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.

Structural Insights into Plant Viruses Revealed by Small-Angle X-ray Scattering and Atomic Force Microscopy.

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.

Atomic force microscopy of spherical intermediates on the pathway to fibril formation of influenza A virus nuclear export protein.

The nuclear export protein of the influenza A virus (NEP) is involved in many important processes of the virus life cycle. This makes it an attractive target for the treatment of a disease caused by a virus. Previously it has been shown, that recombinant variants of NEP are highly prone to aggregation in solution under various conditions with the formation of amyloid-like aggregates. In the present work, the amyloid nature of NEP aggregates was evidenced by Congo red binding assays. Atomic force microscopy has shown that NEP can form two types of spherical nanoparticles, which provide an alternative pathway for the formation of amyloid-like fibrils. Type I of these "fibrillogenic" spheres, formed under physiological conditions, represents the micelle-like particles with height 10-60 nm, which can generate worm-like flexible fibrils with the diameter 2.5-4.0 nm, length 20-500 nm and the Young's modulus ~73 MPa. Type II spherical aggregates with size of about 400-1000 nm, formed at elevated temperatures, includes fractions of drop-like and vesicle-like particles, generating more rigid amyloid-like fibrils with height of ~8 nm, and length of up to 2 µm. The hypothetical mechanism of fibril formation via nanospherical structures was suggested. RESEARCH HIGHLIGHTS: AFM has revealed two types of the influenza A virus nuclear export protein spherical aggregates. They provide an alternative pathway for the formation of amyloid-like fibrils. The mechanism of fibril formation via spherical structures is suggested.

Atomic force microscopy-based approaches for single-molecule investigation of nucleic acid-protein complexes.

The interaction of nucleic acids with proteins plays an important role in many fundamental biological processes in living cells, including replication, transcription, and translation. Therefore, understanding nucleic acid-protein interaction is of high relevance in many areas of biology, medicine and technology. During almost four decades of its existence atomic force microscopy (AFM) accumulated a significant experience in investigation of biological molecules at a single-molecule level. AFM has become a powerful tool of molecular biology and biophysics providing unique information about properties, structure, and functioning of biomolecules. Despite a great variety of nucleic acid-protein systems under AFM investigations, there are a number of typical approaches for such studies. This review is devoted to the analysis of the typical AFM-based approaches of investigation of DNA (RNA)-protein complexes with a major focus on transcription studies. The basic strategies of AFM analysis of nucleic acid-protein complexes including investigation of the products of DNA-protein reactions and real-time dynamics of DNA-protein interaction are categorized and described by the example of the most relevant research studies. The described approaches and protocols have many universal features and, therefore, are applicable for future AFM studies of various nucleic acid-protein systems.

Characterization of the effect of chromium salts on tropocollagen molecules and molecular aggregates.

Though the capability of chromium treatment to improve the stability and mechanical properties of collagen fibrils is well-known, the influence of different chromium salts on collagen molecules (tropocollagen) is not well characterized. In this study, the effect of Cr3+ treatment on the conformation and hydrodynamic properties of collagen was studied using atomic force microscopy (AFM) and dynamic light scattering (DLS). Statistical analysis of contours of adsorbed tropocollagen molecules using the two-dimensional worm-like chain model revealed a reduction of the persistence length (i.e., the increase of flexibility) from ≈72 nm in water to ≈56-57 nm in chromium (III) salt solutions. DLS studies demonstrated an increase of the hydrodynamic radius from ≈140 nm in water to ≈190 nm in chromium (III) salt solutions, which is associated with protein aggregation. The kinetics of collagen aggregation was shown to be ionic strength dependent. Collagen molecules treated with three different chromium (III) salts demonstrated similar properties such as flexibility, aggregation kinetics, and susceptibility to enzymatic cleavage. The observed effects are explained by a model that considers the formation of chromium-associated intra- and intermolecular crosslinks. The obtained results provide novel insights into the effect of chromium salts on the conformation and properties of tropocollagen molecules.

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.

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.

Spatial organization of Dps and DNA-Dps complexes.

DNA co-crystallization with Dps family proteins is a fundamental mechanism, which preserves DNA in bacteria from harsh conditions. Though many aspects of this phenomenon are well characterized, the spatial organization of DNA in DNA-Dps co-crystals is not completely understood, and existing models need further clarification. To advance in this problem we have utilized atomic force microscopy (AFM) as the main structural tool, and small-angle X-scattering (SAXS) to characterize Dps as a key component of the DNA-protein complex. SAXS analysis in the presence of EDTA indicates a significantly larger radius of gyration for Dps than would be expected for the core of the dodecamer, consistent with the N-terminal regions extending out into solution and being accessible for interaction with DNA. In AFM experiments, both Dps protein molecules and DNA-Dps complexes adsorbed on mica or highly oriented pyrolytic graphite (HOPG) surfaces form densely packed hexagonal structures with a characteristic size of about 9 nm. To shed light on the peculiarities of DNA interaction with Dps molecules, we have characterized individual DNA-Dps complexes. Contour length evaluation has confirmed the non-specific character of Dps binding with DNA and revealed that DNA does not wrap Dps molecules in DNA-Dps complexes. Angle analysis has demonstrated that in DNA-Dps complexes a Dps molecule contacts with a DNA segment of ~6 nm in length. Consideration of DNA condensation upon complex formation with small Dps quasi-crystals indicates that DNA may be arranged along the rows of ordered protein molecules on a Dps sheet.

Evidence of (anti)metamorphic properties of modified graphitic surfaces obtained in real time at a single-molecule level.

