Assessment of core-shell nanoparticles surface structure heterogeneity by SAXS contrast variation and ab initio modeling.

For the biomedical applications of nanoparticles, the study of their structure is a major step towards understanding the mechanisms of their interaction with biological environment. Detailed structural analysis of particles' surface is vital for rational design of drug delivery systems. In particular, for core-shell or surface-modified nanoparticles surface structure can be described in terms of shell coating uniformity and shell thickness uniformity around the nanoparticle core. Taken together, these terms can be used to indicate degree of heterogeneity of nanoparticle surface structure. However, characterization of nanoparticle surface structure under physiological conditions is challenging due to limitations of experimental techniques. In this paper, we apply SAXS contrast variation combined with ab initio bead modeling for this purpose. Approach is based on the fact that nanoparticles under study are produced by self-assembly of phospholipid-conjugated molecules that possess moieties with significantly different electron densities enabling SAXS technique to be used to distinguish nanoparticle shell and study its structure. Ab initio single phase and ab initio multiphase modeling based on SAXS curve of nanoparticles in phosphate buffer solution allowed to reconstruct nanoparticle shell coating and assess its uniformity, while serial nanoparticle reconstructions from solutions with gradually increased solvent electron densities revealed relative shell coating thickness around nanoparticle core. Nanoparticle shell structure representation was verified by molecular dynamics simulation and derived full-atom nanoparticle shell structure showed good agreement with SAXS-derived representation. Obtained data indicate that studied nanoparticles exhibit highly heterogeneous surface structure.

Characterization of Tobacco Mosaic Virus Virions and Repolymerized Coat Protein Aggregates in Solution by Small-Angle X-Ray Scattering.

The structure of tobacco mosaic virus (TMV) virions and stacked disk aggregates of TMV coat protein (CP) in solution was analyzed by synchrotron-based small-angle X-ray scattering (SAXS) and negative contrast transmission electron microscopy (TEM). TMV CP aggregates had a unique stability but did not have helical symmetry. According to the TEM data, they were stacked disks associated into transversely striated rod-shaped structures 300 to 800 Å long. According to modeling based on the crystallographic model of the 4-layer TMV CP aggregate (PDB: 1EI7), the stacked disks represented hollow cylinders. The calculated SAXS pattern for the disks was compared to the experimental one over the entire measured range. The best correlation with the SAXS data was found for the model with the repeating central pair of discs; the SAXS curves for the stacked disks were virtually identical irrespectively of the protein isolation method. The positions of maxima on the scatter curves could be used as characteristic features of the studied samples; some of the peaks were assigned to the existing elements of the quaternary structure (periodicity of aggregate structure, virion helix pitch). Low-resolution structural data for the repolymerized TMV CP aggregates in solution under conditions similar to natural were produced for the first time. Analysis of such nano-size objects is essential for their application in biomedicine and biotechnology.

Solution Structure, Self-Assembly, and Membrane Interactions of the Matrix Protein from Newcastle Disease Virus at Neutral and Acidic pH.

Newcastle disease virus (NDV) is an enveloped paramyxovirus. The matrix protein of the virus (M-NDV) has an innate propensity to produce virus-like particles budding from the plasma membrane of the expressing cell without recruiting other viral proteins. The virus predominantly infects the host cell via fusion with the host plasma membrane or, alternatively, can use receptor-mediated endocytic pathways. The question arises as to what are the mechanisms supporting such diversity, especially concerning the assembling and membrane binding properties of the virus protein scaffold under both neutral and acidic pH conditions. Here, we suggest a novel method of M-NDV isolation in physiological ionic strength and employ a combination of small-angle X-ray scattering, atomic force microscopy with complementary structural techniques, and membrane interaction measurements to characterize the solution behavior/structure of the protein as well as its binding to lipid membranes at pH 4.0 and pH 7.0. We demonstrate that the minimal structural unit of the protein in solution is a dimer that spontaneously assembles in a neutral milieu into hollow helical oligomers by repeating the protein tetramers. Acidic pH conditions decrease the protein oligomerization state to the individual dimers, tetramers, and octamers without changing the density of the protein layer and lipid membrane affinity, thus indicating that the endocytic pathway is a possible facilitator of NDV entry into a host cell through enhanced scaffold disintegration.IMPORTANCE The matrix protein of the Newcastle disease virus (NDV) is one of the most abundant viral proteins that regulates the formation of progeny virions. NDV is an avian pathogen that impacts the economics of bird husbandry due to its resulting morbidity and high mortality rates. Moreover, it belongs to the Avulavirus subfamily of the Paramyxoviridae family of Mononegavirales that include dangerous representatives such as respiratory syncytial virus, human parainfluenza virus, and measles virus. Here, we investigate the solution structure and membrane binding properties of this protein at both acidic and neutral pH to distinguish between possible virus entry pathways and propose a mechanism of assembly of the viral matrix scaffold. This work is fundamental for understanding the mechanisms of viral entry as well as to inform subsequent proposals for the possible use of the virus as an adequate template for future drug or vaccine delivery.

