Biomolecular corona is the major obstacle in clinical translation of nanomedicines. To overcome this problem, comprehensive studies of the processes leading to the formation of a biomolecular corona are required. Since such dynamical studies require high spatial and temporal resolution, nanoparticles utilized in it should enable combined experimental and simulation studies. Interactions at nano-bio interface are defined by nanoparticle surface properties such as topography, charge and surface chemistry. Hence, as the preliminary step towards deep understanding of the processes of corona formation it is necessary to develop nanoparticles employing various biocompatible materials and characterize their surface properties. In this work, we applied molecular dynamics simulation to study surface structure of organic core-shell nanoparticles formed by self-assembly of synthetic molecules composed of DOPE lipid, carboxymethylglycine spacer and biotin. Lipid moieties form the hydrophobic core, spacer motifs serve as a hydrophilic shell and biotin residues function as targeting ligand. By mixing such function-spacer-lipid, spacer-lipid and lipid-only constructs at various molar ratios, densities of the ligand and spacer on nanoparticle surface were modified. For convenient analysis of the structure and dynamics of all regions of nanoparticles’ surface, we compiled topography maps based on atomic coordinates. It was shown that an increase in the density of the shell does not reduce exposure of the core, but increases shell average thickness. Biotin, due to its alkyl valeric acid chain and spacer flexibility, is localized primarily near the hydrophobic core and its partial presentation on the surface occurs only in nanoparticles with higher ligand densities. However, an increase in biotin density leads to its clustering. In turn, ligand clustering diminishes stealth properties of the shell and targeting efficiency. Based on nanoparticle surface structures we determined the optimal density of biotin. Experimental studies reported in the literature confirm these conclusions. We also suggest design tips to achieve preferred biotin presentation. Simulation results are consistent with the synchrotron SAXS profile. We believe that such studies will contribute to the better understanding of nano-bio interactions towards the rational design of efficient drug delivery systems.

New promising materials based on a collagen-chitosan matrix containing conjugates of Ag nanoparticles and gramicidin S were obtained for biomedical applications using metal-vapor synthesis. The analysis of the morphology, surface composition and electronic state of the metal in nanocomposites was carried out. Ag atoms 77 K PriOH + Gramicidin S (GramS)-PriOH + collagen-chitosan matrix (CollCh) AgNPs@GramS-AgNPs@GramS/ CollCh PriOH

Capsids of the potexvirus family virions are characterized by the presence on the surface of virions of partially disordered N-terminal protein fragments of various lengths. The present study is devoted to studying the effect of in situ removal of the N-terminal domain of coat protein (CP) on structural organization and physicochemical properties of the potato virus X (PVX) virions. The work considers PVX virions containing an intact Ps-form CP, as well as virions including an in situ degraded Pf-form devoid of 19/21 amino acid residues from the N-end (PVXΔN). Synchrotron small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), tritium bombardment, and several other physicochemical methods were used in the study. Analysis of the images obtained using TEM revealed similarities in the architecture of filamentous PVX and PVXΔN virions. SAXS results demonstrated differences in organization of the capsid of PVX and PVXΔN virions: the latter was characterized by the reduced size of the ordered regions, indicating partial disruption of the structure of the viral protein framework. In addition, based on the SAXS scattering curves, parameters of the spiral packing of virions in solution were calculated, and structural modeling of particles was performed. Modeling results also indicate changes in the structure of the capsid due to removal of the ΔN-peptide. Using information about the secondary structure of the PVX model (PDB ID: 6R7G) and data from our previous studies on tritium labeling of the surface sites of PVX and PVXΔN virions, comparative analysis of the label incorporation profiles into elements of the protein secondary structure was conducted. This approach made it possible to predict localization of the ΔN-peptide above the amino acid residues of neighboring helical subunits (122-129 and 143-153) and demonstrate stabilizing role of this peptide on the overall structure of the virion. Increase in the level of labelling in the C-terminal region after removal of the ΔN-peptide also indicates decrease in the compactness of the virion. In general, the gained knowledge could be useful when using virus-like nanoparticles in biotechnology.