Understanding protein unfolding on a surface is of vital importance in nanoscience, nanobiotechnology, and medicine. Surfaces that retain the native conformation of adsorbed proteins (antimetamorphic surfaces) represent one of the main strategies for creating biocompatible materials, which are in great demand in biotechnology. Though the influence of surfaces on protein conformation has been studied for decades, real-time investigations of protein conformational behavior on a surface obtained at the single-molecule or sub-molecular level are still lacking and remain a challenge. In this work, we apply time-lapse atomic force microscopy (AFM) in aqueous solution to visualize the conformational dynamics of individual model protein molecules (E.coli RNA polymerase, RNAP) adsorbed on modified highly oriented pyrolytic graphite (HOPG) surfaces. We quantitatively characterize the evolution of height and shape of individual RNAP molecules adsorbed on a HOPG surface modified with an oligoglycine-hydrocarbon graphite modifier (GM) during the unfolding process and determine the characteristic unfolding time as ∼7 min. Furthermore, we make a HOPG surface antimetamorphic by modifying it with a denatured RNAP protein layer. Our results provide direct evidence of GM-HOPG-induced RNAP unfolding at the single-molecule level and open new strategies for the development and investigation of antimetamorphic graphitic surfaces.

Label-free sensitive detection of influenza virus using PZT discs with a synthetic sialylglycopolymer receptor layer.

We describe rapid, label-free detection of Influenza A viruses using the first radial mode of oscillations of lead zirconate titanate (PZT) piezoelectric discs with a 2 mm radius and 100 µm thickness fabricated from a piezoelectric membrane. The discs are modified with a synthetic sialylglycopolymer receptor layer, and the coated discs are inserted in a flowing virus suspension. Label-free detection of the virus is achieved by monitoring the disc radial mode resonance frequency shift. Piezo transducers with sialylglycopolymer sensor layers exhibited a long lifetime, a high sensitivity and the possibility of regeneration. We demonstrate positive, label-free detection of Influenza A viruses at concentrations below 105 virus particles per millilitre. We show that label-free, selective, sensitive detection of influenza viruses by home appliances is possible in principle.

Influence of pixelization on height measurement in atomic force microscopy.

Though AFM is capable of obtaining sub-angstrom resolution in z-direction, the accurate height measurement of protruding particles is hindered by raster nature of this technique. In this work using Monte Carlo simulations we have quantified the influence of pixelization on the mean AFM apparent height (hmean) of spheres and cylinders. We have demonstrated that for a zero size AFM probe hmean may be increasing, decreasing function of a pixel size, or has more complex character depending on the standard deviation of a particle size. Therefore, AFM pixelization effects may induce both under- and overestimation of the true diameter. The observed complex behavior of hmean is explained by interplay of two opposing factors: the mismatch of the position of the "highest" pixel to the real topographical maximum and higher registration probabilities of larger particles. Consideration of the AFM probe size results in even bigger pixelization induced drops of hmean, which may amount to ∼50% of the true value. The obtained results contribute to AFM data interpretation and methodological aspects of AFM operation in many fields of nanoscience. In particular, they may be used for estimation of true height of nanoparticles from their AFM images obtained with different (even low) pixel resolution.

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.

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.

Time-Lapse Single-Biomolecule Atomic Force Microscopy Investigation on Modified Graphite in Solution.

Atomic force microscopy (AFM) of biomolecular processes at the single-molecule level can provide unique information for understanding molecular function. In AFM studies of biomolecular processes in solution, mica surfaces are predominantly used as substrates. However, owing to its high surface charge, mica may induce high local ionic strength in the vicinity of its surface, which may shift the equilibrium of studied biomolecular processes such as biopolymer adsorption or protein-DNA interaction. In the search for alternative substrates, we have investigated the behavior of adsorbed biomolecules, such as plasmid DNA and E. coli RNA polymerase σ70 subunit holoenzyme (RNAP), on highly oriented pyrolytic graphite (HOPG) surfaces modified with stearylamine and oligoglycine-hydrocarbon derivative (GM) monolayers using AFM in solution. We have demonstrated ionic-strength-dependent DNA mobility on GM HOPG and nativelike dimensions of RNAP molecules adsorbed on modified HOPG surfaces. We propose an approach to the real-time AFM investigation of transcription on stearylamine monolayers on graphite. We conclude that modified graphite allows us to study biomolecules and biomolecular processes on its surface at controlled ionic strength and may be used as a complement to mica in AFM investigations.

Effect of DNA bending on transcriptional interference in the systems of closely spaced convergent promoters.

BACKGROUND: Over the past years there are increasing evidences that the interplay between two molecules of RNA polymerases, initiating transcription from promoters, oriented in opposite (convergent) directions, can serve as a regulatory factor of gene expression. The data concerning the molecular mechanisms of this so-called transcriptional interference (TI) are not well understood. METHODS: The interaction of RNA polymerase with circular DNA templates, containing the convergent promoters, was investigated in a series of in vitro transcription assays and atomic force microscopy (AFM). RESULTS: In this work, to study the mechanisms of transcription interference a series of plasmids with oppositely oriented closely spaced artificial promoters, recognized by Escherichia coli RNA polymerase, was constructed. The constructs differ in promoter structure and distance between the transcription start sites. We have demonstrated that the transcripts ratio (RNA-R/RNA-L) and morphology of convergent open promoter complexes (OPC) are highly dependent on the interpromoter distance. CONCLUSIONS: The obtained results allowed us to suggest the novel model of TI, which assumes the DNA bending upon binding of RNA polymerase with promoters and explains the phenomenon of complete inactivation of weaker promoter by the stronger one. GENERAL SIGNIFICANCE: The results show that the conformational transitions in DNA helix, associated with DNA bending upon binding of RNA polymerase with promoters, play crucial role in OPC formation in the systems with convergent promoters.

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.