Coexistence of coil and globule domains within a single confined DNA chain.
ABSTRACT: The highly charged DNA chain may be either in an extended conformation, the coil, or condensed into a highly dense and ordered structure, the toroid. The transition, also called collapse of the chain, can be triggered in different ways, for example by changing the ionic conditions of the solution. We observe individual DNA molecules one by one, kept separated and confined inside a protein shell (the envelope of a bacterial virus, 80 nm in diameter). For subcritical concentrations of spermine (4+), part of the DNA is condensed and organized in a toroid and the other part of the chain remains uncondensed around. Two states coexist along the same DNA chain. These 'hairy' globules are imaged by cryo-electron microscopy. We describe the global conformation of the chain and the local ordering of DNA segments inside the toroid.
Project description:DNA release dynamics from layer-by-layer (LbL) films is an important aspect to consider with regards to localized gene delivery systems. The rate of DNA release and the condensation state of DNA during release are of particular interest in the field of gene delivery. A hyperbranched poly(amido amine) (RHB) containing bioreducible disulfide bonds is used to form interpolyelectrolyte complexes with DNA during LbL film assembly. During film disassembly, DNA is released in physiologic conditions due to the reducing nature of the RHB. Uncondensed DNA deposited on the surface was compared to DNA condensed by RHB in polyplex form by using two types of LbL films, RHB/DNA/RHB and polyplex terminated films, RHB/DNA/polyplex. LbL films with up to three layers are used in order to facilitate high-resolution atomic force microscopy (AFM) imaging. X-ray reflectivity, ellipsometry, and Fourier transform infrared spectroscopy are also used. The film disassembly, rearrangement, and release of molecules from the surface due to thiol-disulfide exchange is conducted in reducing dithiothreitol (DTT) solutions. Salt is found to accelerate the overall rate of film disassembly. Additionally, it was found that the polyplex layer disassembles faster than the DNA layer. The predominant intermediate structure is the toroid structure for the polyplex layer and the fiber bundle structure for the DNA layer during film disassembly. This study offers a simple means to modulate DNA release from LbL films by utilizing both condensed and uncondensed DNA in different layers. The study highlights nanostructures, toroids, and bundles as dominant intermediate DNA structures during DNA release from LbL films.
Project description:The Chlorobiales are anoxygenic phototrophs that produce solid, extracellular elemental sulfur globules as an intermediate step in the oxidation of sulfide to sulfate. These organisms must export sulfur while preventing cell encrustation during S0 globule formation; during globule degradation they must find and mobilize the sulfur for intracellular oxidation to sulfate. To understand how the Chlorobiales address these challenges, we characterized the spatial relationships and physical dynamics of Chlorobaculum tepidum cells and S0 globules by light and electron microscopy. Cba. tepidum commonly formed globules at a distance from cells. Soluble polysulfides detected during globule production may allow for remote nucleation of globules. Polysulfides were also detected during globule degradation, probably produced as an intermediate of sulfur oxidation by attached cells. Polysulfides could feed unattached cells, which made up over 80% of the population and had comparable growth rates to attached cells. Given that S0 is formed remotely from cells, there is a question as to how cells are able to move toward S0 in order to attach. Time-lapse microscopy shows that Cba. tepidum is in fact capable of twitching motility, a finding supported by the presence of genes encoding type IV pili. Our results show how Cba. tepidum is able to avoid mineral encrustation and benefit from globule degradation even when not attached. In the environment, Cba. tepidum may also benefit from soluble sulfur species produced by other sulfur-oxidizing or sulfur-reducing bacteria as these organisms interact with its biogenic S0 globules.
Project description:In order to preserve genetic information in stress conditions, bacterial DNA is organized into higher order nucleoid structure. In this paper, with the help of Atomic Force Microscopy, we show the different structural changes in mycobacterial nucleoid at different points of growth in the presence of different concentrations of glucose in the medium. We also observe that in Mycobacterium smegmatis, two different Dps proteins (Dps1 and Dps2) promote two types of nucleoid organizations. At the late stationary phase, under low glucose availability, Dps1 binds to DNA to form a very stable toroid structure. On the other hand, under the same condition, Dps2-DNA complex forms an incompletely condensed toroid and finally forms a further stable coral reef structure in the presence of RNA. This coral reef structure is stable in high concentration of bivalent ion like Mg(2+).
Project description:The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.
Project description:The spermine-induced DNA condensation is a first-order phase transition. Here, we apply a novel technique fluorescence lifetime correlation spectroscopy to analyze this transition in a greater detail. We show that the method allows for the observation of the condensed and uncondensed molecules simultaneously based solely on different fluorescence lifetimes of the intercalating fluorophore PicoGreen in the folded und unfolded domains of DNA. The auto- and cross-correlation functions reveal that a small fraction of the DNA molecules is involved in the dynamic intramolecular equilibrium. Careful inspection of the cross-correlation curves suggests that folding occurs gradually within milliseconds.
