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1.
Nucleic Acids Res ; 51(2): 908-918, 2023 01 25.
Article in English | MEDLINE | ID: mdl-36583341

ABSTRACT

Polyribosomes, the groups of ribosomes simultaneously translating a single mRNA molecule, are very common in both, prokaryotic and eukaryotic cells. Even in early EM studies, polyribosomes have been shown to possess various spatial conformations, including a ring-shaped configuration which was considered to be functionally important. However, a recent in situ cryo-ET analysis of predominant regular inter-ribosome contacts did not confirm the abundance of ring-shaped polyribosomes in a cell cytoplasm. To address this discrepancy, here we analyzed the cryo-ET structure of polyribosomes in diluted lysates of HeLa cells. It was shown that the vast majority of the ribosomes were combined into polysomes and were proven to be translationally active. Tomogram analysis revealed that circular polyribosomes are indeed very common in the cytoplasm, but they mostly possess pseudo-regular structures without specific inter-ribosomal contacts. Although the size of polyribosomes varied widely, most circular polysomes were relatively small in size (4-8 ribosomes). Our results confirm the recent data that it is cellular mRNAs with short ORF that most commonly form circular structures providing an enhancement of translation.


Subject(s)
Protein Biosynthesis , Ribosomes , Humans , HeLa Cells , Polyribosomes/metabolism , Ribosomes/genetics , Ribosomes/metabolism , RNA, Messenger/metabolism , Molecular Conformation
2.
Int J Mol Sci ; 23(10)2022 May 10.
Article in English | MEDLINE | ID: mdl-35628121

ABSTRACT

Dps (DNA-binding protein from starved cells) is well known for the structural protection of bacterial DNA by the formation of highly ordered intracellular assemblies under stress conditions. Moreover, this ferritin-like protein can perform fast oxidation of ferrous ions and subsequently accumulate clusters of ferric ions in its nanocages, thus providing the bacterium with physical and chemical protection. Here, cryo-electron microscopy was used to study the accumulation of iron ions in the nanocage of a Dps protein from Escherichia coli. We demonstrate that Fe2+ concentration in the solution and incubation time have an insignificant effect on the volume and the morphology of iron minerals formed in Dps nanocages. However, an increase in the Fe2+ level leads to an increase in the proportion of larger clusters and the clusters themselves are composed of discrete ~1-1.5 nm subunits.


Subject(s)
Escherichia coli Proteins , Ferritins , Bacterial Outer Membrane Proteins/genetics , Cryoelectron Microscopy , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Ferritins/metabolism , Ions/metabolism , Iron/metabolism
3.
Pharmaceutics ; 14(3)2022 Mar 05.
Article in English | MEDLINE | ID: mdl-35335951

ABSTRACT

Capsules with shells based on nanoparticles of different nature co-assembled at the interface of liquid phases of emulsion are promising carriers of lipophilic drugs. To obtain such capsules, theoretically using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and experimentally using dynamic light-scattering (DLS) and transmission electron microscopy (TEM) methods, the interaction of like-charged silica nanoparticles and detonation nanodiamonds in an aqueous solution was studied and their ratios selected for the formation of submicron-sized colloidosomes. The resulting colloidosomes were modified with additional layers of nanoparticles and polyelectrolytes, applying LbL technology. As a model anti-cancer drug, thymoquinone was loaded into the developed capsules, demonstrating a significant delay of the release as a result of colloidosome surface modification. Fluorescence flow cytometry and confocal laser scanning microscopy showed efficient internalization of the capsules by MCF7 cancer cells. The obtained results demonstrated a high potential for nanomedicine application in the field of the drug-delivery system development.

