Correlation between plant cell wall stiffening and root extension arrest phenotype in the combined abiotic stress of Fe and Al.

The plasticity and growth of plant cell walls (CWs) remain poorly understood at the molecular level. In this work, we used atomic force microscopy (AFM) to observe elastic responses of the root transition zone of 4-day-old Arabidopsis thaliana wild-type and almt1-mutant seedlings grown under Fe or Al stresses. Elastic parameters were deduced from force-distance curve measurements using the trimechanic-3PCS framework. The presence of single metal species Fe2+ or Al3+ at 10 µM exerts no noticeable effect on the root growth compared with the control conditions. On the contrary, a mix of both the metal ions produced a strong root-extension arrest concomitant with significant increase of CW stiffness. Raising the concentration of either Fe2+ or Al3+ to 20 µM, no root-extension arrest was observed; nevertheless, an increase in root stiffness occurred. In the presence of both the metal ions at 10 µM, root-extension arrest was not observed in the almt1 mutant, which substantially abolishes the ability to exude malate. Our results indicate that the combination of Fe2+ and Al3+ with exuded malate is crucial for both CW stiffening and root-extension arrest. However, stiffness increase induced by single Fe2+ or Al3+ is not sufficient for arresting root growth in our experimental conditions.

Cry11Aa and Cyt1Aa exhibit different structural orders in crystal topography.

Cry11Aa and Cyt1Aa are two pesticidal toxins produced by Bacillus thuringiensis subsp. israelensis. To improve our understanding of the nature of their oligomers in the toxic actions and synergistic effects, we performed the atomic force microscopy to probe the surfaces of their natively grown crystals, and used the L-weight filter to enhance the structural features. By L-weight filtering, molecular sizes of the Cry11Aa and Cyt1Aa monomers obtained are in excellent agreement with the three-dimensional structures determined by x-ray crystallography. Moreover, our results show that the layered feature of a structural element distinguishes the topographic characteristics of Cry11Aa and Cyt1Aa crystals, suggesting that the Cry11Aa toxin has a better chance than Cyt1Aa for multimerization and therefore cooperativeness of the toxic actions.

Intrinsically Disordered Tardigrade Proteins Self-Assemble into Fibrous Gels in Response to Environmental Stress.

Tardigrades are remarkable for their ability to survive harsh stress conditions as diverse as extreme temperature and desiccation. The molecular mechanisms that confer this unusual resistance to physical stress remain unknown. Recently, tardigrade-unique intrinsically disordered proteins have been shown to play an essential role in tardigrade anhydrobiosis. Here, we characterize the conformational and physical behaviour of CAHS-8 from Hypsibius exemplaris. NMR spectroscopy reveals that the protein comprises an extended central helical domain flanked by disordered termini. Upon concentration, the protein is shown to successively form oligomers, long fibres, and finally gels constituted of fibres in a strongly temperature-dependent manner. The helical domain forms the core of the fibrillar structure, with the disordered termini remaining highly dynamic within the gel. Soluble proteins can be encapsulated within cavities in the gel, maintaining their functional form. The ability to reversibly form fibrous gels may be associated with the enhanced protective properties of these proteins.

Conditions to minimize soft single biomolecule deformation when imaging with atomic force microscopy.

A recurrent interrogation when imaging soft biomolecules using atomic force microscopy (AFM) is the putative deformation of molecules leading to a bias in recording true topographical surfaces. Deformation of biomolecules comes from three sources: sample instability, adsorption to the imaging substrate, and crushing under tip pressure. To disentangle these causes, we measured the maximum height of a well-known biomolecule, the tobacco mosaic virus (TMV), under eight different experimental conditions positing that the maximum height value is a specific indicator of sample deformations. Six basic AFM experimental factors were tested: imaging in air (AIR) versus in liquid (LIQ), imaging with flat minerals (MICA) versus flat organic surfaces (self-assembled monolayers, SAM), and imaging forces with oscillating tapping mode (TAP) versus PeakForce tapping (PFT). The results show that the most critical parameter in accurately measuring the height of TMV in air is the substrate. In a liquid environment, regardless of the substrate, the most critical parameter is the imaging mode. Most importantly, the expected TMV height values were obtained with both imaging with the PeakForce tapping mode either in liquid or in air at the condition of using self-assembled monolayers as substrate. This study unambiguously explains previous poor results of imaging biomolecules on mica in air and suggests alternative methodologies for depositing soft biomolecules on well organized self-assembled monolayers.

