Molecular Recognition of Proteins through Quantitative Force Maps at Single Molecule Level.

Intermittent jumping force is an operational atomic-force microscopy mode that produces simultaneous topography and tip-sample maximum-adhesion images based on force spectroscopy. In this work, the operation conditions have been implemented scanning in a repulsive regime and applying very low forces, thus avoiding unspecific tip-sample forces. Remarkably, adhesion images give only specific rupture events, becoming qualitative and quantitative molecular recognition maps obtained at reasonably fast rates, which is a great advantage compared to the force-volume modes. This procedure has been used to go further in discriminating between two similar protein molecules, avidin and streptavidin, in hybrid samples. The adhesion maps generated scanning with biotinylated probes showed features identified as avidin molecules, in the range of 40-80 pN; meanwhile, streptavidin molecules rendered 120-170 pN at the selected working conditions. The gathered results evidence that repulsive jumping force mode applying very small forces allows the identification of biomolecules through the specific rupture forces of the complexes and could serve to identify receptors on membranes or samples or be applied to design ultrasensitive detection technologies.

AFM Study of Nanoscale Membrane Perturbation Induced by Antimicrobial Lipopeptide C14 KYR.

Lipopeptides have been extensively studied as potential antimicrobial agents. In this study, we focused on the C14-KYR lipopeptide, a modified version of the KYR tripeptide with myristic acid at the N-terminus. Here, membrane perturbation of live E. coli treated with the parent KYR and C14-KYR peptides was compared at the nanoscale level using AFM imaging. AFM analyses, including average cellular roughness and force spectroscopy, revealed the severe surface disruption mechanism of C14-KYR. A loss of surface roughness and changes in topographic features included membrane shrinkage, periplasmic membrane separation from the cell wall, and cytosolic leakage. Additional evidence from synchrotron radiation FTIR microspectroscopy (SR-FTIR) revealed a marked structural change in the membrane component after lipopeptide attack. The average roughness of the E. coli cell before and after treatment with C14-KYR was 129.2 ± 51.4 and 223.5 ± 14.1 nm, respectively. The average rupture force of the cell treated with C14-KYR was 0.16 nN, four times higher than that of the untreated cell. Our study demonstrates that the mechanistic effect of the lipopeptide against bacterial cells can be quantified through surface imaging and adhesion force using AFM.

Determination of the mechanical properties of polymeric microneedles by micromanipulation.

Precise characterization of the mechanical properties of polymeric microneedles is crucial for their successful penetration into skin and delivery of the loaded active ingredients. However, most available strategies for this purpose are based on compression of the whole patch, which only provide the average rupture force of the needles and can not give information on the variations across individual microneedles in the patch. In this study, we determined the mechanical strength of individual microneedles of two types of hyaluronic acid microneedles with or without loaded model drugs using a micromanipulation technique. The applied force as a function of displacement of the microneedles was recorded, which was used to determine the rupture displacement, rupture force, and then to derive and calculate normal stress-deformation curve, rupture stress and Young's modulus of individual microneedles. The obtained data suggest that the molecular weight of the polymer and the loading of drug into the microneedles can significantly affect the rupture behavior and mechanical properties of the microneedles, which provides a foundation for preparing sufficiently strong microneedles for controlled drug delivery.

Physical 'strength' of the multi-protein chain connecting immune cells: Does the weakest link limit antibody affinity maturation?: The weakest link in the multi-protein chain facilitating antigen acquisition by B cells in germinal centres limits antibody affinity maturation.

The affinities of antibodies (Abs) for their target antigens (Ags) gradually increase in vivo following an infection or vaccination, but reach saturation at values well below those realisable in vitro. This 'affinity ceiling' could in many cases restrict our ability to fight infections and compromise vaccines. What determines the affinity ceiling has been an unresolved question for decades. Here, we argue that it arises from the strength of the chain of protein complexes that is pulled by B cells during the process of Ag acquisition. The affinity ceiling is determined by the strength of the weakest link in the chain. We identify the weakest link and show that the resulting affinity ceiling can explain the Ab affinities realized in vivo, providing a conceptual understanding of Ab affinity maturation. We explore plausible evolutionary underpinnings of the affinity ceiling, examine supporting evidence and alternative hypotheses and discuss implications for vaccination strategies.

