AFM study of potato virus X disassembly induced by movement protein

Recently we have reported that a selective binding of potato virus X (PVX)-coded movement protein (termed TGBp1 MP) to one end of a polar coat protein (CP) helix converted viral RNA into a translatable form and induced a linear destabilization of the whole helical particle. Here, the native PVX virions, RNase-treated (PVX(RNA-DEG)) helical particles lacking intact RNA and their complexes with TGBp1 (TGBp1-PVX and TGBp1-PVX(RNA-DEG)), were examined by atomic force microscopy (AFM). When complexes of the TGBp1 MP with PVX were examined by means of AFM in liquid, no structural reorganization of PVX particles was observed. By contrast, the products of TGBp1-dependent PVX degradation termed "beads-on-string" were formed under conditions of AFM in air. The AFM images of PVX(RNA-DEG) were indistinguishable from images of native PVX particles; however, the TGBp1-dependent disassembly of the CP-helix was triggered when the TGBp1-PVX(RNA-DEG) complexes were examined by AFM, regardless of the conditions used (in air or in liquid). Our data supported the idea that binding of TGBp1 to one end of the PVX CP-helix induced linear destabilization of the whole helical particle, which may lead to its disassembly under conditions of AFM.     

Electric birefringence studies on polyglutamic acid.

The electric birefringence for the aqueous solution of poly-L -glutamic acid (PGA) in the helical form was studied. PGA samples were fractionated by gel column chromatography. PGA showed a positive electric birefringence. The permanent dipole moment of the PGA molecule was suggested to be largely suppressed. The measurements of the intrinsic Kerr constants for various molecular lengths showed that the electric anisotropy (polarizability) of PGA is proportional to the 1.5 power of the length. The electric birefrigence measurement was also carried out in the helix–coil transition region. The Kerr constant of PGA was largely reduced on going from the helical form to the coiled form.     

Atomic force microscopy study of DNA flexibility on short length scales: smooth bending versus kinking

The apparently anomalous flexibility of DNA on short length scales has attracted a lot of attention in recent years. We use atomic force microscopy (AFM) in solution to directly study the DNA bending statistics for small lengths down to one helical turn. The accuracy of experimental estimates could be improved due to a large data volume and a refined algorithm for image processing and measuring bend angles. It is found that, at length scales beyond two helical turns (7 nm), DNA is well described by the harmonic worm-like chain (WLC) model with the bending persistence length of 56 nm. Below this threshold, the AFM data are also described by the WLC model assuming that the accuracy of measured bend angles is limited by the physical width of the double helix. We conclude that the double helical DNA behaves as a uniform elastic rod even at very short length scales. Strong bends due to kinks, melting bubbles and other deviations from the WLC model are statistically negligible.     

Application of atomic force microscopy to the study of micromechanical properties of biological materials

Atomic force microscopy (AFM) has been used to study the micromechanical properties of biological systems. Its unique ability to function both as an imaging device and force sensor with nanometer resolution in both gaseous and liquid environments has meant that AFM has provided unique insights into the mechanical behaviour of tissues, cells and single molecules. As a surface scanning device, AFM can map properties such as adhesion and the Young's modulus of surfaces. As a force sensor and nanoindentor AFM can directly measure properties such as the Young's modulus of surfaces or the binding forces of cells. As a stress-strain gauge AFM can study the stretching of single molecules or fibres and as a nanomanipulator it can dissect biological particles such as viruses or DNA strands. The present paper reviews key research that has demonstrated the versatility of AFM and how it can be exploited to study the micromechanical behaviour of biological materials.     

Applicability of broken-rodlike chain model to conformational analysis of polypeptide chain

In order to check the applicability of the broken-rodlike (BR) chain model, consisting of several rods alternatively joined by flexible random coils, to the conformational analysis of a polypeptide chain in the helix-to-coil transition regions, two relations predicted by the Zimm and Bragg theory and the method with the BR chain model are compared. It is shown that, despite a clear difference between the models employed in the two methods, they give substantially identical results in both probability P(j) that a helical residue is in a helical sequence j units long and averaged helical fraction dependence of the mean-squared radius of gyration. Thus the use of the method with the BR chain model in the conformational analysis of a polypeptide chain could be rationalized, at least, with the same degree of approximation as is assumed in the Zimm and Bragg theory. Using the scattering function for the BR chain model, averaged helical-sequence lengths are evaluated for partially ionized poly(L-glutamic acid) (PGA) in added-salt aqueous solution and nonionized PGA in N-methylacetamide, both in a helical state. As a result, it is shown that the length in the latter molecule is approximately tenfold longer than that in the former one.     

Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex

Saccharomyces cerevisiae Dmc1, a meiosis-specific homologue of RecA, catalyzes homologous pairing and strand exchange during meiotic DNA recombination. The purified budding yeast Dmc1 (ScDmc1) protein exhibits much weaker recombinase activity in vitro as compared to that of the Escherichia coli RecA protein. Using atomic force microscopy (AFM) with carbon nanotube tips, we found ScDmc1 forms rings with an external diameter of 18 nm and a central cavity of 4 nm. In the presence of single-stranded DNA (ssDNA), the majority of the ScDmc1 protein (90%) bound DNA as protein rings; only a small faction (10%) was able to form filamentous structure. In contrast, nearly all RecA proteins form fine helical nucleoprotein filaments with ssDNA under identical conditions. RecA-mediated recombinase activity is initiated through the nucleation of RecA onto ssDNA to form helical nucleoprotein filaments. Our results support the notion that ScDmc1 becomes catalytically active only when it forms a helical nucleoprotein filament with ssDNA.     

Structure of intracellular mature vaccinia virus visualized by in situ atomic force microscopy

Vaccinia virus, the basis of the smallpox vaccine, is one of the largest viruses to replicate in humans. We have used in situ atomic force microscopy (AFM) to directly visualize fully hydrated, intact intracellular mature vaccinia virus (IMV) virions and chemical and enzymatic treatment products thereof. The latter included virion cores, core-enveloping coats, and core substructures. The isolated coats appeared to be composed of a highly cross-linked protein array. AFM imaging of core substructures indicated association of the linear viral DNA genome with a segmented protein sheath forming an extended approximately 16-nm-diameter filament with helical surface topography; enclosure of this filament within a 30- to 40-nm-diameter tubule which also shows helical topography; and enclosure of the folded, condensed 30- to 40-nm-diameter tubule within the core by a wall covered with peg-like projections. Proteins observed attached to the 30- to 40-nm-diameter tubules may mediate folding and/or compaction of the tubules and/or represent vestiges of the core wall and/or pegs. An accessory "satellite domain" was observed protruding from the intact core. This corresponded in size to isolated 70- to 100-nm-diameter particles that were imaged independently and might represent detached accessory domains. AFM imaging of intact virions indicated that IMV underwent a reversible shrinkage upon dehydration (as much as 2.2- to 2.5-fold in the height dimension), accompanied by topological and topographical changes, including protrusion of the satellite domain. As shown here, the chemical and enzymatic dissection of large, asymmetrical virus particles in combination with in situ AFM provides an informative complement to other structure determination techniques.     

Characterization by Atomic Force Microscopy of Alzheimer Paired Helical Filaments under Physiological Conditions

Paired helical filaments (PHF) is an aberrant structure present in the brain of Alzheimer's disease patients which has been correlated with their degree of dementia. In order to determine the structure of PHF, several studies have been performed using atomic force microscopy (AFM). However, those studies have the limitation that they have not been done in solution and the sample could be far from the real physiological conditions. In this work we present an AFM analysis of PHF in liquid environment and we compare that analysis with that performed in dry conditions. PHF imaging in liquid was only possible by using jumping mode AFM as the imaging technique. Jumping mode AFM images of PHF in solution show first, a notable increase in the absolute values of the height of the filament, and second, a smaller ratio between the height measured at the upper and at the lower part of the PHF. Direct comparison of the experimental data with structural models has been performed. From this we conclude that the PHF structure is compatible with two coupled ribbons with an overall height of 20 nm and a width of 10 nm.     

Localization and force analysis at the single virus particle level using atomic force microscopy

Atomic force microscopy (AFM) is a vital instrument in nanobiotechnology. In this study, we developed a method that enables AFM to simultaneously measure specific unbinding force and map the viral glycoprotein at the single virus particle level. The average diameter of virus particles from AFM images and the specificity between the viral surface antigen and antibody probe were integrated to design a three-stage method that sets the measuring area to a single virus particle before obtaining the force measurements, where the influenza virus was used as the object of measurements. Based on the purposed method and performed analysis, several findings can be derived from the results. The mean unbinding force of a single virus particle can be quantified, and no significant difference exists in this value among virus particles. Furthermore, the repeatability of the proposed method is demonstrated. The force mapping images reveal that the distributions of surface viral antigens recognized by antibody probe were dispersed on the whole surface of individual virus particles under the proposed method and experimental criteria; meanwhile, the binding probabilities are similar among particles. This approach can be easily applied to most AFM systems without specific components or configurations. These results help understand the force-based analysis at the single virus particle level, and therefore, can reinforce the capability of AFM to investigate a specific type of viral surface protein and its distributions.     

