Atomic force microscopy investigation of DNA denaturation on a highly oriented pyrolytic graphite surface.

Understanding of DNA interaction with carbonaceous surfaces (including graphite, graphene and carbon nanotubes) is important for the development of DNA-based biosensors and other biotechnological devices. Though many issues related to DNA adsorption on graphitic surfaces have been studied, some important aspects of DNA interaction with graphite remain unclear. In this work, we use atomic force microscopy (AFM) equipped with super-sharp cantilevers to analyze the morphology and conformation of relatively long DNA molecule adsorbed on a highly oriented pyrolytic graphite (HOPG) surface. We have revealed the effect of DNA embedding into an organic monolayer of N,N'-(decane-1,10-diyl)-bis(tetraglycinamide) (GM), which may "freeze" DNA conformation on a HOPG surface during drying. The dependence of the mean squared point-to-point distance on the contour length suggests that DNA adsorbs on a bare HOPG by a "kinetic trapping" mechanism. For the first time, we have estimated the unfolded fraction of DNA upon contact with a HOPG surface (24 ± 5 %). The obtained results represent a novel experimental model for investigation of the conformation and morphology of DNA adsorbed on graphitic surfaces and provide with a new insight into DNA interaction with graphite.

Self-assembling amyloid-like nanostructures from SARS-CoV-2 S1, S2, RBD and N recombinant proteins.

Self-assembling nanoparticles (saNP) and nanofibers were found in the recombinant coronavirus SARS-CoV-2 S1, S2, RBD and N proteins purified by affinity chromatography using Ni Sepharose. Scanning electron (SEM), atomic force (AFM) microscopy on mica or graphite surface and in liquid as well as dynamic light scattering (DLS) revealed nanostructures of various sizes. AFM in liquid cell without drying on the surface showed mean height of S1 saNP 80.03 nm, polydispersity index (PDI) 0.006; for S2 saNP mean height 93.32 nm, PDI = 0.008; for N saNP mean height 16.71 nm, PDI = 0.99; for RBD saNP mean height 16.25 nm, PDI = 0.55. Ratios between the height and radius of each saNP in the range 0.1-0.5 suggested solid protein NP but not vesicles with internal empty spaces. The solid but not empty structures of the protein saNP were also confirmed by STEM after treatment of saNP with the standard contrasting agent uranyl acetate. The saNP remained stable after multiple freeze-thaw cycles in water and hyperosmotic solutions for 2 years at -20 °C. Receptor-mediated penetration of the SARS-CoV-2 S1 and RBD saNP in the African green mokey kidney Vero cells with the specific receptors for ß-coronavirus reproduction was more efficient compared to unspecific endocytosis into MDCK cells without the specific receptors. Amyloid-like structures were revealed in the SARS-CoV-2 S1, S2, RBD and N saNP by means of their interaction with Thioflavin T and Congo Red dyes. Taken together, spontaneous formation of the amyloid-like self-assembling nanostructures due to the internal affinity of the SARS-CoV-2 virion proteins might induce proteinopathy in patients, including conformational neurodegenerative diseases, change stability of vaccines and diagnostic systems.

Features of DNA-Montmorillonite Binding Visualized by Atomic Force Microscopy.

In the present work, complexes of DNA with nano-clay montmorillonite (Mt) were investigated by means of atomic force microscopy (AFM) under various conditions. In contrast to the integral methods of analysis of the sorption of DNA on clay, AFM allowed us to study this process at the molecular level in detail. DNA molecules in the deionized water were shown to form a 2D fiber network weakly bound to both Mt and mica. The binding sites are mostly along Mt edges. The addition of Mg2+ cations led to the separation of DNA fibers into separate molecules, which bound mainly to the edge joints of the Mt particles according to our reactivity estimations. After the incubation of DNA with Mg2+, the DNA fibers were capable of wrapping around the Mt particles and were weakly bound to the Mt edge surfaces. The reversible sorption of nucleic acids onto the Mt surface allows it to be used for both RNA and DNA isolation for further reverse transcription and polymerase chain reaction (PCR). Our results show that the strongest binding sites for DNA are the edge joints of Mt particles.

Stable Enzymatic Nanoparticles from Nucleases, Proteases, Lipase and Antioxidant Proteins with Substrate-Binding and Catalytic Properties.