4.
Carbohydr Polym ; 282: 119106, 2022 Apr 15.
Article in English | MEDLINE | ID: mdl-35123742

ABSTRACT

Alginate hydrogels with embedded rigid percolating network of halloysite clay nanotubes were evaluated as a novel ink for 3D printing. Hydrophilic alginate macromolecules adsorbing on halloysite stabilize the network of the nanotubes and form their own network of interlaced polymer chains. The effect of halloysite content on the structure and properties of the hydrogels was studied by rheometry, thermogravimetric analysis, FTIR-spectroscopy, dynamic light scattering, transmission electron microscopy, and 3D cryo-electron microscopy. Hydrogels demonstrate a very pronounced shear-thinning at extrusion and rather quick viscosity recovery after extrusion assigned to rapid rearrangement of the network structure promoted by mobile alginate chains. Even at low volume fractions (up to 0.054) the nanotubes reinforce the hydrogel increasing its storage modulus up to 650 Pa and inducing the appearance of yield stress. These properties make the alginate/halloysite hydrogels promising for the application in 3D printing for fabrication of green and sustainable nanocomposite materials made from natural components.

5.
Int J Mol Sci ; 22(11)2021 Jun 03.
Article in English | MEDLINE | ID: mdl-34205216

ABSTRACT

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg2+ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe2+ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.


Subject(s)
Bacterial Outer Membrane Proteins/ultrastructure , DNA/ultrastructure , Escherichia coli Proteins/ultrastructure , Iron/chemistry , Magnesium/chemistry , Amino Acid Sequence/drug effects , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Cations/chemistry , Cryoelectron Microscopy , DNA/chemistry , DNA/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/ultrastructure , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Scattering, Small Angle , X-Ray Diffraction
6.
Int J Mol Sci ; 22(3)2021 Feb 02.
Article in English | MEDLINE | ID: mdl-33540542

ABSTRACT

The existence of a complete oxidative phosphorylation system (OXPHOS) supercomplex including both electron transport system and ATP synthases has long been assumed based on functional evidence. However, no structural confirmation of the docking between ATP synthase and proton pumps has been obtained. In this study, cryo-electron tomography was used to reveal the supramolecular architecture of the rat heart mitochondria cristae during ATP synthesis. Respirasome and ATP synthase structure in situ were determined using subtomogram averaging. The obtained reconstructions of the inner mitochondrial membrane demonstrated that rows of respiratory chain supercomplexes can dock with rows of ATP synthases forming oligomeric ordered clusters. These ordered clusters indicate a new type of OXPHOS structural organization. It should ensure the quickness, efficiency, and damage resistance of OXPHOS, providing a direct proton transfer from pumps to ATP synthase along the lateral pH gradient without energy dissipation.


Subject(s)
Mitochondria, Heart/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proton-Translocating ATPases/metabolism , Proton Pumps/metabolism , Animals , Cryoelectron Microscopy , Electron Transport , Mitochondria, Heart/ultrastructure , Mitochondrial Membranes/ultrastructure , Mitochondrial Proton-Translocating ATPases/ultrastructure , Oxidative Phosphorylation , Protein Conformation , Proton Pumps/ultrastructure , Rats , Rats, Wistar
7.
Biomolecules ; 10(1)2019 12 26.
Article in English | MEDLINE | ID: mdl-31888079

ABSTRACT

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization-one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps-DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps-DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.


Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Crystallography, X-Ray , DNA/chemistry , Electron Microscope Tomography , Scattering, Small Angle , Amino Acid Sequence , Models, Molecular , Nucleic Acid Conformation , Protein Conformation
8.
Biochimie ; 156: 196-205, 2019 Jan.
Article in English | MEDLINE | ID: mdl-30385318

ABSTRACT

Human plasma butyrylcholinesterase (BChE) is an endogenous bioscavenger that hydrolyzes numerous medicamentous and poisonous esters and scavenges potent organophosphorus nerve agents. BChE is thus a marker for the diagnosis of OP poisoning. It is also considered a therapeutic target against Alzheimer's disease. Although the X-ray structure of a partially deglycosylated monomer of human BChE was solved 15 years ago, all attempts to determine the 3D structure of the natural full-length glycosylated tetrameric human BChE have been unsuccessful so far. Here, a combination of three complementary structural methods-single-particle cryo-electron microscopy, molecular dynamics and small-angle X-ray scattering-were implemented to elucidate the overall structural and spatial organization of the natural tetrameric human plasma BChE. A 7.6 ŠcryoEM map clearly shows the major features of the enzyme: a dimer of dimers with a nonplanar monomer arrangement, in which the interconnecting super helix complex PRAD-(WAT)4-peptide C-terminal tail is located in the center of the tetramer, nearly perpendicular to its plane, and is plunged deep between the four subunits. Molecular dynamics simulations allowed optimization of the geometry of the molecule and reconstruction of the structural features invisible in the cryoEM density, i.e., glycan chains and glycan interdimer contact areas, as well as intermonomer disulfide bridges at the C-terminal tail. Finally, SAXS data were used to confirm the consistency of the obtained model with the experimental data. The tetramer organization of BChE is unique in that the four subunits are joined at their C-termini through noncovalent contacts with a short polyproline-rich peptide. This tetramer structure could serve as a model for the design of highly stable glycosylated tetramers.