Polyelectrolyte films based on polysaccharides of different conformations: effects on multilayer structure and mechanical properties

Ultrathin films were prepared with cationic poly(allylamine hydrochloride) (PAH) and two anionic polysaccharides, iota- and lambda-carrageenan, of similar chemical composition but different conformations using the layer-by-layer (LbL) technique. The study of aqueous solutions of carrageenans confirms that iota-carrageenan is at room temperature in helical conformation while lambda-carrageenan is in random coil conformation. Characterization of the multilayers by ellipsometry, circular dichroism, and AFM revealed that iota-carrageenan keeps its helical conformation within the films while lambda-carrageenan chains are in random coil conformation. Investigation of the mechanical properties of the films by performing nanoindentation experiments using force spectroscopy showed clear differences between the two films based on carrageenans of different conformations.     

Effects of a lipid environment on the fibrillogenic pathway of the N-terminal polypeptide of human apolipoprotein A-I, responsible for in vivo amyloid fibril formation

In amyloidosis associated with apolipoprotein A-I (ApoA-I), heart amyloid deposits are mainly constituted by the 93-residue ApoA-I N-terminal region. A recombinant form of the amyloidogenic polypeptide, named [1-93]ApoA-I, shares conformational properties and aggregation propensity with its natural counterpart. The polypeptide, predominantly in a random coil state at pH 8.0, following acidification to pH 4.0 adopts a helical/molten globule transient state, which leads to formation of aggregates. Here we provide evidence that fibrillogenesis occurs also in physiologic-like conditions. At pH 6.4, [1-93]ApoA-I was found to assume predominantly an α-helical state, which undergoes aggregation at 37°C over time at a lower rate than at pH 4.0. After 7 days at pH 6.4, protofibrils were observed by atomic force microscopy (AFM). Using a multidisciplinary approach, including circular dichroism (CD), fluorescence, electrophoretic, and AFM analyses, we investigated the effects of a lipid environment on the conformational state and aggregation propensity of [1-93]ApoA-I. Following addition of the lipid-mimicking detergent Triton X-100, the polypeptide was found to be in a helical state at both pH 8.0 and 6.4, with no conformational transition occurring upon acidification. These helical conformers are stable and do not generate aggregated species, as observed by AFM after 21 days. Similarly, analyses of the effects of cholesterol demonstrated that this natural ApoA-I ligand induces formation of α-helix at physiological concentrations at both pH 8.0 and 6.4. Zwitterionic, positively charged, and negatively charged liposomes were found to affect [1-93]ApoA-I conformation, inducing helical species. Our data support the idea that lipids play a key role in [1-93]ApoA-I aggregation in vivo.     

Measuring the interaction forces between protein inclusion bodies and an air bubble using an atomic force microscope

Interaction forces between protein inclusion bodies and an air bubble have been quantified using an atomic force microscope (AFM). The inclusion bodies were attached to the AFM tip by covalent bonds. Interaction forces measured in various buffer concentrations varied from 9.7 nN to 25.3 nN (+/- 4-11%) depending on pH. Hydrophobic forces provide a stronger contribution to overall interaction force than electrostatic double layer forces. It also appears that the ionic strength affects the interaction force in a complex way that cannot be directly predicted by DLVO theory. The effects of pH are significantly stronger for the inclusion body compared to the air bubble. This study provides fundamental information that will subsequently facilitate the rational design of flotation recovery system for inclusion bodies. It has also demonstrated the potential of AFM to facilitate the design of such processes from a practical viewpoint.     

The application of atomic force microscopy to the study of living vertebrate cells in culture

Atomic force microscopy (AFM), a relatively new variant of scanning probe microscopy developed for the material sciences, is becoming an increasingly important tool in other disciplines. In this review I describe in nontechnical terms some of the basic aspects of using AFM to study living vertebrate cells. Although AFM has some unusual attributes such as an ability to be used with living cells, AFM also has attributes that make its use in cell biology a real challenge. This review was written to encourage researchers in the biological and biomedical sciences to consider AFM as a potential (and potent) tool for their cell biological research.     

Seawater at the nanoscale: marine gel imaged by atomic force microscopy

The present study introduces atomic force microscopy (AFM) as a tool for characterization of marine gel network and marine biopolymers self-assembly, not accessible by other techniques. AFM imaging of marine gel samples collected in summers 2003 and 2004 in the northern Adriatic Sea provided insight into molecular organization of gel network and associations between polysaccharide fibrils in the network. Initial stages of biopolymers self-assembly were visualized by AFM in a phytoplankton bloom experiment performed in the same aquatorium. Based on AFM imaging and differential scanning calorimetry, the marine gel is characterized as a thermoreversible physical gel and the dominant mode of gelation as crosslinking of polysaccharide fibrils by hydrogen bonding which results in helical structures and their associations. Direct deposition of whole seawater on freshly cleaved mica followed by rinsing was the procedure that caused the least impact on the original structures of biopolymer assemblies in seawater.     