The structural study of plant viruses is of great importance to reduce the damage caused by these agricultural pathogens and to support their biotechnological applications. Nowadays, X-ray crystallography, NMR spectroscopy and cryo-electron microscopy are well accepted methods to obtain the 3D protein structure with the best resolution. However, for large and complex supramolecular structures such as plant viruses, especially flexible filamentous ones, there are a number of technical limitations to resolving their native structure in solution. In addition, they do not allow us to obtain structural information about dynamics and interactions with physiological partners. For these purposes, small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) are well established. In this review, we have outlined the main principles of these two methods and demonstrated their advantages for structural studies of plant viruses of different shapes with relatively high spatial resolution. In addition, we have demonstrated the ability of AFM to obtain information on the mechanical properties of the virus particles that are inaccessible to other experimental techniques. We believe that these under-appreciated approaches, especially when used in combination, are valuable tools for studying a wide variety of helical plant viruses, many of which cannot be resolved by classical structural methods.

The results of investigation of DNA-protein complexes by two independent structural methods, namely, molecular dynamics (MD) and small-angle X-ray scattering (SAXS), were compared. Molecular dynamics is a computational method enabling the visualization of the behavior of macromolecules in real media, which is based on the laws of physics, but it is limited by numerous simplifications. Small-angle X-ray scattering is an X-ray method, which allows the reconstruction of the three-dimensional structure of a system in solution from a one-dimensional small-angle scattering profile, but it faces the problem of ambiguity in solving inverse problems. The use of structural characteristics of the complexes determined by SAXS for the validation of 3D structural models generated by MD simulations made it possible to significantly reduce the ambivalence of theoretical predictions and demonstrated the efficiency of a combination of MD simulations and SAXS for solving problems of structural biology.

The oligomeric state of the nucleoid-associated protein IHF (integration host factor) plays a significant role in the organization and compaction of the bacterial nucleoid and also in the evolution of bacterial resistance to unfavorable environmental conditions, in particular to antibiotics. Although IHF was identified more than 25 years ago, the molecular mechanisms of its participation in these processes are poorly understood. Using small-angle X-ray scattering, it was demonstrated for the first time that there are different oligomeric states of IHF in an aqueous medium depending on the presence of metal cations. It was found that the presence of Mg2+ and K+ ions hinders the formation of high-order oligomers of IHF. The results of this study may be useful in the development of strategies against bacterial resistance to drugs.

Biomolecular corona is the major obstacle to the clinical translation of nanomedicines. Since corona formation is governed by molecular interactions at the nano-bio interface, nanoparticle surface properties such as topography, charge and surface chemistry can be tuned to manipulate biomolecular corona formation. To this end, as the first step towards a deep understanding of the processes of corona formation, it is necessary to develop nanoparticles employing various biocompatible materials and characterize their surface structure and dynamics at the molecular level. In this work, we applied molecular dynamics simulation to study the surface structure of organic core-shell nanoparticles formed by the self-assembly of synthetic molecules composed of a DOPE lipid, a carboxymethylglycine spacer and biotin. Lipid moieties form the hydrophobic core, spacer motifs serve as a hydrophilic shell and biotin residues function as a targeting ligand. By mixing such function-spacer-lipid, spacer-lipid and lipid-only constructs at various molar ratios, densities of the ligand and spacer on the nanoparticle surface were modified. For convenient analysis of the structure and dynamics of all regions of the nanoparticle surface, we compiled topography maps based on atomic coordinates. It was shown that an increase in the density of the shell does not reduce exposure of the core, but increases shell average thickness. Biotin, due to its alkyl valeric acid chain and spacer flexibility, is localized primarily near the hydrophobic core and its partial presentation on the surface occurs only in nanoparticles with higher ligand densities. However, an increase in biotin density leads to its clustering. In turn, ligand clustering diminishes the stealth properties of the shell and targeting efficiency. Based on nanoparticle surface structures, we determined the optimal density of biotin. Experimental studies reported in the literature confirm these conclusions. We also suggest design tips to achieve the preferred biotin presentation. Simulation results are consistent with the synchrotron SAXS profile. We believe that such studies will contribute to a better understanding of nano-bio interactions towards the rational design of efficient drug delivery systems.