Atomic force microscope, molecular imaging, and analysis.

Image visibility is a central issue in analyzing all kinds of microscopic images. An increase of intensity contrast helps to raise the image visibility, thereby to reveal fine image features. Accordingly, a proper evaluation of results with current imaging parameters can be used for feedback on future imaging experiments. In this work, we have applied the Laplacian function of image intensity as either an additive component (Laplacian mask) or a multiplying factor (Laplacian weight) for enhancing image contrast of high-resolution AFM images of two molecular systems, an unknown protein imaged in air, provided by AFM COST Action TD1002 (http://www.afm4nanomedbio.eu/), and tobacco mosaic virus (TMV) particles imaged in liquid. Based on both visual inspection and quantitative representation of contrast measurements, we found that the Laplacian weight is more effective than the Laplacian mask for the unknown protein, whereas for the TMV system the strengthened Laplacian mask is superior to the Laplacian weight. The present results indicate that a mathematical function, as exemplified by the Laplacian function, may yield varied processing effects with different operations. To interpret the diversity of molecular structure and topology in images, an explicit expression for processing procedures should be included in scientific reports alongside instrumental setups.

Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules.

BACKGROUND: Synchrotron radiation facilities are pillars of modern structural biology. Small-Angle X-ray scattering performed at synchrotron sources is often used to characterize the shape of biological macromolecules. A major challenge with high-energy X-ray beam on such macromolecules is the perturbation of sample due to radiation damage. RESULTS: By employing atomic force microscopy, another common technique to determine the shape of biological macromolecules when deposited on flat substrates, we present a protocol to evaluate and characterize consequences of radiation damage. It requires the acquisition of images of irradiated samples at the single molecule level in a timely manner while using minimal amounts of protein. The protocol has been tested on two different molecular systems: a large globular tetremeric enzyme (ß-Amylase) and a rod-shape plant virus (tobacco mosaic virus). Radiation damage on the globular enzyme leads to an apparent increase in molecular sizes whereas the effect on the long virus is a breakage into smaller pieces resulting in a decrease of the average long-axis radius. CONCLUSIONS: These results show that radiation damage can appear in different forms and strongly support the need to check the effect of radiation damage at synchrotron sources using the presented protocol.

Adepth: New Representation and its implications for atomic depths of macromolecules.

We applied the signed distance function (SDF) for representing the depths of atoms in a macromolecule. The calculations of SDF values were performed on grid points in a rectangular box that accommodates the macromolecule. The depth for an atom inside the molecule was then obtained as a result of tri-linear interpolation of SDF values at the nearest grid points surrounding the atom. For testing the performance of present program Adepth, we have constructed an artificial molecule whose atomic depths are known as the gold standard for accuracy assessments. On average, our results showed that Adepth reached an accuracy of 1.6% at 0.5 Å of grid spacing, whereas the current reference server DEPTH reached 7.5%. The Adepth program provides both depth and height representations; it is capable of computing iso-surfaces for atomic depths and presenting graphical view of macromolecular shape at some distance away from the surface. Web interface is available at http://biodev.cea.fr/adepth.

Conformational dynamics of individual antibodies using computational docking and AFM.