[Structure of Potato Virus A Coat Protein Particles and Their Dissociation].

This paper reports on a complex structural analysis of the potato virus A coat protein using a set of complementary physico-chemical methods. We have demonstrated previously that this protein does not exist as individual subunits in solution and undergoes association into oligomers with subsequent transition to ß-conformation. The purpose of the present work was to study the possible mechanisms of this transformation and to search for methods that dissociate protein oligomers. To analyze the low resolution protein structure in solution, small-angle X-ray scattering was used. Stable particles representing clusters of 30 coat protein subunits were present even in an aqueous salt solution with a high ionic strength and pH (pH 10.5; 0.5 M NaCl). The particles did not dissociate in the presence of 10 mM dextran sulfates (15 and 100 kDa). Dissociation in the presence of 5.2 mM sodium dodecyl sulfate results in the formation of the subunit-detergent complexes consisting of 10-12 small particles joined together like "beads on a string". Similar effects of sodium dodecyl sulfate were shown for serum albumins (bovine and human). Denaturation of the potato virus A coat protein molecules occurs in the presence of detergent concentrations that are seven times lower than that in albumins (5.2 and 35 mM), which confirms low stability of the potato virus A coat protein. Using spectral methods, preservation of the secondary structure and loss of the tertiary structure of the protein in its complex with sodium dodecyl sulfate have been demonstrated. Possible mechanism for protein particle formation through the interaction between unordered terminal domains and their transformation into ß-structures has been suggested.

[Superparamagnetic Cobalt Ferrite Nanoparticles "Blow up" Spatial Ordering of Double-stranded DNA Molecules].

The formation of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules, handled by positively charged superparamagnetic cobalt ferrite nanoparticles, as well as action of these nanoparticles on DNA dispersion, are considered. The binding of magnetic nanoparticles to the linear double-stranded DNA in solution of high ionic strength (0.3 M NaCl) and subsequent phase exclusion of these complexes from polyethylene glycol-containing solutions lead to their inability to form dispersions, whose particles do possess the spatially twisted arrangement of neighboring double-stranded DNA molecules. The action of magnetic nanoparticles on DNA dispersion (one magnetic nanoparticle per one double-stranded DNA molecule) results in such "perturbation" of DNA structure at sites of magnetic nanoparticles binding that the regular spatial structure of DNA dispersion particles "blows up"; this process is accompanied by disappearance of both abnormal optical activity and characteristic Bragg maximum on the small-angle X-ray scattering curve. Allowing with the fact that the physicochemical properties of the DNA liquid-crystalline dispersion particles reflect features of spatial organization of these molecules in chromosomes of primitive organisms, it is possible, that the found effect can have the relevant biological consequences.

[Linear clusters of gold nanoparticles in quasinematic layers of DNA liquid-crystalline dispersion particles].

The effects of small size (approximately 2 nm) gold nanoparticles on the properties of particles of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules were analyzed. It has been shown that gold nanoparticles induce two different processes. First, they facilitate reorganization of the spatial cholesteric structure of dispersion particles to nematic one. This process is accompanied by the fast decrease in the amplitude of abnormal band in the CD spectrum. Second, they can form ensembles consisting of gold nanoparticles. This process is accompanied by the development and displacement of surface plasmon resonance band in the visible region of the absorption spectrum. The appearance of this band is analyzed by considering two different models of the formation of ensembles consisting of gold nanoparticles. By small-angle X-ray scattering we performed structural analysis of phases formed by DNA cholesteric liquid-crystalline dispersion particles treated with gold nanoparticles. As a result of this study it was possible to prove the formation of linear clusters of gold nanoparticles in the "free space" between the adjacent DNA molecules fixed in the quasinematic layers of liquid-crystalline particles. It has been hypothesized that the formation of linear clusters of gold nanoparticles is most likely related to DNA molecules, ordered in the spatial structure of quasinematic layers, and the toxicity of these nanoparticles in biological systems hypothesized.