Subject(s)
Butyrylcholinesterase/chemistry , Molecular Dynamics Simulation , Cryoelectron Microscopy , Humans , Protein Structure, Quaternary , Scattering, Small Angle , X-Ray Diffraction
9.
Nanotechnology ; 30(3): 035603, 2019 Jan 18.
Article in English | MEDLINE | ID: mdl-30422813

ABSTRACT

We have designed sensors based on Ag nanoparticles synthesized in situ on the vaterite beads. In this article we demonstrate an approach to produce size controllable spherical and elliptical vaterite particles and discuss time-dependent in situ Ag nanoparticles synthesis and its potential effect on surface-enhanced Raman scattering. The time dependent silver reduction synthesis in inorganic porous particles allows to regulate the number and size of Ag nanoparticles. It is shown that the irregular surface and high porosity of vaterite particles and large amount (surface filling factor) of the Ag nanoparticles are the critical parameters to increase the SERS signal to 104 times. Such inorganic composites have a huge potential in medical applications; soon they provide an opportunity to study intracellular processes in vivo. The detailed characterization of the microstructure of these composites was studied by scanning and transmission electron microscopy, including 3D visualization and energy dispersive x-ray microanalysis.

10.
Phys Rev Lett ; 101(16): 164801, 2008 Oct 17.
Article in English | MEDLINE | ID: mdl-18999674

ABSTRACT

Beam deflection due to axial channeling in a silicon crystal bent along the 111 axis was observed with 400 GeV/c protons at the CERN Super Proton Synchrotron. The condition for doughnut scattering of protons by the atomic strings of the crystal was attained. Such a condition allowed one to observe a beam deflection of 50 murad with about 30% efficiency. The contribution of hyperchanneled states of protons to the observed beam deflection was less than 2% according to simulation results.

11.
Rev Sci Instrum ; 79(2 Pt 1): 023303, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18315289

ABSTRACT

A high performance apparatus has been designed and built by the H8-RD22 collaboration for the study of channeling and volume reflection phenomena in the interaction of 400 GeV/c protons with bent silicon crystals, during the 2006 data taking in the external beamline H8 of the CERN SPS. High-quality silicon short crystals were bent by either anticlastic or quasimosaic effects. Alignment with the highly parallel (8 murad divergence) proton beam was guaranteed through a submicroradian goniometric system equipped with both rotational and translational stages. Particle tracking was possible by a series of silicon microstrip detectors with high-resolution and a parallel plate gas chamber, triggered by various scintillating detectors located along the beamline. Experimental observation of volume reflection with 400 GeV/c protons proved true with a deflection angle of (10.4+/-0.5) murad with respect to the unperturbed beam, with a silicon crystal whose (111) planes were parallel to the beam.

12.
Phys Rev Lett ; 98(15): 154801, 2007 Apr 13.
Article in English | MEDLINE | ID: mdl-17501358

ABSTRACT

The volume reflection phenomenon was detected while investigating 400 GeV proton interactions with bent silicon crystals in the external beam H8 of the CERN Super Proton Synchrotron. Such a process was observed for a wide interval of crystal orientations relative to the beam axis, and its efficiency exceeds 95%, thereby surpassing any previously observed value. These observations suggest new perspectives for the manipulation of high-energy beams, e.g., for collimation and extraction in new-generation hadron colliders, such as the CERN Large Hadron Collider.

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