Molecular recognition between a receptor and a ligand requires a certain level of flexibility in macromolecules. In this study, we aimed at analyzing the conformational variability of receptors portrayed by monoclonal antibodies that have been individually imaged using atomic force microscopy (AFM). Individual antibodies were chemically coupled to activated mica surface, and they have been imaged using AFM in ambient conditions. The resulting topographical surface of antibodies was used to assemble the three subunits constituting antibodies: two antigen-binding fragments and one crystallizable fragment using a surface-constrained computational docking approach. Reconstructed structures based on 10 individual topographical surfaces of antibodies are presented for which separation and relative orientation of the subunits were measured. When compared with three X-ray structures of antibodies present in the protein data bank database, results indicate that several arrangements of the reconstructed subunits are comparable with those of known structures. Nevertheless, no reconstructed structure superimposes adequately to any particular X-ray structure consequence of the antibody flexibility. We conclude that high-resolution AFM imaging with appropriate computational reconstruction tools is adapted to study the conformational dynamics of large individual macromolecules deposited on mica.

Measuring external primary cell wall elasticity of seedling roots using atomic force microscopy.

Stiffness plays a central action in plant cell extension. Here, we present a protocol to detect changes in stiffness on the external epidermal cell wall of living plant roots using atomic force microscopy (AFM). We provide generalized instructions for collecting force-distance curves and analysis of stiffness using contact-based mechanical model. With this protocol, and some initial training in AFM, a user is able to perform indentation experiments on 4- and 5-day-old Arabidopsis thaliana and determine stiffness properties. For complete details on the use and execution of this protocol, please refer to Godon et al.1.

Nano-structural stiffness measure for soft biomaterials of heterogeneous elasticity.

Measuring the structural stiffness aims to reveal the impact of nanostructured components or various physiological circumstances on the elastic response of material to an external indentation. With a pyramidal tip at a nano-scale, we employed the atomic force microscopy (AFM) to indent the surfaces of two compositions of polyacrylamide gels with different softness and seedling roots of Arabidopsis thaliana. We found that the stiffness-depth curve derived from the measured force exhibits a heterogeneous character in elasticity. According to the tendency of stiffness-depth curve, we decomposed the responding force into depth-impact (FC), Hookean (FH) and tip-shape (FS) components, called trimechanic, where FS and its gradient should be offset at the surface or subsurfaces of the indented material. Thereby, trimechnic theory allows us to observe how the three restoring nanomechanics change with varied depth. Their strengths are represented by the respective spring constants (kC, kH, kS) of three parallel-connected spring (3PCS) analogs to differentiate restoring nanomechansims of indented materials. The effective Young's modulus Ê and the total stiffness kT (= kH + kS) globally unambiguously distinguish the softness between the two gel categories. Data fluctuations were observed in the elasticity parameters of individual samples, reflecting nanostructural variations in the gel matrix. Similar tendencies were found in the results from growing plant roots, though the data fluctuations are expectedly much more dramatic. The zone-wise representation of stiffness by the trimechanic-3PCS framework demonstrates a stiffness measure that reflects beneath nanostructures encountered by deepened depth. The trimechanic-3PCS framework can apply any mechanical model of power-law based force-depth relationship and is compatible with thin layer corrections. It provides a new paradigm for analyzing restoring nanomechanics of soft biomaterials in response to indenting forces.

Single and multiple bonds in (strept)avidin-biotin interactions.

Thanks to Dynamic Force Spectroscopy (DFS) and developments of massive data analysis tools, such as YieldFinder, Atomic Force Microscopy (AFM) becomes a powerful method for analyzing long lifetime ligand-receptor interactions. We have chosen the well-known system, (strept)avidin-biotin complex, as an experimental model due to the lack of consensus on interpretations of the rupture force spectrum (Walton et al., 2008). We present new measurements of force-displacement curves for the (strept)avidin-biotin complex. These data were analyzed using the YieldFinder software based on the Bell-Evans formalism. In addition, the Williams model was adopted to interpret the bonding state of the system. Our results indicate the presence of at least two energy barriers in two loading rate regimes. Combining with structural analysis, the energy barriers can be interpreted in a novel physico-chemical context as one inner barrier for H-bond ruptures ( <1 Å), and one outer barrier for escaping from the binding pocket which is blocked by the side chain of a symmetry-related Trp120 in the streptavidin tetramer. In each loading rate regime, the presence of multiple parallel bonds was implied by the Williams model. Interestingly, we found that in literature different terms created for addressing the apparent discrepancies in the results of avidin-biotin interactions can be reconciled by taking into account multiple parallel bonds.