Sustained interest in the use of renewable resources for the production of medical materials has stimulated research on bacterial cellulose (BC) and nanocomposites based on it. New Ag-containing nanocomposites were obtained by modifying various forms of BC with Ag nanoparticles prepared by metal–vapor synthesis (MVS). Bacterial cellulose was obtained in the form of films (BCF) and spherical BC beads (SBCB) by the Gluconacetobacter hansenii GH-1/2008 strain under static and dynamic conditions. The Ag nanoparticles synthesized in 2-propanol were incorporated into the polymer matrix using metal-containing organosol. MVS is based on the interaction of extremely reactive atomic metals formed by evaporation in vacuum at a pressure of 10−2 Pa with organic substances during their co-condensation on the cooled walls of a reaction vessel. The composition, structure, and electronic state of the metal in the materials were characterized by transmission and scanning electron microscopy (TEM, SEM), powder X-ray diffraction (XRD), small-angle X-ray scattering (SAXS) and X-ray photoelectron spectroscopy (XPS). Since antimicrobial activity is largely determined by the surface composition, much attention was paid to studying its properties by XPS, a surface-sensitive method, at a sampling depth about 10 nm. C 1s and O 1s spectra were analyzed self-consistently. XPS C 1s spectra of the original and Ag-containing celluloses showed an increase in the intensity of the C-C/C-H groups in the latter, which are associated with carbon shell surrounding metal in Ag nanoparticles (Ag NPs). The size effect observed in Ag 3d spectra evidenced on a large proportion of silver nanoparticles with a size of less than 3 nm in the near-surface region. Ag NPs in the BC films and spherical beads were mainly in the zerovalent state. BC-based nanocomposites with Ag nanoparticles exhibited antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli bacteria and Candida albicans and Aspergillus niger fungi. It was found that AgNPs/SBCB nanocomposites are more active than Ag NPs/BCF samples, especially against Candida albicans and Aspergillus niger fungi. These results increase the possibility of their medical application.

The amino acid sequences of the coat proteins (CPs) of the potexviruses potato virus X (PVX) and alternanthera mosaic virus (AltMV) share 40

The search for materials for a new generation of wound coatings is important due to the increase in antibiotic-resistant microorganisms and the number of patients with untreatable chronic purulent wounds. Metal nanoparticles, specifically silver nanoparticles, have antimicrobial activity and do not induce known bacterial resistance. To obtain new Ag-containing nanocomposites, type I collagen was extracted by an enzyme–acid method from cattle tendons. Silver nanoparticles were obtained by an environmentally safe method, metal-vapor synthesis (MVS), which enables obtaining metal nanoparticles without impurities. For this, metal vapors were cocondensed in a vacuum of 10−2 Pa on the walls of a quartz reactor cooled to 77 K using acetone as an organic dispersion medium. The composition of the collagen surface was determined by XPS using the spectra of C1s, N1s, and O1s. The presence of a peak with a binding energy of approximately 368.57 eV in the Ag 3d5/2 spectrum indicates the state of Ag0 silver atoms in the nanocomposite. SEM images showed that collagen contributes to the effective stabilization of Ag nanoparticles with an average size of 13.0 ± 3.5 nm. It was found that collagen is non-toxic and biocompatible with skin cells and fibroblasts. The collagen–Ag nanoparticle nanocomposites exhibited antimicrobial activity against bacteria Bacillus subtilis, Escherichia coli, and fungi Aspergillus niger.

Au and Fe nanoparticles and their conjugates with the drug methotrexate were obtained by an environmentally safe method of metal–vapor synthesis (MVS). The materials were characterized by transmission and scanning electron microscopy (TEM, SEM), X-ray photoelectron spectroscopy (XPS), and small-angle X-ray scattering using synchrotron radiation (SAXS). The use of acetone as an organic reagent in the MVS makes it possible to obtain Au and Fe particles with an average size of 8.3 and 1.8 nm, respectively, which was established by TEM. It was found that Au, both in the NPs and the composite with methotrexate, was in the Au0, Au+ and Au3+ states. The Au 4f spectra for Au-containing systems are very close. The effect of methotrexate was manifested in a slight decrease in the proportion of the Au0 state—from 0.81 to 0.76. In the Fe NPs, the main state is the Fe3+ state, and the Fe2+ state is also present in a small amount. The analysis of samples by SAXS registered highly heterogeneous populations of metal nanoparticles coexisting with a wide proportion of large aggregates, the number of which increased significantly in the presence of methotrexate. For Au conjugates with methotrexate, a very wide asymmetric fraction with sizes up to 60 nm and a maximum of ~4 nm has been registered. In the case of Fe, the main fraction consists of particles with a radius of 4.6 nm. The main fraction consists of aggregates up to 10 nm. The size of the aggregates varies in the range of 20–50 nm. In the presence of methotrexate, the number of aggregates increases. The cytotoxicity and anticancer activity of the obtained nanomaterials were determined by MTT and NR assays. Fe conjugates with methotrexate showed the highest toxicity against the lung adenocarcinoma cell line and Au nanoparticles loaded with methotrexate affected the human colon adenocarcinoma cell line. Both conjugates displayed lysosome-specific toxicity against the A549 cancer cell line after 120 h of culture. The obtained materials may be promising for the creation of improved agents for cancer treatment.