Limited membrane permeability and biodegradation hamper the intracellular delivery of the free natural or recombinant enzymes necessary for compensatory therapy. Nanoparticles (NP) provide relative protein stability and unspecific endocytosis-mediated cellular uptake. Our objective was the fabrication of NP from 7 biomedicine-relevant enzymes, including DNase I, RNase A, trypsin, chymotrypsin, catalase, horseradish peroxidase (HRP) and lipase, the analysis of their conformation stability and enzymatic activity as well as possible toxicity for eukaryotic cells. The enzymes were dissolved in fluoroalcohol and mixed with 40% ethanol as an anti-solvent with subsequent alcohol evaporation at high temperature and low pressure. The shapes and sizes of NP were determined by scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). Enzyme conformations in solutions and in NP were compared using circular dichroism (CD) spectroscopy. The activity of the enzymes was assayed with specific substrates. The cytotoxicity of the enzymatic NP (ENP) was studied by microscopic observations and by using an MTT test. Water-insoluble ENP of different shapes and sizes in a range 50-300 nm consisting of 7 enzymes remained stable for 1 year at +4 °C without any cross-linking. CD spectroscopy of the ENP permitted us to reveal changes in proportions of α-helixes, ß-turns and random coils in comparison with fresh enzyme solutions in water. Despite the minor conformation changes of the proteins in the ENP, the enzymes retained their substrate-binding and catalytic properties. Among the studied bioactive ENP, only DNase NP were highly toxic for 3 cell lines with granulation in 1 day posttreatment, whereas other NP were less toxic (if any). Taken together, the enzymes in the stable ENP retained their catalytic activity and might be used for intracellular delivery.

Single-molecule AFM study of hyaluronic acid softening in electrolyte solutions.

Investigation of hyaluronic acid (HA) morphology and mechanical properties at a single-molecule level is important for the development of HA based biomaterials. We have developed the atomic force microscopy (AFM) based approach for quantitative characterization of conformation of HA molecules. HA molecules adsorbed on a modified graphitic surface form oriented linear segments. Conformation of HA molecules can be considered as two-dimensional quasi-projection of a three-dimensional conformation locally straightened by a substrate. The persistence length and Young's modulus of biomolecules estimated using wormlike chain model decrease from 15.7 to 9.9 nm, and from ∼21 to ∼13 GPa, respectively, when KCl concentration increases from 0 to 100 mM. The dependence of the persistence length on ionic strength supports the Odijk-Skolnick-Fixman model of polyelectrolyte stiffening in electrolyte solution. The obtained results represent a new insight into the conformation and mechanical characteristics of HA molecules and complement the characterization of this biopolymer by bulk methods.

Activation of Neutrophils by Mucin-Vaterite Microparticles.

Nano- and microparticles enter the body through the respiratory airways and the digestive system, or form as biominerals in the gall bladder, salivary glands, urinary bladder, kidney, or diabetic pancreas. Calcium, magnesium, and phosphate ions can precipitate from biological fluids in the presence of mucin as hybrid nanoparticles. Calcium carbonate nanocrystallites also trap mucin and are assembled into hybrid microparticles. Both mucin and calcium carbonate polymorphs (calcite, aragonite, and vaterite) are known to be components of such biominerals as gallstones which provoke inflammatory reactions. Our study was aimed at evaluation of neutrophil activation by hybrid vaterite-mucin microparticles (CCM). Vaterite microparticles (CC) and CCM were prepared under standard conditions. The diameter of CC and CCM was 3.3 ± 0.8 µm and 5.8 ± 0.7 µm, with ƺ-potentials of -1 ± 1 mV and -7 ± 1 mV, respectively. CC microparticles injured less than 2% of erythrocytes in 2 h at 1.5 mg mL-1, and no hemolysis was detected with CCM; this let us exclude direct damage of cellular membranes by microparticles. Activation of neutrophils was analyzed by luminol- and lucigenin-dependent chemiluminescence (Lum-CL and Luc-CL), by cytokine gene expression (IL-6, IL-8, IL-10) and release (IL-1ß, IL-6, IL-8, IL-10, TNF-α), and by light microscopy of stained smears. There was a 10-fold and higher increase in the amplitude of Lum-CL and Luc-CL after stimulation of neutrophils with CCM relative to CC. Adsorption of mucin onto prefabricated CC microparticles also contributed to activation of neutrophil CL, unlike mucin adsorption onto yeast cell walls (zymosan); adsorbed mucin partially suppressed zymosan-stimulated production of oxidants by neutrophils. Preliminary treatment of CCM with 0.1-10 mM NaOCl decreased subsequent activation of Lum-CL and Luc-CL of neutrophils depending on the used NaOCl concentration, presumably because of the surface mucin oxidation. Based on the results of ELISA, incubation of neutrophils with CCM downregulated IL-6 production but upregulated that of IL-8. IL-6 and IL-8 gene expression in neutrophils was not affected by CC or CCM according to RT2-PCR data, which means that post-translational regulation was involved. Light microscopy revealed adhesion of CC and CCM microparticles onto the neutrophils; CCM increased neutrophil aggregation with a tendency to form neutrophil extracellular traps (NETs). We came to the conclusion that the main features of neutrophil reaction to mucin-vaterite hybrid microparticles are increased oxidant production, cell aggregation, and NET-like structure formation, but without significant cytokine release (except for IL-8). This effect of mucin is not anion-specific since particles of powdered kidney stone (mainly calcium oxalate) in the present study or calcium phosphate nanowires in our previous report also activated Lum-CL and Luc-CL response of neutrophils after mucin sorption.