Mechanical properties and cytoskeletal protein changes after anln deficiency / 中国组织工程研究

BACKGROUND: To date, ANLN has definite roles in altering cell shape, regulating cell-cell junction integrity in interphase and stabilizing actomyosin contractile rings in cytokinesis, but its effects on cell mechanical properties and on cytoskeletal proteins have rarely been reported. OBJECTIVE: To investigate the effect of ANLN deletion on the mechanical properties and cytoskeleton of interphase Hela cells. METHODS: Surface elastic modulus and membrane rupture force of normal untreated Hela cells and ANLN RNA stably knocked down Hela cells were measured by atomic force microscopy. We screened for the cells that stably expressed mCherry-Myosin II A, and observed the distribution characteristics of cytoskeletal proteins by laser scanning confocal microscopy. RESULTS AND CONCLUSION: (1) The elastic modulus of Hela cells with ANLN stably knocked down was significantly higher than that of normal Hela cells, and the elastic modulus of normal cells were more prone to polar distribution (gradually decreasing between the two poles) than that of ANLN knockdown Hela cells. However, there was no significant difference in the membrane rupture force at the long-axis edge region between the two groups. (2) Myosin IIA lowly expressed in the marginal region of ANLN knockdown cells. (3) The actin fibers tended to be scattered in the near-bottom cell-cell junction region of the ANLN knockdown group, and there were no obvious intracellular fibers bundles arranging along the long axis. The cell gap tended to enlarge in the middle layer. To conclude, ANLN knockdown cells have the greatest impact in the marginal region, the deficiency of ANLN leads to a more frequent remodeling in the cell marginal region, and the cells need to accumulate more cytoskeletal proteins and binding proteins to stabilize the cell state, resulting in higher modulus of elastics.

An estimate to the first approximation of microtubule rupture force.

Microtubule mechanical properties are essential for understanding basic cellular processes, including cell motility and division, but the forces that result in microtubule rupture or breakage have not yet been measured directly. These forces are essential to understand the mechanical properties of the cytoskeleton and responses by cells to both normal conditions and stress caused by injury or disease. Here we estimate the force required to rupture a microtubule by analyzing kinesin-14 Ncd motor-induced microtubule breakage in ensemble motility assays. We model the breakage events as caused by Ncd motors pulling or pushing on single microtubules that are clamped at one end by other motors attached to the glass surface. The number of pulling or pushing Ncd motors is approximated from the length of the microtubule bound to the surface and the forces produced by the pulling or pushing motors are estimated from forces produced by the Ncd motor in laser-trap assays, reported by others. Our analysis provides an estimate, to the first approximation, of ~ 500 pN for the minimal force required to rupture a 13-pf microtubule. The value we report is close to the forces estimated from microtubule stretching/fragmentation experiments and overlaps with the forces applied by AFM in microtubule indentation assays that destabilize microtubules and break microtubule protofilaments. It is also consistent with the forces required to disrupt protein noncovalent bonds in force spectroscopy experiments. These findings are relevant to microtubule deformation and breakage caused by cellular tension in vivo.

Identification of novel protein domain for sialyloligosaccharide binding essential to Mycoplasma mobile gliding.

Sialic acids, terminal structures of sialylated glycoconjugates, are widely distributed in animal tissues and are often involved in intercellular recognitions, including some bacteria and viruses. Mycoplasma mobile, a fish pathogenic bacterium, binds to sialyloligosaccharide (SO) through adhesin Gli349 and glides on host cell surfaces. The amino acid sequence of Gli349 shows no similarity to known SO-binding proteins. In the present study, we predicted the binding part of Gli349, produced it in Escherichia coli and proved its binding activity to SOs of fetuin using atomic force microscopy. Binding was detected with a frequency of 10.3% under retraction speed of 400 nm/s and was shown to be specific for SO, as binding events were competitively inhibited by the addition of free 3'-sialyllactose. The histogram of the unbinding forces showed 24 pN and additional peaks. These results suggested that the distal end of Gli349 constitutes a novel sialoadhesin domain and is directly involved in the gliding mechanism of M. mobile.

Mechanical properties of P-selectin PSGL-1 bonds.

The accurate determination of the mechanical properties of P-selectin and PSGL-1 is crucial for design and optimization of applications utilizing such bonds, e.g. biosensors and targeted drug delivery systems, as adhesion and mechanical interactions play a critical role in several key functions of biological cells. In current work, the spring constant and rupture force of a single P-selectin PSGL-1 ligand receptor bond and the Young's modulus of a layer made of these ligand receptors are reported. The work-of-adhesion of the P-selectin PSGL-1 interface is also characterized. In the reported experiments, PSGL-1 coated particles are deposited on a P-selectin coated substrate and their transient nanometer scale out-of-plane displacements are acquired employing a laser Doppler vibrometer as they are excited by an ultrasonic field. From the spectral response of a single particle, the resonance frequencies of its vibrational motion are identified, and with help of a particle adhesion model, the average rupture force and stiffness of a single P-selectin PSGL-1 ligand receptor are determined as Frupt = 171 ± 56 pN and kb = 0.56 ± 0.04 mN/m, respectively. Furthermore, the Young's modulus and work-of-adhesion of a layer of P-selectin PSGL-1 ligand receptors are extracted as E = 28.74 ± 3.96 MPa and WA = 70.0 ± 8.0 mJ/m2, respectively. Unlike Atomic Force Microscopy (AFM) and other probe-based techniques, the reported approach eliminates the need for direct contact with the sample, which could compromise the accuracy of the results by imposing unspecified additional contact interactions. Further, the current technique can be employed for measurements under various fluid flow conditions.

