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.

Direction Matters: Monovalent Streptavidin/Biotin Complex under Load.

Novel site-specific attachment strategies combined with improvements of computational resources enable new insights into the mechanics of the monovalent biotin/streptavidin complex under load and forced us to rethink the diversity of rupture forces reported in the literature. We discovered that the mechanical stability of this complex depends strongly on the geometry in which force is applied. By atomic force microscopy-based single molecule force spectroscopy we found unbinding of biotin to occur beyond 400 pN at force loading rates of 10 nN/s when monovalent streptavidin was tethered at its C-terminus. This value is about twice as high than that for N-terminal attachment. Steered molecular dynamics simulations provided a detailed picture of the mechanics of the unbinding process in the corresponding force loading geometries. Using machine learning techniques, we connected findings from hundreds of simulations to the experimental results, identifying different force propagation pathways. Interestingly, we observed that depending on force loading geometry, partial unfolding of N-terminal region of monovalent streptavidin occurs before biotin is released from the binding pocket.

Force sensing by the vascular protein von Willebrand factor is tuned by a strong intermonomer interaction.

The large plasma glycoprotein von Willebrand factor (VWF) senses hydrodynamic forces in the bloodstream and responds to elevated forces with abrupt elongation, thereby increasing its adhesiveness to platelets and collagen. Remarkably, forces on VWF are elevated at sites of vascular injury, where VWF's hemostatic potential is important to mediate platelet aggregation and to recruit platelets to the subendothelial layer. Adversely, elevated forces in stenosed vessels lead to an increased risk of VWF-mediated thrombosis. To dissect the remarkable force-sensing ability of VWF, we have performed atomic force microscopy (AFM)-based single-molecule force measurements on dimers, the smallest repeating subunits of VWF multimers. We have identified a strong intermonomer interaction that involves the D4 domain and critically depends on the presence of divalent ions, consistent with results from small-angle X-ray scattering (SAXS). Dissociation of this strong interaction occurred at forces above [Formula: see text]50 pN and provided [Formula: see text]80 nm of additional length to the elongation of dimers. Corroborated by the static conformation of VWF, visualized by AFM imaging, we estimate that in VWF multimers approximately one-half of the constituent dimers are firmly closed via the strong intermonomer interaction. As firmly closed dimers markedly shorten VWF's effective length contributing to force sensing, they can be expected to tune VWF's sensitivity to hydrodynamic flow in the blood and to thereby significantly affect VWF's function in hemostasis and thrombosis.

From genes to protein mechanics on a chip.

Single-molecule force spectroscopy enables mechanical testing of individual proteins, but low experimental throughput limits the ability to screen constructs in parallel. We describe a microfluidic platform for on-chip expression, covalent surface attachment and measurement of single-molecule protein mechanical properties. A dockerin tag on each protein molecule allowed us to perform thousands of pulling cycles using a single cohesin-modified cantilever. The ability to synthesize and mechanically probe protein libraries enables high-throughput mechanical phenotyping.

C-5 Propynyl Modifications Enhance the Mechanical Stability of DNA.

Increased thermal or mechanical stability of DNA duplexes is desired for many applications in nanotechnology or -medicine where DNA is used as a programmable building block. Modifications of pyrimidine bases are known to enhance thermal stability and have the advantage of standard base-pairing and easy integration during chemical DNA synthesis. Through single-molecule force spectroscopy experiments with atomic force microscopy and the molecular force assay we investigated the effect of pyrimidines harboring C-5 propynyl modifications on the mechanical stability of double-stranded DNA. Utilizing these complementary techniques, we show that propynyl bases significantly increase the mechanical stability if the DNA is annealed at high temperature. In contrast, modified DNA complexes formed at room temperature and short incubation times display the same stability as non-modified DNA duplexes.

Nanoscale arrangement of proteins by single-molecule cut-and-paste.

Protein-based nanostructures are key to the organization of life and it is their precise arrangement, which determines their specific functions. A single-molecule approach for the directed assembly of protein arrangements allows for a controlled composition of systems based on protein components. Applying antibodies and antigenic peptide tags we utilized the Single-Molecule Cut-and-Paste (SMC&P) technique for the handling of single proteins. Protein-DNA complexes could be arranged to complex patterns with the functionality of the protein part remaining unimpaired.

The regulatory domain of the RIG-I family ATPase LGP2 senses double-stranded RNA.

RIG-I and MDA5 sense cytoplasmic viral RNA and set-off a signal transduction cascade, leading to antiviral innate immune response. The third RIG-I-like receptor, LGP2, differentially regulates RIG-I- and MDA5-dependent RNA sensing in an unknown manner. All three receptors possess a C-terminal regulatory domain (RD), which in the case of RIG-I senses the viral pattern 5'-triphosphate RNA and activates ATP-dependent signaling by RIG-I. Here we report the 2.6 A crystal structure of LGP2 RD along with in vitro and in vivo functional analyses and a homology model of MDA5 RD. Although LGP2 RD is structurally related to RIG-I RD, we find it rather binds double-stranded RNA (dsRNA) and this binding is independent of 5'-triphosphates. We identify conserved and receptor-specific parts of the RNA binding site. Latter are required for specific dsRNA binding by LGP2 RD and could confer pattern selectivity between RIG-I-like receptors. Our data furthermore suggest that LGP2 RD modulates RIG-I-dependent signaling via competition for dsRNA, another pattern sensed by RIG-I, while a fully functional LGP2 is required to augment MDA5-dependent signaling.