DeStripe: frequency-based algorithm for removing stripe noises from AFM images.

BACKGROUND: Atomic force microscopy (AFM) is a relatively recently developed technique that shows a promising impact in the field of structural biology and biophysics. It has been used to image the molecular surface of membrane proteins at a lateral resolution of one nanometer or less. An immediate obstacle of characterizing surface features in AFM images is stripe noise. To better interpret structures at a sub-domain level, pre-processing of AFM images for removing stripe noises is necessary. Noise removal can be performed in either spatial or frequency domain. However, denoising processing in the frequency domain is a better solution for preserving edge sharpness. RESULTS: We have developed a denoising protocol, called DeStripe, for AFM bio-molecular images that are contaminated with heavy and fine stripes. This program adopts a divide-and-conquer approach by dividing the Fourier spectrum of the image into central and off-center regions for noisy pixels detection and intensity restoration; it is also applicable to other images interfered with high-density stripes such as those acquired by the scanning electron microscope. The denoising effect brought by DeStripe provides better visualization for image objects without introducing additional artifacts into the restored image. CONCLUSIONS: The DeStripe denoising effect on AFM images is illustrated in the present work. It allows extracting extended information from the topographic measurements and implicitly enhances the molecular features in the image. All the presented images were processed by DeStripe with the raw image as the only input without any requirement for other prior information. A web service, http://biodev.cea.fr/destripe, is available for running DeStripe.

Nanoscale surface structures of DNA bound to Deinococcus radiodurans HU unveiled by atomic force microscopy.

The Deinococcus radiodurans protein HU (DrHU) was shown to be critical for nucleoid activities, yet its functional and structural properties remain largely unexplored. We have applied atomic force microscopy (AFM) imaging to study DrHU binding to pUC19-DNA in vitro and analyzed the topographic structures formed at the nanoscale. At the single-molecule level, AFM imaging allows visualization of super-helical turns on naked DNA surfaces and characterization of free DrHU molecules observed as homodimers. When enhancing the molecular surface structures of AFM images by the Laplacian weight filter, the distribution of bound DrHUs was visibly varied as a function of the DrHU/DNA molar ratio. At a low molar ratio, DrHU binding was found to reduce the volume of condensed DNA configuration by about 50%. We also show that DrHU is capable of bridging distinct DNA segments. Moreover, at a low molar ratio, the binding orientation of individual DrHU dimers could be perceived on partially "open" DNA configuration. At a high molar ratio, DrHU stiffened the DNA molecule and enlarged the spread of the open DNA configuration. Furthermore, a lattice-like pattern could be seen on the surface of DrHU-DNA complex, indicating that DrHU multimerization had occurred leading to the formation of a higher order architecture. Together, our results show that the functional plasticity of DrHU in mediating DNA organization is subject to both the conformational dynamics of DNA molecules and protein abundance.

Structure-activity relationships in peptide-antibody complexes: implications for epitope prediction and development of synthetic peptide vaccines.

Interaction modes and molecular surface properties for both peptide- and protein-antibody complexes have been investigated. Datasets were constituted from the IMGT database and consisted of 37 peptide-antibody (PEPT) and 155 protein-antibody (PROT) complexes. A computer approach was developed to analyze the surface of peptides and proteins using a level set method which allows the characterization of shape complementarity using surface curvature. We found that in both datasets, the interacting surfaces of the two binding partners, exhibited a moderate degree of shape complementarity at the molecular level but not at the atomic level. We also evaluated the structural similarity between peptides bound to antibodies and the corresponding regions in the 3D structures of the cognate proteins. We found that no more than 25% of Phipsi; dihedral angles were conserved between the corresponding regions in peptides and proteins. We also superimposed the parent protein structure onto that of the bound peptides and visually looked for the presence of bumps or clashes between the cognate protein and the antibody. Except for antibodies possessing neutralizing activity and for those bound to a peptide longer than 30 residues, no superimposition in peptide-antibody complexes was found to be bump or clash-free. These findings indicate that studies restricted to continuous epitopes are unlikely to provide the information needed to design short linear peptides that could be expected to mimic satisfactorily the discontinuous epitopes of native proteins and be successful as synthetic vaccines.