SAXS-data-based structural modeling of DNA-gadolinium complexes fixed in particles of cholesteric liquid-crystalline dispersions.

Structure of cholesteric liquid-crystalline dispersions (CLCDs) formed by double-stranded DNA molecules and treated with gadolinium salts was studied by small-angle X-ray scattering (SAXS). The obtained SAXS data open the way for structural modeling of these complexes to obtain a reasonable explanation for the correlated decrease in amplitude of an abnormal negative band in the circular dichroism (CD) spectra and the characteristic Bragg peak in the experimental small-angle X-ray scattering curves observed on treatment of CLCD by gadolinium salts. Model simulations of different kinds of structural organizations of the DNA-gadolinium complex were performed using novel SAXS data analysis methods in combination with several new, complementary modeling techniques, enabling us to build low-resolution three-dimensional structural models of DNA-gadolinium complexes fixed in CLCD particles. The obtained models allow us to suggest that a change takes place in the helical twist of quasinematic layers formed by these molecules at high concentrations of gadolinium salt. This change in the twist can be used to explain the experimentally observed increase in amplitude of an abnormal band in the CD spectra of DNA CLCD.

[Formation of the regular, optical isotropic phase of low molecular weight DNA molecules modified by cis-dichlorodiammineplatinum]. / Obrazovanie uporiadochennoi, opticheski izotropnoi fazy molekul DNK nizkoi molekuliarnoi massy, modifitsirovannykh tsis-dikhlorodiamminplatinoi.

It is shown that condensation of DNA molecules of low molecular mass (less than 1 X 10(6) in NaClO4-containing solution of poly(ethylene glycol) brings about formation of cholesteric liquid crystal phase; pattern of this phase is presented. It has been found by means of X-ray analysis and polarization microscopy that at certain level of modification with cis-dichlorodiammineplatinum (II) the DNA molecules instead of cholesteric structure form an ordered optically isotropic phase. The problem about the causes of the formation of such phase and about the pattern of spatial organisation of adjacent DNA molecules in it remains open.

[Kinetics of formation of optically active liquid-crystalline microphases of low molecular weight DNA]. / Kinetika formirovaniia opticheski aktivnykh zhidkokristallicheskikh mikrofaz nizkomolekuliarnoi DNK.

Studies were carried out of compactization kinetics of low molecular DNA by means of CD and correlation spectroscopy.

[Rheological properties of solutions of purified hemoglobin and blood]. / Reologicheskie svoistva rastvorov ochishchennogo gemoglobina v sochetanii s krov'iu.

The results of studying rheological properties of solutions of purified hemoglobin and its mixtures with blood indicate low relative viscosity of the solutions, as well as an appreciable decrease, when the blood is diluted with these solutions, of the indicators of its viscosity and yield stress without impairment of the blood suspension stability. This allows the conclusion about prospectiveness of further studies aimed at the design of hemocorrectors on the basis of extraerythrocytic hemoglobin.

[Correlation between conformation distortion of the DNA sugar-phosphate backbone and high optical activity of its compact form]. / O sviazi mezhdu konformatsionnoi neodnorodnost'iu struktury dvukhtsepochechnoi DNK i vysokoi opticheskoi aktivnost'iu ee kompaktnoi formy.

Different physico-chemical methods (CD, ORD, small-angle X-ray diffraction, etc) were used for investigating the properties of the DNA compact particles formed in PEG-containing water-salt solutions. It has been shown that small-angle reflection, characteristic of the DNA compact particles, changes from 36.8 A (CPEG = 140 mg/ml) to 25 A (CPEG = 300 mg/ml). The maximal optical activity (the intense negative CD-band and optical rotation [alpha] = 60 000 degrees) are inherent properties of the DNA compact particles formed at CPEG 120--180 mg/ml. The high optical activity points to the twist of DNA chromophores through the DNA molecule resulting in a long-rang pitch (P approximately 2000A). Such macroscopic superhelical structure (diameter 40--30 A) is due to conformational distortion of the DNA double-helix with alternating "left" and "right" orientation of chromophoes. Disappearance of conformation distortion is accompanied by disappearance of the high optical activity of the DNA compact particles and results in a small-angle reflection of 25 A. Taking into account the reasons of formation of the optically-active DNA compact particles conditions are suggested to conserve high optical activity at CPEG equal to 400 mg/ml.