HTHP: a novel class of hexameric, tyrosine-coordinated heme proteins.

We have cloned, expressed, isolated and characterized a hexameric tyrosine-coordinated heme protein (HTHP) from the marine bacterium Silicibacter pomeroyi. HTHP shows peroxidase and catalase activity and has a high thermal stability. As-isolated HTHP has absorption maxima at 407, 495, 504, 532 and 622 nm wavelength. Upon reduction maxima at 430, 564 and 596 nm wavelength are discernible. The crystal structure of HTHP reveals a hexameric, ring-like arrangement of six monomers. Each monomer binds a solvent accessible heme group, which is stabilized by the interaction of three neighboring monomers. The pocket around the heme distal side is positively charged due to three conserved arginine residues in direct vicinity. The heme iron is penta-coordinated with a tyrosine residue as proximal ligand. The coordinating hydroxyl-group of the tyrosine ligand interacts with the guanidinium group of a nearby arginine residue, an arrangement closely resembling the catalytic dyad found in monofunctional heme-containing catalases and coral allene oxide synthases, which are b-type cytochromes with tyrosine coordination trans to an empty coordination site. Despite the similarity in heme coordination HTHP is functionally and structurally unrelated to catalases and other heme-containing proteins. Its hexameric arrangement, solvent accessible heme binding pocket and heme coordination by tyrosine render HTHP a unique protein with unusual properties. A database search against complete and incomplete genomes shows that the 76 amino acid residues sequence of HTHP is unrelated to characterized proteins, but is homologous to orfs found in a phylogenetically diverse set of bacteria with sequence identities of 30-76%. We therefore propose that HTHP is the prototype of a new class of heme proteins.

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 Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10-6 s-1 range.

Monovalent Strep-Tactin for strong and site-specific tethering in nanospectroscopy.

Strep-Tactin, an engineered form of streptavidin, binds avidly to the genetically encoded peptide Strep-tag II in a manner comparable to streptavidin binding to biotin. These interactions have been used in protein purification and detection applications. However, in single-molecule studies, for example using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), the tetravalency of these systems impedes the measurement of monodispersed data. Here, we introduce a monovalent form of Strep-Tactin that harbours a unique binding site for Strep-tag II and a single cysteine that allows Strep-Tactin to specifically attach to the atomic force microscope cantilever and form a consistent pulling geometry to obtain homogeneous rupture data. Using AFM-SMFS, the mechanical properties of the interaction between Strep-tag II and monovalent Strep-Tactin were characterized. Rupture forces comparable to biotin:streptavidin unbinding were observed. Using titin kinase and green fluorescent protein, we show that monovalent Strep-Tactin is generally applicable to protein unfolding experiments. We expect monovalent Strep-Tactin to be a reliable anchoring tool for a range of single-molecule studies.

Tip localization of an atomic force microscope in transmission microscopy with nanoscale precision.

Since the atomic force microscope (AFM) has evolved into a general purpose platform for mechanical experiments at the nanoscale, the need for a simple and generally applicable localization of the AFM cantilever in the reference frame of an optical microscope has grown. Molecular manipulations like in single molecule cut and paste or force spectroscopy as well as tip mediated nanolithography are prominent examples for the broad variety of applications implemented to date. In contrast to the different kinds of superresolution microscopy where fluorescence is used to localize the emitter, we, here, employ the absorbance of the tip to localize its position in transmission microscopy. We show that in a low aperture illumination, the tip causes a significant reduction of the intensity in the image plane of the microscope objective when it is closer than a few hundred nm. By independently varying the z-position of the sample slide, we could verify that this diffraction limited image of the tip is not caused by a near field effect but is rather caused by the absorbance of the transmitted light in the low apex needle-like tip. We localized the centroid position of this tip image with a precision of better than 6 nm and used it in a feedback loop to position the tip into nano-apertures of 110 nm radius. Single-molecule force spectroscopy traces on the unfolding of individual green fluorescent proteins within the nano-apertures showed that their center positions were repeatedly approached with very high fidelity leaving the specific handle chemistry on the tip's surface unimpaired.

A force-based, parallel assay for the quantification of protein-DNA interactions.

Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

Parallel force assay for protein-protein interactions.

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

Protein-DNA chimeras for nano assembly.

In synthetic biology, "understanding by building" requires exquisite control of the molecular constituents and their spatial organization. Site-specific coupling of DNA to proteins allows arrangement of different protein functionalities with emergent properties by self-assembly on origami-like DNA scaffolds or by direct assembly via Single-Molecule Cut & Paste (SMC&P). Here, we employed the ybbR-tag/Sfp system to covalently attach Coenzyme A-modified DNA to GFP and, as a proof of principle, arranged the chimera in different patterns by SMC&P. Fluorescence recordings of individual molecules proved that the proteins remained folded and fully functional throughout the assembly process. The high coupling efficiency and specificity as well as the negligible size (11 amino acids) of the ybbR-tag represent a mild, yet versatile, general and robust way of adding a freely programmable and highly selective attachment site to virtually any protein of interest.

A mutation of human cytochrome c enhances the intrinsic apoptotic pathway but causes only thrombocytopenia.

We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.