Project description:Alpha-1-antitrypsin deficiency is a disease of the liver and lung where accumulation of misfolded ATZ protein forms large protein aggregates or globules that result in significant cellular stess. From Birth to 2 months of age, virtually every hepatocyte has numerous globules of the mutant ATZ protien. At 2 months of age small colonies of globule free hepatocytes arise and over time the liver becomes largely globule free. this analysis aims to identify differential gene expression profiles in the globule free vs the globule containing cells to idenitfy key factors and pathways regulating clearance of the mutant ATZ protien. We used Laser Capture Microdisection to isolate RNA from ATZ-globule-free hepatocytes and ATZ-globule-containing hepatocytes and subjected them to microarray analysis to detail the differential global gene expression profiles. Overall design: PIZ mice 3 months of age were sacrificed and their livers were mounted in OCT compound and fresh frozen in methylbutane. Liver sections were placed on slides and counterstained with Cresyl Violet to visualize globules. The slides were then subjected to laser capture micro-disection of globule free and globule containing areas.
Project description:Native molten globules are the most folded kind of intrinsically disordered proteins. Little is known about the mechanism by which native molten globules bind to their cognate ligands to form fully folded complexes. The nuclear coactivator binding domain (NCBD) of CREB binding protein is particularly interesting in this respect as structural studies of its complexes have shown that NCBD folds into two remarkably different states depending on the ligand being ACTR or IRF-3. The ligand-free state of NCBD was characterized in order to understand the mechanism of folding upon ligand binding. Biophysical studies show that despite the molten globule nature of the domain, it contains a small cooperatively folded core. By NMR spectroscopy, we have demonstrated that the folded core of NCBD has a well ordered conformer with specific side chain packing. This conformer resembles the structure of the NCBD in complex with the protein ligand, ACTR, suggesting that ACTR binds to prefolded NCBD molecules from the ensemble of interconverting structures.
Project description:Axonopathy is critical in the early pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Axonal swellings such as globules and spheroids are a distinct feature of axonopathy and our recent study showed that transgenic (tg) mice expressing DLB-linked P123H ?-synuclein (P123H ?S) were characterized by P123H ?S-immunoreactive axonal swellings (P123H ?S-globules). Therefore, the objectives of this study were to evaluate ?-synuclein (?S)-immunoreactive axonal swellings (?S-globules) in the brains of tg mice expressing human wild-type ?S and to compare them with the globules in P123H ?S tg mice.In ?S tg mice, ?S-globules were formed in an age-dependent manner in various brain regions, including the thalamus and basal ganglia. These globules were composed of autophagosome-like membranous structures and were reminiscent of P123H ?S-globules in P123H ?S tg mice. In the ?S-globules, frequent clustering and deformation of mitochondria were observed. These changes were associated with oxidative stress, based on staining of nitrated ?S and 4-hydroxy-2-nonenal (4-HNE). In accord with the absence of mitochondria in the P123H ?S-globules, staining of nitrated ?S and 4-HNE in these globules was weaker than that for ?S-globules. Leucine-rich repeat kinase 2 (LRRK2), the PARK8 of familial PD, was detected exclusively in ?S-globules, suggesting a specific role of this molecule in these globules.Lysosomal pathology was similarly observed for both ?S- and P123H ?S-globules, while oxidative stress was associated with the ?S-globules, and to a lesser extent with the P123H ?S-globules. Other pathologies, such as mitochondrial alteration and LRRK2 accumulation, were exclusively detected for ?S-globules. Collectively, both ?S- and P123H ?S-globules were formed through similar but distinct pathogenic mechanisms. Our findings suggest that synuclein family members might contribute to diverse axonal pathologies.
Project description:Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin's molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an ?-? parallel protein.
Project description:The molecular mechanism underlying milk fat globule secretion in mammary epithelial cells ostensibly involves the formation of complexes between plasma membrane butyrophilin and cytosolic xanthine oxidoreductase. These complexes bind adipophilin in the phospholipid monolayer of milk secretory granules, the precursors of milk fat globules, enveloping the nascent fat globules in a layer of plasma membrane and pinching them off the cell. However, using freeze-fracture immunocytochemistry, we find these proteins in locations other than those previously inferred. Significantly, butyrophilin in the residual plasma membrane of the fat globule envelope is concentrated in a network of ridges that are tightly apposed to the monolayer derived from the secretory granule, and the ridges coincide with butyrophilin labeling in the globule monolayer. Therefore, we propose that milk fat globule secretion is controlled by interactions between plasma membrane butyrophilin and butyrophilin in the secretory granule phospholipid monolayer rather than binding of butyrophilin-xanthine oxidoreductase complexes to secretory granule adipophilin.