASQ2 to aggregate, suggesting that CASQ2 has a preferential binding to Ca2+ over all other metals. We additionally found that transition metals (having higher co-ordinated bonding ability than Ca2+) can also initiate folding and promote aggregation of CASQ2. These studies led us to suggest that folding and formation of higher-order structures depends on cationic properties such as co-ordinate bonding ability and ionic radius. Among the CPVT mutants studied, the L167H mutation disrupts the Ca2+-dependent folding and, when folding is achieved by Mn2+, L167H can undergo aggregation in a Ca2+-dependent manner. Interestingly, domain III mutants (D307H and P308L) lost their selectivity to Ca2+ and could be aggregated in the presence of Mg2+. In conclusion, these studies suggest that CPVT mutations modify CASQ2 behaviour, including folding, aggregation/polymerization and selectivity towards Ca2+.

Effects of blending poly(D,L-lactide) with poly(ethylene glycol) on the higher-oder crystalline structures of poly(ethylene glycol) as revealed by small-angle X-ray scattering

Effects of blending poly(lactic acid) (PLA) with poly(ethylene glycol) (PEG) on higher-order crystalline structures of PEG were examined using small-angle X-ray scattering (SAXS). For this purpose, the fact that two polymers are both crystalline makes situtation much complicated. To simplify, non-crystalline PLA is suitable. Thus, we used poly(D,L-lactic acid) (DLPLA), which is random copolymer comprising D- and L-lactic acid moieties. Multiple scattering peaks arising from the regular crystalline lamellar structure were observed for the PEG homopolymer and the blends. Surprisingly, the structure is much more regular for the blend DLPLA/PEG at composition of 20/80 wt.% than for the PEG homopolymer. Also for this blend sample as well as for a PEG homopolymer, very peculiar SAXS profiles were observed just 1°C below Tm of PEG. This is found to be a particle scattering of plate-like objects, which has never been reported for polymer blends or crystalline polymers. Futhermore, it was found that there was strong hysteresis of the higher-order structure formation.

Preparation of Hybrid Film of Polyaniline and Organically Pillared Zirconium Phosphate Nanosheet by Electrodeposition

α-Zirconium phosphate was chemically modified with 1,2-bis(dimethylchlorosilyl)ethane to graft organic chain, and then it was used as host material for inorganic nanosheet-polyaniline hybrid. The grafted α-zirconium phosphate was exfoliated in an acetonitrile solution with tetrabutylammonium salt and aniline. The electrodeposition in the presence of aniline was performed, and then it resulted in a formation of higher-order structure in which phosphate nanosheet was propped up by 1,2-bis(dimethylchlorosilyl)ethane with intrusion of polyaniline into the nanospace. The gravimetric capacitance of the α-zirconium phosphate without grafts and polyaniline hybrid film was around 194 F/g with the base on the amount of polyaniline mass. On the other hand, the α-zirconium phosphate nanosheet with grafts and polyaniline hybrid film provided larger capacitance of around 350 F/g in maximum. The nanospace formed by grafted phosphate nanosheet with 1,2-bis(dimethylchlorosilyl)ethane molecules gives increased amounts of polyaniline included and diffusion paths for ions.

Molecular behavior of DNA origami in higher-order self-assembly.

DNA-based self-assembly is a unique method for achieving higher-order molecular architectures made possible by the fact that DNA is a programmable information-coding polymer. In the past decade, two main types of DNA nanostructures have been developed: branch-shaped DNA tiles with small dimensions (commonly up to ∼20 nm) and DNA origami tiles with larger dimensions (up to ∼100 nm). Here we aimed to determine the important factors involved in the assembly of DNA origami superstructures. We constructed a new series of rectangular-shaped DNA origami tiles in which parallel DNA helices are arranged in a zigzag pattern when viewed along the DNA helical axis, a design conceived in order to relax an intrinsic global twist found in the original planar, rectangular origami tiles. Self-associating zigzag tiles were found to form linear arrays in both diagonal directions, while planar tiles showed significant growth in only one direction. Although the series of zigzag tiles were designed to promote two-dimensional array formation, one-dimensional linear arrays and tubular structures were observed instead. We discovered that the dimensional aspect ratio of the origami unit tiles and intertile connection design play important roles in determining the final products, as revealed by atomic force microscopy imaging. This study provides insight into the formation of higher-order structures from self-assembling DNA origami tiles, revealing their unique behavior in comparison with conventional DNA tiles having smaller dimensions.

ArabidopsisVILLIN5, an Actin Filament Bundling and Severing Protein, Is Necessary for Normal Pollen Tube Growth

A dynamic actin cytoskeleton is essential for pollen germination and tube growth. However, the molecular mechanisms underlying the organization and turnover of the actin cytoskeleton in pollen remain poorly understood. Villin plays a key role in the formation of higher-order structures from actin filaments and in the regulation of actin dynamics in eukaryotic cells. It belongs to the villin/gelsolin/fragmin superfamily of actin binding proteins and is composed of six gelsolin-homology domains at its core and a villin headpiece domain at its C terminus. Recently, several villin family members from plants have been shown to sever, cap, and bundle actin filaments in vitro. Here, we characterized a villin isovariant, Arabidopsis thaliana VILLIN5 (VLN5), that is highly and preferentially expressed in pollen. VLN5 loss-of-function retarded pollen tube growth and sensitized actin filaments in pollen grains and tubes to latrunculin B. In vitro biochemical analyses revealed that VLN5 is a typical member of the villin family and retains a full suite of activities, including barbed-end capping, filament bundling, and calcium-dependent severing. The severing activity was confirmed with time-lapse evanescent wave microscopy of individual actin filaments in vitro. We propose that VLN5 is a major regulator of actin filament stability and turnover that functions in concert with oscillatory calcium gradients in pollen and therefore plays an integral role in pollen germination and tube growth.

Monitoring of duplex and triplex formation by 19F NMR using oligodeoxynucleotides possessing 5-fluorodeoxyuridine unit as 19F signal transmitter

We prepared oligodeoxynucleotides (ODNs) possessing a 5-fluorodeoxyuridine (5-FU) unit as a 19F-signal transmitter, and characterized their structures including single strand, duplex, and triplex using 19F NMR. The change in chemical shift induced by incorporation of 5-FU into the ODNs and the formation of higher order structures allowed monitoring of structural changes. Data from UV melting experiments and CD spectra were consistent with the spectral changes in the NMR studies. These 19F-labeled ODNs may be promising molecular probes for the identification of DNA structures in complicated biological conditions.