Single‐Molecule Manipulation in Zero‐Mode Waveguides

The mechanobiology of receptor–ligand interactions and force‐induced enzymatic turnover can be revealed by simultaneous measurements of force response and fluorescence. Investigations at physiologically relevant high labeled substrate concentrations require total internal reflection fluorescence microscopy or zero mode waveguides (ZMWs), which are difficult to combine with atomic force microscopy (AFM). A fully automatized workflow is established to manipulate single molecules inside ZMWs autonomously with noninvasive cantilever tip localization. A protein model system comprising a receptor–ligand pair of streptavidin blocked with a biotin‐tagged ligand is introduced. The ligand is pulled out of streptavidin by an AFM cantilever leaving the receptor vacant for reoccupation by freely diffusing fluorescently labeled biotin, which can be detected in single‐molecule fluorescence concurrently to study rebinding rates. This work illustrates the potential of the seamless fusion of these two powerful single‐molecule techniques

Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry

The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin's tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1: 1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Delta x(0) = 0.38 nm and a zero-force off-rate k(off,0) in the 10(-6) s(-1) range

Conformational Changes and Flexibility of DNA Devices Observed by Small-Angle X-ray Scattering

Self-assembled DNA origami nanostructures enable the creation of precisely defined shapes at the molecular scale. Dynamic DNA devices that are capable of switching between defined conformations could afford completely novel functionalities for diagnostic, therapeutic, or engineering applications. Developing such objects benefits strongly from experimental feedback about conformational changes and 3D structures, ideally in solution, free of potential biases from surface attachment or labeling. Here, we demonstrate that small-angle X-ray scattering (SAXS) can quantitatively resolve the conformational changes of a DNA origami two-state switch device as a function of the ionic strength of the solution. In addition, we show how SAXS data allow for refinement of the predicted idealized three-dimensional structure of the DNA object using a normal mode approach based on an elastic network model. The results reveal deviations from the idealized design geometries that are otherwise difficult to resolve. Our results establish SAXS as a powerful tool to investigate conformational changes and solution structures of DNA origami and we anticipate our methodology to be broadly applicable to increasingly complex DNA and RNA devices

Different Vinculin Binding Sites Use the Same Mechanism to Regulate Directional Force Transduction

Vinculin is a universal adaptor protein that transiently reinforces the mechanical stability of adhesion complexes. It stabilizes mechanical connections that cells establish between the actomyosin cytoskeleton and the extracellular matrix via integrins or to neighboring cells via cadherins, yet little is known regarding its mechanical design. Vinculin binding sites (VBSs) from different nonhomologous actin-binding proteins use conserved helical motifs to associate with the vinculin head domain. We studied the mechanical stability of such complexes by pulling VBS peptides derived from talin, α-actinin, and Shigella IpaA out of the vinculin head domain. Experimental data from atomic force microscopy single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations both revealed greater mechanical stability of the complex for shear-like than for zipper-like pulling configurations. This suggests that reinforcement occurs along preferential force directions, thus stabilizing those cytoskeletal filament architectures that result in shear-like pulling geometries. Large force-induced conformational changes in the vinculin head domain, as well as protein-specific fine-tuning of the VBS sequence, including sequence inversion, allow for an even more nuanced force response.ISSN:0006-3495ISSN:1542-008

Unfolding forces of ddFLN4 and unbinding forces of biotin and mSA for different pulling velocities.

<p>The distribution of the forces of the first (transparent bars in the background) and second (semi-transparent bars) step of the ddFLN4 unfolding gives rise to two distinct peaks at approximately 85 pN and 75 pN. The biotin:mSA unbinding forces (opaque bars) are distributed more broadly but exhibit a clear maximum at about 200 pN depending on the applied force loading rate. The experiment was carried out with a cantilever with a spring constant of 73.9 pN/nm. The dashed lines show independent fits of Bell-Evans distributions to the force histograms.</p

Possible pulling geometries for SA of different valencies.

<p>(a) Crystal structure of mSA (pdb identification code 5TO2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188722#pone.0188722.ref015" target="_blank">15</a>], overlaid with 1MK5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188722#pone.0188722.ref016" target="_blank">16</a>] to show the position of biotin). The functional subunit (green) with biotin (red) bound is stabilized by the three non-functional subunits (grey). Black arrows show the direction of the applied load for the AFM-based SMFS measurement. (b) Tetravalent SA consists of four functional subunits (green balls) each possessing a biotin (red triangles) binding site. In previous experiments, SA has been attached to a biotinylated surface resulting in a variety of possible pulling geometries: Across the strong interface, across the weak interface or diagonally across the tetramer. Having several functional binding pockets available, multiple binding to surface or cantilever can also occur. Black arrows indicate the pulling direction, black dotted lines possible ways force propagates through the molecule. (c) Attaching the tetravalent SA molecule covalently to the surface gives also rise to diverse pulling geometries. (d) In our experiments, we employ mSA consisting of one functional (green ball) and three non-functional subunits that are unable to bind biotin (grey balls). Having mSA tethered by a single N-terminal cysteine in the functional subunit, we pull biotin out of the binding pocket. The force only propagates through a single subunit.</p

Investigation of the mechanical stability of the biotin:mSA binding with a well-defined pulling geometry.

<p>The functionalized cantilever tip is approached to the surface and a bond between biotin (red triangle) and mSA (green and gray balls) is formed. First, only the PEG (grey lines) spacers are stretched, when retracting the cantilever with constant speed from the surface. At forces of about 60 pN, the ddFLN4 (blue) unfolds in a characteristic two-step process that is used to identify single-molecule interactions. PEG spacers and the polypeptide chain are then further stretched until biotin unbinds from mSA under the applied load. The force drops and ddFLN4 folds back into its native state. As an example, one of the recorded force-distance curves (pulled at 800 nm/s) is shown in blue. More force-distance curves are shown in the supplement (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188722#pone.0188722.s002" target="_blank">S2 Appendix</a>).</p

Overlay of force-extension curves and transformation into contour length space.

<p>(a) The 575 force-extension curves for which the characteristic unfolding pattern of ddFLN4 was visible are overlaid. We fit the three parts of the curve independently with the worm-like chain polymer model (black lines). (b) Using the mean persistence length of the worm-like chain fits, each point of the force extension curve is translated into contour length space. From the histogram, the contour lengths of the stretched constructs corresponding to the three parts of the force curve are determined.</p

Bell-Evans plot of unfolding and unbinding forces.

<p>For all specific single-molecule interactions, the unbindig forces of biotin:mSA (circles) and the forces of the first (diamonds) and second (squares) step of the ddFLN4 unfolding are plotted against the loading rates at the corresponding force peak. The data are equal to the one shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188722#pone.0188722.g006" target="_blank">Fig 6</a> and the same color code is used. The dashed lines are linear fits to the centers of gravity (shown as filled circles, diamonds and squares) of the distributions of forces and loading rates, respectively. The colored crosses indicate the corresponding standard deviations.</p