Mechanical properties of bean grains (BRSMG Majestic) coated with carnauba wax / Propriedades mecânicas de grãos de feijão cultivar BRSMG Majestoso revestidos com cera de carnaúba

ABSTRACT: The objective of this research was to determine the mechanical properties of beans (BRSMG Majestuoso) coated with carnauba wax solution. Grains with 0.1481 (d.b.) moisture content was used. Treatments 0, 1 and 2 were respectively defined as the control sample, application of the wax diluted 1/1 (carnauba wax solution/water) and application of carnauba wax without dilution. Uniaxial compression tests were performed in the universal test apparatus, TA HD Texture Analyzer with 500 N load cell. Compression tests were carried out during the storage period of in-nature grains, in those that underwent the hydration process at 40 and 50 °C of temperature and in grains during the cooking process. Only the storage time had a significant effect (P<0.05) on the rupture force and the proportional deformity module. Hydration and cooking of the grains provided a reduction in the values of the studied variables.

RESUMO: Objetivou-se com este trabalho determinar as propriedades mecânicas de grãos de feijão cultivar BRSMG Majestoso revestidos com cera de carnaúba. Foram utilizados grãos com teor de água de aproximadamente 0,1481 decimal b.s. (base seca), tratados com solução de cera de carnaúba. Parte destes destinou-se à amostra testemunha, uma a aplicação de cera diluída na proporção de 1/1 (solução de cera de carnaúba/água) e outra a aplicação apenas de solução de cera de carnaúba, tratamentos 0, 1 e 2, respectivamente. Foram realizados ensaios de compressão uniaxiais no aparelho universal de testes, TA. HD Texture Analyser, com célula de carga de 500 N. Os testes de compressão foram realizados ao longo do período de armazenamento nos grãos in natura, naqueles que passaram pelo processo de hidratação nas temperaturas de 40 e 50 °C e em grãos ao longo do processo de cocção. Apenas o tempo de armazenamento teve um efeito significativo (P<0,05) sobre a força de ruptura e o módulo proporcional de deformidade. A hidratação e a cocção dos grãos proporcionaram redução nos valores das variáveis estudadas.

Single-Cell Acoustic Force Spectroscopy: Resolving Kinetics and Strength of T Cell Adhesion to Fibronectin.

Assessing the strength and kinetics of molecular interactions of cells with the extracellular matrix is fundamental to understand cell adhesion processes. Given the relevance of these processes, there is a strong need for physical methods to quantitatively assess the mechanism of cell adhesion at the single-cell level, allowing discrimination of cells with different behaviors. Here we introduce single-cell acoustic force spectroscopy (scAFS), an approach that makes use of acoustic waves to exert controlled forces, up to 1 nN, to hundreds of individual cells in parallel. We demonstrate the potential of scAFS by measuring adhesion forces and kinetics of CD4+ T lymphocytes (CD4) to fibronectin. We determined that CD4 adhesion is accelerated by interleukin-7, their main regulatory cytokine, whereas CD4 binding strength remains the same. Activation of these cells likely increases their chance to bind to the vessel wall in the blood flow to infiltrate inflamed tissues and locally coordinate the immune response.

Exposure to Bordetella pertussis adenylate cyclase toxin affects integrin-mediated adhesion and mechanics in alveolar epithelial cells.

BACKGROUND INFORMATION: The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown. RESULTS: In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure <1 h. At 30 min of exposure, CyaA toxin has a minimal effect on cell viability (>95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 µm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin-specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho-physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions). CONCLUSIONS: MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in intracellular cAMP in these target cells. SIGNIFICANCE: These results suggest that mechanical and adhesion properties of the cells appear as pertinent markers of cytotoxicity of CyaA toxin.

Fast and accurate determination of the relative binding affinities of small compounds to HIV-1 protease using non-equilibrium work.

The fast pulling ligand (FPL) out of binding cavity using non-equilibrium molecular dynamics (MD) simulations was demonstrated to be a rapid, accurate and low CPU demand method for the determination of the relative binding affinities of a large number of HIV-1 protease (PR) inhibitors. In this approach, the ligand is pulled out of the binding cavity of the protein using external harmonic forces, and the work of pulling force corresponds to the relative binding affinity of HIV-1 PR inhibitor. The correlation coefficient between the pulling work and the experimental binding free energy of R=-0.95 shows that FPL results are in good agreement with experiment. It is thus easier to rank the binding affinities of HIV-1 PR inhibitors, that have similar binding affinities because the mean error bar of pulling work amounts to δW=7%. The nature of binding is discovered using the FPL approach. © 2016 Wiley Periodicals, Inc.