Targeting of Silver Cations, Silver-Cystine Complexes, Ag Nanoclusters, and Nanoparticles towards SARS-CoV-2 RNA and Recombinant Virion Proteins.

Background: Nanosilver possesses antiviral, antibacterial, anti-inflammatory, anti-angiogenesis, antiplatelet, and anticancer properties. The development of disinfectants, inactivated vaccines, and combined etiotropic and immunomodulation therapy against respiratory viral infections, including COVID-19, remains urgent. Aim: Our goal was to determine the SARS-CoV-2 molecular targets (genomic RNA and the structural virion proteins S and N) for silver-containing nanomaterials. Methods: SARS-CoV-2 gene cloning, purification of S2 and N recombinant proteins, viral RNA isolation from patients' blood samples, reverse transcription with quantitative real-time PCR ((RT)2-PCR), ELISA, and multiplex immunofluorescent analysis with magnetic beads (xMAP) for detection of 17 inflammation markers. Results: Fluorescent Ag nanoclusters (NCs) less than 2 nm with a few recovered silver atoms, citrate coated Ag nanoparticles (NPs) with diameters of 20-120 nm, and nanoconjugates of 50-150 nm consisting of Ag NPs with different protein envelopes were constructed from AgNO3 and analyzed by means of transmission electron microscopy (TEM), atomic force microscopy (AFM), ultraviolet-visible light absorption, and fluorescent spectroscopy. SARS-CoV-2 RNA isolated from COVID-19 patients' blood samples was completely cleaved with the artificial RNase complex compound Li+[Ag+2Cys2-(OH-)2(NH3)2] (Ag-2S), whereas other Ag-containing materials provided partial RNA degradation only. Treatment of the SARS-CoV-2 S2 and N recombinant antigens with AgNO3 and Ag NPs inhibited their binding with specific polyclonal antibodies, as shown by ELISA. Fluorescent Ag NCs with albumin or immunoglobulins, Ag-2S complex, and nanoconjugates of Ag NPs with protein shells had no effect on the interaction between coronavirus recombinant antigens and antibodies. Reduced production of a majority of the 17 inflammation biomarkers after treatment of three human cell lines with nanosilver was demonstrated by xMAP. Conclusion: The antiviral properties of the silver nanomaterials against SARS-CoV-2 coronavirus differed. The small-molecular-weight artificial RNase Ag-2S provided exhaustive RNA destruction but could not bind with the SARS-CoV-2 recombinant antigens. On the contrary, Ag+ ions and Ag NPs interacted with the SARS-CoV-2 recombinant antigens N and S but were less efficient at performing viral RNA cleavage. One should note that SARS-CoV-2 RNA was more stable than MS2 phage RNA. The isolated RNA of both the MS2 phage and SARS-CoV-2 were more degradable than the MS2 phage and coronavirus particles in patients' blood, due to the protection with structural proteins. To reduce the risk of the virus resistance, a combined treatment with Ag-2S and Ag NPs could be used. To prevent cytokine storm during the early stages of respiratory infections with RNA-containing viruses, nanoconjugates of Ag NPs with surface proteins could be recommended.