In response to adverse environmental factors, Escherichia coli cells actively produce Dps proteins which form ordered complexes (biocrystals) with bacterial DNA to protect the genome. The effect of biocrystallization has been described extensively in the scientific literature; furthermore, to date, the structure of the Dps–DNA complex has been established in detail in vitro using plasmid DNA. In the present work, for the first time, Dps complexes with E. coli genomic DNA were studied in vitro using cryo-electron tomography. We demonstrate that genomic DNA forms one-dimensional crystals or filament-like assemblies which transform into weakly ordered complexes with triclinic unit cells, similar to what is observed for plasmid DNA. Changing such environmental factors as pH and KCl and MgCl2 concentrations leads to the formation of cylindrical structures.

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.

The structures of hydrophobic membrane proteins are studied using matrices, which serve as models of cell membranes and are formed by the appropriate amphiphilic molecules, e.g., by surfactant or lipid molecules. To study the structure of a protein incorporated into an artificial membrane, first of all it is necessary to determine the structure of the membrane. The ELLLIP and ELLMIC algorithms were previously developed to address this issue by small-angle X-ray scattering. These algorithms allow the construction of models of ellipsoidal vesicles based on the atomic structure of a lipid or surfactant monomer. However, the results of modeling depend, to a large extent, on the subjective assessment of the initial values of the structural parameters of the matrices and may be wrong due to the ambiguity in the solution of such problems. Here, we present an independent approach to the determination of the initial sizes of model membranes for their subsequent structural modeling, which is based on the analysis of the pair-distance distribution functions derived directly from the small-angle X-ray scattering curve.

Structure and function of bacterial nucleoid is controlled by the nucleoid-associated proteins (NAP). In any phase of growth, various NAPs, acting sequentially, condense nucleoid and facilitate formation of its transcriptionally active structure. However, in the late stationary phase, only one of the NAPs, Dps protein, is strongly expressed, and DNA–protein crystals are formed that transform nucleoid into a static, transcriptionally inactive structure, effectively protected from the external influences. Discovery of crystal structures in living cells and association of this phenomenon with the bacterial resistance to antibiotics has aroused great interest in studying this phenomenon. The aim of this work is to obtain and compare structures of two related NAPs (HU and IHF), since they are the ones that accumulate in the cell at the late stationary stage of growth, which precedes formation of the protective DNA–Dps crystalline complex. For structural studies, two complementary methods were used in the work: small-angle X-ray scattering (SAXS) as the main method for studying structure of proteins in solution, and dynamic light scattering as a complementary one. To interpret the SAXS data, various approaches and computer programs were used (in particular, the evaluation of structural invariants, rigid body modeling and equilibrium mixture analysis in terms of the volume fractions of its components were applied), which made it possible to determine macromolecular characteristics and obtain reliable 3D structural models of various oligomeric forms of HU and IHF proteins with 2 nm resolution typical for SAXS. It was shown that these proteins oligomerize in solution to varying degrees, and IHF is characterized by the presence of large oligomers consisting of initial dimers arranged in a chain. An analysis of the experimental and published data made it possible to hypothesize that just before the Dps expression, it is IHF that forms toroidal structures previously observed in vivo and prepares the platform for formation of DNA–Dps crystals. The results obtained are necessary for further investigation of the phenomenon of biocrystal formation in bacterial cells and finding ways to overcome resistance of various pathogens to external conditions.

Nucleoid-associated proteins (NAPs) control the structure and functions of bacterial nucleoid. Histone-like HU proteins are most abundant NAPs in dividing bacterial cells. Previously, structural ensembles of conformations of HU proteins from pathogenic mycoplasmas Spiroplasma melliferum and Mycoplasma gallisepticum were obtained using NMR spectroscopy. A structural study of these mycoplasma proteins is performed by small-angle X-ray scattering (SAXS). The occurrence of individual conformations from the ensemble, obtained by NMR, is estimated from the scattering data on HU protein solutions. In particular, an approach based on characterization of equilibrium mixtures in terms of volume fractions of their components was applied. The general shape of the proteins and their oligomeric state are independently confirmed using ab initio bead modelling. The flexibility of DNA-binding protein domains is analyzed by the ensemble optimization method, which is based on comparison of the structural characteristics of conformations fitting the SAXS data to the distribution of these characteristics in a randomly generated set. The results obtained give a new insight on the variability of the structure of HU proteins, which is necessary for their functioning.