On the Operational Aspects of Measuring Nanoparticle Sizes.

Nanoparticles are defined as elementary particles with a size between 1 and 100 nm for at least 50% (in number). They can be made from natural materials, or manufactured. Due to their small sizes, novel toxicological issues are raised and thus determining the accurate size of these nanoparticles is a major challenge. In this study, we performed an intercomparison experiment with the goal to measure sizes of several nanoparticles, in a first step, calibrated beads and monodispersed SiO2 Ludox®, and, in a second step, nanoparticles (NPs) of toxicological interest, such as Silver NM-300 K and PVP-coated Ag NPs, Titanium dioxide A12, P25(Degussa), and E171(A), using commonly available laboratory techniques such as transmission electron microscopy, scanning electron microscopy, small-angle X-ray scattering, dynamic light scattering, wet scanning transmission electron microscopy (and its dry state, STEM) and atomic force microscopy. With monomodal distributed NPs (polystyrene beads and SiO2 Ludox®), all tested techniques provide a global size value amplitude within 25% from each other, whereas on multimodal distributed NPs (Ag and TiO2) the inter-technique variation in size values reaches 300%. Our results highlight several pitfalls of NP size measurements such as operational aspects, which are unexpected consequences in the choice of experimental protocols. It reinforces the idea that averaging the NP size from different biophysical techniques (and experimental protocols) is more robust than focusing on repetitions of a single technique. Besides, when characterizing a heterogeneous NP in size, a size distribution is more informative than a simple average value. This work emphasizes the need for nanotoxicologists (and regulatory agencies) to test a large panel of different techniques before making a choice for the most appropriate technique(s)/protocol(s) to characterize a peculiar NP.

Multi-template approach to modeling engineered disulfide bonds.

The key issue for disulfide bond engineering is to select the most appropriate location in the protein. By surveying the structure of experimentally engineered disulfide bonds, we found about half of them that have geometry incompatible with any native disulfide bond geometry. To improve the current prediction methods that tend to apply either ideal geometrical or energetical criteria to single three-dimensional structures, we have combined a novel computational protocol with the usage of multiple protein structures to take into account protein backbone flexibility. The multiple structures can be selected from either independently determined crystal structures for identical proteins, models of nuclear magnetic resonance experiments, or crystal structures of homology-related proteins. We have validated our approach by comparing the predictions with known disulfide bonds. The accuracy of prediction for native disulfide bonds reaches 99.6%. In a more stringent test on the reported engineered disulfide bonds, we have obtained a success rate of 93%. Our protocol also determines the oxido-reduction state of a predicted disulfide bond and the corresponding mutational cost. From the energy ranking, the user can easily choose top predicted sites for mutagenesis experiments. Our method provides information about local stability of the engineered disulfide bond surroundings.

Identification of functionally important residues in proteins using comparative models.

Rational design in protein engineering leads to significant progresses in medicinal chemistry research. It alleviates the difficulty of exploring unreasonable biological functions. Combining with analysis of biophysical-chemical properties, a three-dimensional (3D) structure provides fruitful information for rational design by revealing functionally important residues. Comparative (homology) modeling, one of the 3D structural prediction techniques, takes advantage of that homologous proteins share similarity in their 3D structures despite the lack of sequence similarity. Of the most value, 3D models provide functional clues even though the function may have been modified during evolution. We illustrate here two applications to medicinal chemistry research where comparative models made a significant improvement on the understanding of relevant biological functions of two proteins. These multiple collaborative projects involve the identification of solvent-exposed residues in a membrane anchoring domain of human coagulation factor V, and revealing critical residues in the interfaces of an antibody and a polynuclear aromatic hydrocarbon ligand. Since the protocol of comparative modeling technique we employed is essential to proposing useful hypotheses for experimental testing, we also present our methodology underlying our modeling programs. Our results show that inaccuracies in comparative models do not hamper functional evaluation as long as an in depth analysis of 3D structures is performed.

