A Flexible Nanoarray Approach for the Assembly and Probing of Molecular Complexes

Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid β peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid β peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers’ rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment.     

Microcapillary reactors using solid-phase DNA sequencing for direct sample introduction into slab gels

Solid-phase micro-reactors have been prepared in glass capillaries for DNA sequencing applications using slab gel electrophoresis, which consisted of a fused silica capillary (i.d. = 100 microns; o.d. = 365 microns; length = 15 cm; volume = 1.2 microL) that contained a covalently bound biotin molecule. With the addition of streptavidin to the capillary, an anchoring site was produced for the tethering of biotinylated DNA sequencing templates to the wall of the capillary. Using a four-lane, single dye primer chemistry sequencing strategy, the individual tracts were prepared in the capillaries using cycle sequencing (20 thermal cycles) on a PCR-generated lambda-bacteriophage template (about 1000 bp). The dye label in this case was a fluorescent tag that displayed emission properties in the near-IR and could be processed on an automated sequencer. The read length was found to be 589 bases, which was determined primarily by the fractionating power of the gel. It was also found that the tethering system was very stable to typical cycle sequencing conditions, with the amount of tethered DNA lost amounting to 40% after 120 thermal cycles. The ability to use dye terminator chemistry was also investigated by using a near-IR dye-labeled terminator (ddGTP). It was found that the quality of the ladder that was generated was comparable to that obtained in a conventional sample preparation format. However, ethanol precipitation was required before gel loading to remove excess terminator.     

DNA-tethered membranes formed by giant vesicle rupture

We have developed a strategy for preparing tethered lipid bilayer membrane patches on solid surfaces by DNA hybridization. In this way, the tethered membrane patch is held at a controllable distance from the surface by varying the length of the DNA used. Two basic strategies are described. In the first, single-stranded DNA strands are immobilized by click chemistry to a silica surface, whose remaining surface is passivated to prevent direct assembly of a solid supported bilayer. Then giant unilamellar vesicles (GUVs) displaying the antisense strand, using a DNA–lipid conjugate developed in earlier work [Chan, Y.-H.M., van Lengerich, B., et al., 2008. Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing. Biointerphases 3 (2), FA17–FA21], are allowed to tether, spread and rupture to form tethered bilayer patches. In the second, a supported lipid bilayer displaying DNA using the DNA–lipid conjugate is first assembled on the surface. Then GUVs displaying the antisense strand are allowed to tether, spread and rupture to form tethered bilayer patches. The essential difference between these methods is that the tethering hybrid DNA is immobile in the first, while it is mobile in the second. Both strategies are successful; however, with mobile DNA hybrids as tethers, the patches are unstable, while in the first strategy stable patches can be formed. In the case of mobile tethers, if different length DNA hybrids are present, lateral segregation by length occurs and can be visualized by fluorescence interference contrast microscopy making this an interesting model for interactions that occur in cell junctions. In both cases, lipid mobility is high and there is a negligible immobile fraction. Thus, these architectures offer a flexible platform for the assembly of lipid bilayers at a well-defined distance from a solid support.     

Force spectroscopy of the double-tethered concanavalin-A mannose bond

We present the measurement of the force required to rupture a single protein-sugar bond using a methodology that provides selective discrimination between specific and nonspecific binding events and helps verify the presence of a single functional molecule on the atomic force microscopy tip. In particular, the interaction force between a polymer-tethered concanavalin-A protein (ConA) and a similarly tethered mannose carbohydrate was measured as 47 +/- 9 pN at a bond loading rate of approximately 10 nN/s. Computer simulations of the polymer molecular configurations were used to determine the angles that the polymers could sweep out during binding and, in conjunction with mass spectrometry, used to separate the angular effects from the effects due to a distribution of tether lengths. We find that when using commercially available polymer tethers that vary in length from 19 to 29 nm, the angular effects are relatively small and the rupture distributions are dominated by the 10-nm width of the tether length distribution. In all, we show that tethering both a protein and its ligand allows for the determination of the single-molecule bond rupture force with high sensitivity and includes some validation for the presence of a single-tethered functional molecule on the atomic force microscopy tip.     