Directly measuring single-molecule heterogeneity using force spectroscopy.

One of the most intriguing results of single-molecule experiments on proteins and nucleic acids is the discovery of functional heterogeneity: the observation that complex cellular machines exhibit multiple, biologically active conformations. The structural differences between these conformations may be subtle, but each distinct state can be remarkably long-lived, with interconversions between states occurring only at macroscopic timescales, fractions of a second or longer. Although we now have proof of functional heterogeneity in a handful of systems-enzymes, motors, adhesion complexes-identifying and measuring it remains a formidable challenge. Here, we show that evidence of this phenomenon is more widespread than previously known, encoded in data collected from some of the most well-established single-molecule techniques: atomic force microscopy or optical tweezer pulling experiments. We present a theoretical procedure for analyzing distributions of rupture/unfolding forces recorded at different pulling speeds. This results in a single parameter, quantifying the degree of heterogeneity, and also leads to bounds on the equilibration and conformational interconversion timescales. Surveying 10 published datasets, we find heterogeneity in 5 of them, all with interconversion rates slower than 10 s(-1) Moreover, we identify two systems where additional data at realizable pulling velocities is likely to find a theoretically predicted, but so far unobserved crossover regime between heterogeneous and nonheterogeneous behavior. The significance of this regime is that it will allow far more precise estimates of the slow conformational switching times, one of the least understood aspects of functional heterogeneity.

A new approach to analyze the dynamic strength of eggs.

The mechanical behavior of eggshell was determined in terms of average rupture force and corresponding deformation. For the experiment, we selected goose eggs (Anser anser f. domestica). Samples of eggs were compressed along their x-axis and z-axis. The effect of the loading orientation can be described in terms of the eggshell contour curvature. Two different experimental methods were used: compression between two plates (loading rates up to 5 mm/s) and the Hopkinson split pressure bar technique. This second method enables achieving loading rates up to about 17 m/s. The response of goose eggs to this high loading rate was characterized also by simultaneous measurement of the eggshell surface displacements using a laser vibrometer and by the measurement of both circumferential and meridian strains.

Effect of the loading rate on compressive properties of goose eggs.

The resistance of goose (Anser anser f. domestica) eggs to damage was determined by measuring the average rupture force, specific deformation and rupture energy during their compression at different compression speeds (0.0167, 0.167, 0.334, 1.67, 6.68 and 13.36 mm/s). Eggs have been loaded between their poles (along X axis) and in the equator plane (Z axis). The greatest amount of force required to break the eggs was required when eggs were loaded along the X axis and the least compression force was required along the Z axis. This effect of the loading orientation can be described in terms of the eggshell contour curvature. The rate sensitivity of the eggshell rupture force is higher than that observed for the Japanese quail's eggs.

A Self-Adaptive Steered Molecular Dynamics Method Based on Minimization of Stretching Force Reveals the Binding Affinity of Protein-Ligand Complexes.

Binding affinity prediction of protein-ligand complexes has attracted widespread interest. In this study, a self-adaptive steered molecular dynamics (SMD) method is proposed to reveal the binding affinity of protein-ligand complexes. The SMD method is executed through adjusting pulling direction to find an optimum trajectory of ligand dissociation, which is realized by minimizing the stretching force automatically. The SMD method is then used to simulate the dissociations of 19 common protein-ligand complexes which are derived from two homology families, and the binding free energy values are gained through experimental techniques. Results show that the proposed SMD method follows a different dissociation pathway with lower a rupture force and energy barrier when compared with the conventional SMD method, and further analysis indicates the rupture forces of the complexes in the same protein family correlate well with their binding free energy, which reveals the possibility of using the proposed SMD method to identify the active ligand.

Some physical and mechanical properties of roasted Zerun wheat.

Some physical and mechanical properties of roasted Zerun wheat were investigated in the moisture range from 8.80 % to 23.40 % wet basis. Mechanical properties were evaluated by examining the effect of moisture content upon the grain rupture force, energy and Weibull parameters. Length, width, thickness, porosity and angle of repose increased nonlinearly from 6.09 to 6.36 mm; 4.17 to 4.18 mm; 2.66 to 2.78 mm; 37.71 % to 39.09 % and 33.02° to 37.90°, respectively when moisture content increased. The Weibull distribution fits the data for rupture force and energy. The Weibull modulus and scale parameter for rupture force varied between 3.88 and 6.20; 26.61 and 44.24N, respectively. The Weibull modulus for energy increased from 2.15 to 3.24 with increased in moisture content. Measured mechanical properties of grains showed that the brittleness and fragile structure of the roasted grain gradually lost its characteristic crispiness and become soft and ductile above 13.78 % moisture content.