Myeloperoxidase-induced fibrinogen unfolding and clotting.

Due to its unique properties and high biomedical relevance fibrinogen is a promising protein for the development of various matrixes and scaffolds for biotechnological applications. Fibrinogen molecules may form extensive clots either upon specific cleavage by thrombin or in thrombin-free environment, for example, in the presence of different salts. Here, we report the novel type of non-conventional fibrinogen clot formation, which is mediated by myeloperoxidase and takes place even at low fibrinogen concentrations (<0.1 mg/ml). We have revealed fibrillar nature of myeloperoxidase-mediated fibrinogen clots, which differ morphologically from fibrin clots. We have shown that fibrinogen clotting is mediated by direct interaction of myeloperoxidase molecules with the outer globular regions of fibrinogen molecules followed by fibrinogen unfolding from its natural trinodular to a fibrillar structure. We have demonstrated a major role of the Debye screening effect in regulating of myeloperoxidase-induced fibrinogen clotting, which is facilitated by small ionic strength. While fibrinogen in an aqueous solution with myeloperoxidase undergoes changes, the enzymatic activity of myeloperoxidase is not inhibited in excess of fibrinogen. The obtained results open new insights into fibrinogen clotting, give new possibilities for the development of fibrinogen-based functional biomaterials, and provide the novel concepts of protein unfolding.

Anomalous Laterally Stressed Kinetically Trapped DNA Surface Conformations.

HIGHLIGHTS: DNA kinking is inevitable for the highly anisotropic 1D-1D electrostatic interaction with the one-dimensionally periodically charged surface. The double helical structure of the DNA kinetically trapped on positively charged monomolecular films comprising the lamellar templates is strongly laterally stressed and extremely perturbed at the nanometer scale. The DNA kinetic trapping is not a smooth 3D-> 2D conformational flattening but is a complex nonlinear in-plane mechanical response (bending, tensile and unzipping) driven by the physics beyond the scope of the applicability of the linear worm-like chain approximation. Up to now, the DNA molecule adsorbed on a surface was believed to always preserve its native structure. This belief implies a negligible contribution of lateral surface forces during and after DNA adsorption although their impact has never been elucidated. High-resolution atomic force microscopy was used to observe that stiff DNA molecules kinetically trapped on monomolecular films comprising one-dimensional periodically charged lamellar templates as a single layer or as a sublayer are oversaturated by sharp discontinuous kinks and can also be locally melted and supercoiled. We argue that kink/anti-kink pairs are induced by an overcritical lateral bending stress (> 30 pNnm) inevitable for the highly anisotropic 1D-1D electrostatic interaction of DNA and underlying rows of positive surface charges. In addition, the unexpected kink-inducing mechanical instability in the shape of the template-directed DNA confined between the positively charged lamellar sides is observed indicating the strong impact of helicity. The previously reported anomalously low values of the persistence length of the surface-adsorbed DNA are explained by the impact of the surface-induced low-scale bending. The sites of the local melting and supercoiling are convincingly introduced as other lateral stress-induced structural DNA anomalies by establishing a link with DNA high-force mechanics. The results open up the study in the completely unexplored area of the principally anomalous kinetically trapped DNA surface conformations in which the DNA local mechanical response to the surface-induced spatially modulated lateral electrostatic stress is essentially nonlinear. The underlying rich and complex in-plane nonlinear physics acts at the nanoscale beyond the scope of applicability of the worm-like chain approximation.

Molecular patterns of oligopeptide hydrocarbons on graphite.

Graphitic materials including graphene, carbon nanotubes and fullerenes, are promising for use in nanotechnology and biomedicine. Non-covalent functionalization by peptides and other organic molecules allows changing the properties of graphitic surfaces in a controlled manner and represents a big potential for fundamental research and applications. Recently described oligopeptide-hydrocarbon derivative N,N'-(decane-1,10-diyl)bis(tetraglycineamide) (GM) is highly prospective for the development of graphitic interfaces in biosensor application as well as in structural biology for improving the quality of high-resolution atomic force microscopy (AFM) visualization of individual biomacromolecules. However, molecular organization of GM on graphitic surfaces is still unknown. In this work, the molecular model of GM at the water/highly oriented pyrolytic graphite (HOPG) interface has been developed basing on the high-resolution AFM and full-atom molecular modeling data. This model explains two periodicities observed in AFM images by GM self-assembly on a HOPG surface with formation of the stacks with the lateral shifts. The obtained results reveal the particular patterns and dynamics of GM molecules adsorbed on graphite and unravel the puzzle of peptide self-assembly on graphitic surfaces.