Modification of collagen with a natural cross-linker, procyanidin

We have investigated the modification of collagen with a natural plant polyphenol, procyanidin under acidic conditions. Fourier transform infrared spectroscopy (FTIR) and Atomic force microscopy (AFM) studies demonstrate that the hydrogen bond interactions between collagen and procyanidin does not destroy the triple helix conformation of collagen, and the fibril aggregation occurs because of the cross-linking with procyanidin. The water contact angle (WCA) tests indicate that the hydrophobicity of the procyanidin modified collagen films can be improved. Whereas, the water vapor permeability (WVP) of the films decrease with the increasing procyanidin content due to the formation of denser structure. Moreover, differential scanning calorimetry (DSC) and thermogravimetric (TG) measurements reveal that the collagen/procyanidin films have improved thermal stability in comparison with pure collagen. The present study reveals that procyanidin stabilizes collagen as a cross-linker and preserves its triple helical structure.     

Degradation of N5-(2-hydroxyethyl)-L-glutamine and L-glutamic acid homopolymers and copolymers by papain

The rate of degradation of poly[N5-(2-hydroxyethyl)-L-glutamine] (PHEG), poly(L-glutamic acid) (PGA) and poly[HEG-co-GA] random copolymers by papain was measured in the pH range 4.0-7.5, employing the gel permeation chromatography method. The effect of the degree of ionization on the polymer conformation was measured by circular dichroism (c.d.). PHEG, which is uncharged, had a random coil conformation and an almost constant degradation rate within the whole pH interval. The ionization of PGA increased with increasing pH and was accompanied by conformational transition from helix to random coil. The hydrolysis of PGA by papain depended on pH with the optimum at about pH 5, indicating that both the high content of helix (at pH less than 5) and increasing charge density (at pH greater than 5), decreased the degradation rate. Contrary to PGA, pH profiles of the degradation rate of poly[HEG-co-GA] copolymers are monotonous and do not decrease at pH less than 5. In the copolymers the HEG residues act as a helix breaker and limit the formation of helical conformation. The role of structural features of a macromolecular substrate, i.e. the charge, helical conformation and the nature of amino acid residues, in the interaction between enzyme and polymer is discussed.     

Thermodynamic parameters of helix-coil transition in polypeptide chains. I. Poly-(L-glutamic acid)

The helix-coil transitions for poly(L -glutamic acid) (PGA) in 0.2M NaCl and in its mixture with dioxane were studied by the methods of spectropolarimetry, viscometry, and potentiometric titration at different temperatures from 8 to 50°C. The enthalpy and entropy differences between the helical and coillike states of uncharged PGA molecules were determined from the curves of potentiometric titration. The temperature dependence of the cooperativity parameter σ was determined by two methods: from the sharpness of transition and from the dependence of the intrinsic viscosity on the helical content in the transition region. In 0.2MNaCl, σ= (2.5 ± 0.5) × 10^?3 and practically does not depend on temperature, i.e., the cooperativity of the helix-coil transition is connected mainly with the entropy decrease in initiating helical regions (ΔS_i ≈ ?12 is mole of helical regions). On the contrary, initiation of a helical region in the water-organic solvent mixture is accompanied by a considerable enthalpy increase.     

Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or “tapping mode” AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming.PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials.The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.     

Elucidation of the effect of aptamer immobilization strategies on the interaction between cell and its aptamer using atomic force spectroscopy

The immobilization strategy of cell‐specific aptamers is of great importance for studying the interaction between a cell and its aptamer. However, because of the difficulty of studying living cell, there have not been any systematic reports about the effect of immobilization strategies on the binding ability of an immobilized aptamer to its target cell. Because atomic force spectroscopy (AFM) could not only be suitable for the investigation of living cell under physiological conditions but also obtains information reflecting the intrinsic properties of individuals, the effect of immobilization strategies on the interaction of aptamer/human hepatocarcinoma cell Bel‐7404 was successively evaluated using AFM here. Two different immobilization methods, including polyethylene glycol immobilization method and glutaraldehyde immobilization method were used, and the factors, such as aptamer orientation, oligodeoxythymidine spacers and dodecyl spacers, were investigated. Binding events measured by AFM showed that a similar unbinding force was obtained regardless of the change of the aptamer orientation, the immobilization method, and spacers, implying that the biophysical characteristics of the aptamer at the molecular level remain undisturbed. However, it showed that the immobilization orientation, immobilization method, and spacers could alter the binding probability of aptamer/Bel‐7404 cell. Presumably, these factors may affect the accessibility of the aptamer toward its target cell. These results may provide valuable information for aptamer sensor platforms including ultrasensitive biosensor design. Copyright © 2015 John Wiley & Sons, Ltd.