Enveloped viruses, which include such pathogens as SARS-CoV-2, influenza virus, HIV, and many others, represent a family of severe pathogens. These enveloped viruses share common structural proteins that are essential to their assembly, budding, and fusion to target cell membrane. In all enveloped viruses, the structural proteins with the highest copy number are their capsid or matrix proteins, which simultaneously are the most conserved proteins between all the different viral strains of any one virus. However, none of these proteins yet serves as a target for antiviral drugs for any enveloped virus. We used different biophysical and structural methods to demonstrated that all capsid or matrix proteins studied to date, irrespective of origin, use common physicochemical mechanisms to regulate the assembly of the viral scaffold and budding of progeny virions. These mechanisms include surface activity, binding affinity, and insertion into the lipid membrane. Thus, despite the sequence differences of matrix and capsid proteins of different viruses, their functional activities fall into a few possible actions, and these functional activities are common for all enveloped viruses. Thus, our findings open a new way for the search for possible antiviral drugs, targeted at viral protein assembly and lipid-protein interactions. This work was supported by the Russian Foundation for Basic Researches (project #20-54-14006). Enveloped viruses, which include such pathogens as SARS-CoV-2, influenza virus, HIV, and many others, represent a family of severe pathogens. These enveloped viruses share common structural proteins that are essential to their assembly, budding, and fusion to target cell membrane. In all enveloped viruses, the structural proteins with the highest copy number are their capsid or matrix proteins, which simultaneously are the most conserved proteins between all the different viral strains of any one virus. However, none of these proteins yet serves as a target for antiviral drugs for any enveloped virus. We used different biophysical and structural methods to demonstrated that all capsid or matrix proteins studied to date, irrespective of origin, use common physicochemical mechanisms to regulate the assembly of the viral scaffold and budding of progeny virions. These mechanisms include surface activity, binding affinity, and insertion into the lipid membrane. Thus, despite the sequence differences of matrix and capsid proteins of different viruses, their functional activities fall into a few possible actions, and these functional activities are common for all enveloped viruses. Thus, our findings open a new way for the search for possible antiviral drugs, targeted at viral protein assembly and lipid-protein interactions. This work was supported by the Russian Foundation for Basic Researches (project #20-54-14006).

A solution of a histone-like protein from Spiroplasma melliferum (HUSpm) was examined by small-angle X-ray scattering (SAXS). The experimental SAXS curve was compared with those calculated for the HUSpm structures from the PDB databank obtained by both X-ray diffraction analysis and nuclear magnetic resonance spectroscopy. The model of the HUSpm structure in solution, which best agrees with the experimental SAXS data, has a shorter distance between the centers of mass of the HUSpm monomers compared to the crystal structure, indicating that the HUSpm monomers can be located closer to each other in solution than in the crystalline state.

DNA-binding protein from starved cells (Dps) takes a special place among dodecamer mini-ferritins. Its most important function is protection of bacterial genome from various types of destructive external factors via in cellulo Dps-DNA co-crystallization. This protective response results in the emergence of bacterial resistance to antibiotics and other drugs. The protective properties of Dps have attracted a significant attention of researchers. However, Dps has another equally important functional role. Being a ferritin-like protein, Dps acts as an iron depot and protects bacterial cells from the oxidative damage initiated by the excess of iron. Here we investigated formation of iron oxide nanoparticles in the internal cavity of the Dps dodecamer. We used anomalous small-angle X-ray scattering as the main research technique, which allows to examine the structure of metal-containing biological macromolecules and to analyze the size distribution of metal nanoparticles formed in them. The contributions of protein and metal components to total scattering were distinguished by varying the energy of the incident X-ray radiation near the edge of the metal atom absorption band (the K-band for iron). We examined Dps specimens containing 50, 500, and 2000 iron atoms per protein dodecamer. Analysis of the particle size distribution showed that, depending on the iron content in the solution, the size of the nanoparticles formed inside the protein molecule was 2 to 4 nm and the growth of metal nanoparticles was limited by the size of the protein inner cavity. We also found some amount of iron ions in the Dps surface layer. This layer is very important for the protein to perform its protective functions, since the surface-located N-terminal domains determine the nature of interactions between Dps and DNA. In general, the results obtained in this work can be useful for the next step in studying the Dps phenomenon, as well as in creating biocompatible and solution-stabilized metal nanoparticles.

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called “intrinsically disordered” nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552.