Toehold-Mediated Selective Assembly of Compact Discrete DNA Nanostructures.

Production of small discrete DNA nanostructures containing covalent junctions requires reliable methods for the synthesis and assembly of branched oligodeoxynucleotide (ODN) conjugates. This study reports an approach for self-assembly of hard-to-obtain primitive discrete DNA nanostructures-"nanoethylenes", dimers formed by double-stranded oligonucleotides using V-shaped furcate blocks. We scaled up the synthesis of V-shaped oligonucleotide conjugates using pentaerythritol-based diazide and alkyne-modified oligonucleotides using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and optimized the conditions for "nanoethylene" formation. Next, we designed nanoethylene-based "nanomonomers" containing pendant adapters. They demonstrated smooth and high-yield spontaneous conversion into the smallest cyclic product, DNA tetragon aka "nano-methylcyclobutane". Formation of DNA nanostructures was confirmed using native polyacrylamide gel electrophoresis (PAGE) and atomic force microscopy (AFM) and additionally studied by molecular modeling. The proposed facile approach to discrete DNA nanostructures using precise adapter-directed association expands the toolkit for the realm of DNA origami.

In Situ Single-Molecule AFM Investigation of Surface-Induced Fibrinogen Unfolding on Graphite.

Fibrinogen adsorption plays a key role in important biological processes, such as blood coagulation and foreign body reaction, which determine the biocompatibility of a material. Fibrinogen conformation on a surface is one of the main factors triggering these processes. Understanding the conformational dynamics of fibrinogen molecules adsorbed on solid surfaces is, therefore, of great interest in biomedicine and may contribute to the development of new biomaterials. In this work, unfolding of fibrinogen molecules adsorbed on a model surface (highly oriented pyrolytic graphite modified with an oligoglycine-hydrocarbon graphite modifier) is directly visualized using time-lapse atomic force microscopy. A gradual transformation of native-like fibrinogen molecules into fibrillar structures is observed at a timescale of several minutes. This transformation is accompanied by a decrease in molecular height from 4-5 to 1-2 nm. Independent unfolding of different fibrinogen domains is demonstrated. The obtained results provide a new, direct insight into the unfolding of individual fibrinogen molecules on a surface and give new opportunities for the development of graphite-based biosensors and biomaterials.

Efficient silica synthesis from tetra(glycerol)orthosilicate with cathepsin- and silicatein-like proteins.

Silicateins play a key role in biosynthesis of spicules in marine sponges; they are also capable to catalyze formation of amorphous silica in vitro. Silicateins are highly homologous to cathepsins L - a family of cysteine proteases. Molecular mechanisms of silicatein activity remain controversial. Here site-directed mutagenesis was used to clarify significance of selected residues in silica polymerization. A number of mutations were introduced into two sponge proteins - silicatein A1 and cathepsin L from Latrunculia oparinae, as well as into human cathepsin L. First direction was alanine scanning of the proposed catalytic residues. Also, reciprocal mutations were introduced at selected positions that differ between cathepsins L and silicateins. Surprisingly, all the wild type and mutant proteins were capable to catalyze amorphous silica formation with a water-soluble silica precursor tetra(glycerol)orthosilicate. Some mutants possessed several-fold enhanced silica-forming activity and can potentially be useful for nanomaterial synthesis applications. Our findings contradict to the previously suggested mechanisms of silicatein action via a catalytic triad analogous to that in cathepsins L. Instead, a surface-templated biosilification by silicateins and related proteins can be proposed.

Protein nanoparticles with ligand-binding and enzymatic activities.