Model of a ternary complex between activated factor VII, tissue factor and factor IX.

Upon binding to tissue factor, FVIIa triggers coagulation by activating vitamin K-dependent zymogens, factor IX (FIX) and factor X (FX). To understand recognition mechanisms in the initiation step of the coagulation cascade, we present a three-dimensional model of the ternary complex between FVIIa:TF:FIX. This model was built using a full-space search algorithm in combination with computational graphics. With the known crystallographic complex FVIIa:TF kept fixed, the FIX docking was performed first with FIX Gla-EGF1 domains, followed by the FIX protease/EGF2 domains. Because the FIXa crystal structure lacks electron density for the Gla domain, we constructed a chimeric FIX molecule that contains the Gla-EGF1 domains of FVIIa and the EGF2-protease domains of FIXa. The FVIIa:TF:FIX complex has been extensively challenged against experimental data including site-directed mutagenesis, inhibitory peptide data, haemophilia B database mutations, inhibitor antibodies and a novel exosite binding inhibitor peptide. This FVIIa:TF:FIX complex provides a powerful tool to study the regulation of FVIIa production and presents new avenues for developing therapeutic inhibitory compounds of FVIIa:TF:substrate complex.

Nanoscale structural features determined by AFM for single virus particles.

In this work, we propose "single-image analysis", as opposed to multi-image averaging, for extracting valuable information from AFM images of single bio-particles. This approach allows us to study molecular systems imaged by AFM under general circumstances without restrictions on their structural forms. As feature exhibition is a resolution correlation, we have performed AFM imaging on surfaces of tobacco mosaic virus (TMV) to demonstrate variations of structural patterns with probing resolution. Two AFM images were acquired with the same tip at different probing resolutions in terms of pixel width, i.e., 1.95 and 0.49 nm per pixel. For assessment, we have constructed an in silico topograph based on the three-dimensional crystal structure of TMV as a reference. The prominent artifacts observed in the AFM-determined shape of TMV were attributed to tip convolutions. The width of TMV rod was systematically overestimated by ~10 nm at both probing resolutions of AFM. Nevertheless, the effects of tip convolution were less severe in vertical orientation so that the estimated height of TMV by AFM imaging was in close agreement with the in silico X-ray topograph. Using dedicated image processing algorithms, we found that at low resolution (i.e., 1.95 nm per pixel), the extracted surface features of TMV can be interpreted as a partial or full helical repeat (three complete turns with ~7.0 nm in length), while individual protein subunits (~2.5 nm) were perceivable only at high resolution. The present study shows that the scales of revealed structural features in AFM images are subject to both probing resolution and processing algorithms for image analysis.

Is the coexistence of thromboembolic events and Factor VII deficiency fortuitous?

Over 30 thromboembolic events have been reported in factor VII (FVII) deficiency either associated with previously asymptomatic forms or bleeding diathesis. Whether this coexistence is fortuitous or not is still a mater of debate. Nevertheless, it is well admitted that (i) thrombotic events occurring in FVII-deficient patients with any apparent triggering factors are very rare, (ii) surgical procedures, replacement therapy (especially containing activated factors) but also the presence of an antiphospholipid syndrome are frequently associated with these particular thrombotic events, (iii) in the same way, R304Q and A294V FVII variants appear to be more prevalent than other FVII equally frequent mutations and finally (iv) low FVII coagulant activity levels do not protect against thrombosis. Therefore, peri-operative thrombotic prophylaxis should be relevant for these particular FVII-deficient patients. However, safety, treatment modalities and specific indications of such an antithrombotic prophylaxis remain to be established.