PURPOSE: To develop a general method for NP fabrication from various proteins with maintenance of biological activity. METHODS: A novel general approach for producing protein nanoparticles (NP) by nanoprecipitation of the protein solutions in 1,1,1,3,3,3-hexafluoroisopropanol is described. Protein NP sizes and shapes were analyzed by dynamic light scattering, scanning electron and atomic force microscopy (SEM and AFM). Chemical composition of the NP was confirmed using ultraviolet (UV) spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and circular dichroism (CD). Biological properties of the NP were analyzed in ELISA, immunofluorescent analysis and lysozyme activity assay. RESULTS: Water-insoluble NP were constructed from globular (bovine serum albumin (BSA), lysozyme, immunoglobulins), fibrillar (fibrinogen) proteins and linear polylysines by means of nanoprecipitation of protein solutions in fluoroalcohols. AFM and SEM revealed NP sizes of 20-250 nm. The NP chemical structure was confirmed by UV spectroscopy, protease digestion and EDX spectroscopy. CD spectra revealed a stable secondary structure of proteins in NP. The UV spectra, microscopy and SDS-PAA gel electrophoresis (PAGE) proved the NP stability at +4°C for 7 months. Co-precipitation of proteins with fluorophores or nanoprecipitation of pre-labeled BSA resulted in fluorescent NP that retained antigenic structures as shown by their binding with specific antibodies. Moreover, NP from monoclonal antibodies could bind with the hepatitis B virus antigen S. Besides that, lysozyme NP could digest bacterial cellular walls. CONCLUSION: Thus, the water-insoluble, stable protein NP were produced by nanoprecipitation without cross-linking and retained ligand-binding and enzymatic activities.

High-resolution atomic force microscopy visualization of metalloproteins and their complexes.

BACKGROUND: Metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP) and lactoferrin (LF) play an important role in regulation of inflammation and oxidative stress in vertebrates. It was previously shown that these proteins may work synergetically as antimicrobial and anti-inflammatory agents by forming complexes, such as MPO-CP and LF-CP. However, interaction of metalloprotein molecules with each other has never been characterized at a single-molecule level. METHODS: In this study, the pairwise interactions of MPO, CP and LF molecules were investigated at a single-molecule level using high-resolution atomic force microscopy (AFM). Highly oriented pyrolytic graphite surface (HOPG) modified with oligoglycine-hydrocarbon graphite modifier (GM) was used as a substrate for protein deposition. RESULTS: The procedure for reliable AFM investigation of metalloproteins and their complexes has been developed. Using this procedure, we have visualized, for the first time, single MPO, CP and LF molecules, characterized the morphology of MPO-CP and LF-CP complexes and confirmed the absence of direct contacts between MPO and LF molecules. Moreover, we have revealed the novel chainlike shape of MPO-CP conjugates. CONCLUSIONS: GM-HOPG was shown to be a convenient substrate for AFM investigation of metalloproteins and their complexes. Direct AFM visualization of MPO-CP and LF-CP complexes, on the one hand, complements previous data obtained from the "bulk techniques" and, on the other hand, provides new insight into the ultrastructure of MPO-CP complexes. GENERAL SIGNIFICANCE: The obtained results contribute to the better understanding of regulation of inflammation and oxidation stress mediated by collaborative action of the metalloproteins such as MPO, CP and LF.

Polymorphism of G4 associates: from stacks to wires via interlocks.

We examined the assembly of DNA G-quadruplexes (G4s) into higher-order structures using atomic force microscopy, optical and electrophoretic methods, NMR spectroscopy and molecular modeling. Our results suggest that parallel blunt-ended G4s with single-nucleotide or modified loops may form different types of multimers, ranging from stacks of intramolecular structures and/or interlocked dimers and trimers to wires. Decreasing the annealing rate and increasing salt or oligonucleotide concentrations shifted the equilibrium from intramolecular G4s to higher-order structures. Control antiparallel and hybrid G4s demonstrated no polymorphism or aggregation in our experiments. The modification that mimics abasic sites (1',2'-dideoxyribose residues) in loops enhanced the oligomerization/multimerization of both the 2-tetrad and 3-tetrad G4 motifs. Our results shed light on the rules that govern G4 rearrangements. Gaining control over G4 folding enables the harnessing of the full potential of such structures for guided assembly of supramolecular DNA structures for nanotechnology.

Thermal denaturation of fibrinogen visualized by single-molecule atomic force microscopy.

Fibrinogen denaturation is an important phenomenon in biology and medicine. It has been previously investigated with bulk methods and characterized by parameters, which refer to big protein ensembles. Here we provide a new insight into fibrinogen denaturation with a high-resolution single-molecule atomic force microscopy (AFM). The ultrastructure of individual fibrinogen molecules was studied after heating or extended contact with the highly oriented pyrolytic graphite surface (HOPG) modified with oligoglycine-hydrocarbon graphite modifier (GM). Fibrinogen heating to 65 °C and 90 °C resulted in the formation of various shapes containing fibrillar and globular structures, which were attributed to the monomers and small aggregates of fibrinogen. Fibrinogen unfolded by the extended (10 min) incubation on GM-HOPG surface in water revealed a different morphology. It contained fibrillar structures only, and their organization reflected the initial native structure of fibrinogen: typically, six polypeptide chains connected by multiple disulfide bonds were seen. A combination of two morphologies - globular aggregates with dense fibrillar networks - has been revealed for thermally denatured protein adsorbed on a GM-HOPG surface with extended (10 min) rinsing with water. The obtained results provide better understanding of fibrinogen unfolding induced by different factors and are important for improvement of biomedical applications, such as fibrinogen-based protein matrixes and carbon-based biomaterials.

A water-soluble precursor for efficient silica polymerization by silicateins.

Silicateins, the spicule-forming proteins from marine demosponges capable to polymerize silica, are popular objects of biomineralization studies due to their ability to form particles varied in shape and composition under physiological conditions. Despite the occurrence of the many approaches to nanomaterial synthesis using silicateins, biochemical properties of this protein family are poorly characterized. The main reason for this is that tetraethyl orthosilicate (TEOS), the commonly used silica acid precursor, is almost insoluble in water and thus is poorly available for the protein. To solve this problem, we synthesized new water-soluble silica precursor, tetra(glycerol)orthosilicate (TGS), and characterized biochemical properties of the silicatein A1 from marine sponge Latrunculia oparinae. Compared to TEOS, TGS ensured much greater activity of silicatein and was less toxic for the mammalian cell culture. We evaluated optimum conditions for the enzyme - pH range, temperature and TGS concentration. We concluded that TGS is a useful silica acid precursor that can be used for silica particles synthesis and in vivo applications.

Morphometric characterization of fibrinogen's αC regions and their role in fibrin self-assembly and molecular organization.

The flexible C-terminal parts of fibrinogen's Aα chains named the αC regions have been shown to play a role in fibrin self-assembly, although many aspects of their structure and functions remain unknown. To examine the involvement of the αC regions in the early stages of fibrin formation, we used high-resolution atomic force microscopy to image fibrinogen and oligomeric fibrin. Plasma-purified full-length human fibrinogen or des-αC fibrinogen lacking most of the αC regions, untreated or treated with thrombin, was imaged. Up to 80% of the potentially existing αC regions were visualized and quantified; they were highly heterogeneous in their length and configurations. Conversion of fibrinogen to fibrin was accompanied by an increase in the incidence and length of the αC regions as well as transitions from more compact conformations, such as a globule on a string, to extended and more flexible offshoots. Concurrent dynamic turbidimetry, confocal microscopy, and scanning electron microscopy revealed that trimming of the αC regions slowed down fibrin formation, which correlated with longer protofibrils, thinner fibers, and a denser network. No structural distinctions, except for the incidence of the αC regions, were revealed in the laterally aggregated protofibrils made of the full-length or des-αC fibrinogens, suggesting a pure kinetic effect of the αC regions on the fibrin architecture. This work provides a structural molecular basis for the promoting role of the αC regions in the early stages of fibrin self-assembly and reveals this stage of fibrin formation as a potential therapeutic target to modulate the structure and mechanical properties of blood clots.

AFM visualization at a single-molecule level of denaturated states of proteins on graphite.

Different graphitic materials are either already used or believed to be advantageous in biomedical and biotechnological applications, e.g., as biomaterials or substrates for sensors. Most of these applications or associated important issues, such as biocompatibility, address the problem of adsorption of protein molecules and, in particular the conformational state of the adsorbed protein molecule on graphite. High-resolution AFM demonstrates highly oriented pyrolytic graphite (HOPG) induced denaturation of four proteins of blood plasma, such as ferritin, fibrinogen, human serum albumin (HSA) and immunoglobulin G (IgG), at a single molecule level. Protein denaturation is accompanied by the decrease of the heights of protein globules and spreading of the denatured protein fraction on the surface. In contrast, the modification of HOPG with the amphiphilic oligoglycine-hydrocarbon derivative monolayer preserves the native-like conformation and provides even more mild conditions for the protein adsorption than typically used mica. Protein unfolding on HOPG may have universal character for "soft" globular proteins.