Enhanced Tethered-Particle Motion Analysis Reveals Viscous Effects

Tethered-particle motion experiments do not require expensive or technically complex hardware, and increasing numbers of researchers are adopting this methodology to investigate the topological effects of agents that act on the tethering polymer or the characteristics of the polymer itself. These investigations depend on accurate measurement and interpretation of changes in the effective length of the tethering polymer (often DNA). However, the bead size, tether length, and buffer affect the confined diffusion of the bead in this experimental system. To evaluate the effects of these factors, improved measurements to calibrate the two-dimensional range of motion (excursion) versus DNA length were carried out. Microspheres of 160 or 240 nm in radius were tethered by DNA molecules ranging from 225 to 3477 basepairs in length in aqueous buffers containing 100 mM potassium glutamate and 8 mM MgCl₂ or 10 mM Tris-HCl and 200 mM KCl, with or without 0.5% Tween added to the buffer, and the motion was recorded. Different buffers altered the excursion of beads on identical DNA tethers. Buffer with only 10 mM NaCl and >5 mM magnesium greatly reduced excursion. Glycerol added to increase viscosity slowed confined diffusion of the tethered beads but did not change excursion. The confined-diffusion coefficients for all tethered beads were smaller than those expected for freely diffusing beads and decreased for shorter tethers. Tethered-particle motion is a sensitive framework for diffusion experiments in which small beads on long leashes most closely resemble freely diffusing, untethered beads.     

A toolbox for generating single‐stranded DNA in optical tweezers experiments

Essential genomic transactions such as DNA‐damage repair and DNA replication take place on single‐stranded DNA (ssDNA) or require specific single‐stranded/double‐stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single‐molecule studies of DNA–protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to dsDNA, only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single‐molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force‐induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force‐induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA. © 2013 Wiley Periodicals, Inc. Biopolymers 99:611–620, 2013.     

Curvature‐sensitive trans‐assembly of human Atg8‐family proteins in autophagy‐related membrane tethering

In macroautophagy, de novo formation of the double membrane‐bound organelles, termed autophagosomes, is essential for engulfing and sequestering the cytoplasmic contents to be degraded in the lytic compartments such as vacuoles and lysosomes. Atg8‐family proteins have been known to be responsible for autophagosome formation via membrane tethering and fusion events of precursor membrane structures. Nevertheless, how Atg8 proteins act directly upon autophagosome formation still remains enigmatic. Here, to further gain molecular insights into Atg8‐mediated autophagic membrane dynamics, we study the two representative human Atg8 orthologs, LC3B and GATE‐16, by quantitatively evaluating their intrinsic potency to physically tether lipid membranes in a chemically defined reconstitution system using purified Atg8 proteins and synthetic liposomes. Both LC3B and GATE‐16 retained the capacities to trigger efficient membrane tethering at the protein‐to‐lipid molar ratios ranging from 1:100 to 1:5,000. These human Atg8‐mediated membrane‐tethering reactions require trans‐assembly between the membrane‐anchored forms of LC3B and GATE‐16 and can be reversibly and strictly controlled by the membrane attachment and detachment cycles. Strikingly, we further uncovered distinct membrane curvature dependences of LC3B‐ and GATE‐16‐mediated membrane tethering reactions: LC3B can drive tethering more efficiently than GATE‐16 for highly curved small vesicles (e.g., 50 nm in diameter), although GATE‐16 turns out to be a more potent tether than LC3B for flatter large vesicles (e.g., 200 and 400 nm in diameter). Our findings establish curvature‐sensitive trans‐assembly of human Atg8‐family proteins in reconstituted membrane tethering, which recapitulates an essential subreaction of the biogenesis of autophagosomes in vivo.     

Enhanced Tethered-Particle Motion Analysis Reveals Viscous Effects

Tethered-particle motion experiments do not require expensive or technically complex hardware, and increasing numbers of researchers are adopting this methodology to investigate the topological effects of agents that act on the tethering polymer or the characteristics of the polymer itself. These investigations depend on accurate measurement and interpretation of changes in the effective length of the tethering polymer (often DNA). However, the bead size, tether length, and buffer affect the confined diffusion of the bead in this experimental system. To evaluate the effects of these factors, improved measurements to calibrate the two-dimensional range of motion (excursion) versus DNA length were carried out. Microspheres of 160 or 240 nm in radius were tethered by DNA molecules ranging from 225 to 3477 basepairs in length in aqueous buffers containing 100 mM potassium glutamate and 8 mM MgCl₂ or 10 mM Tris-HCl and 200 mM KCl, with or without 0.5% Tween added to the buffer, and the motion was recorded. Different buffers altered the excursion of beads on identical DNA tethers. Buffer with only 10 mM NaCl and >5 mM magnesium greatly reduced excursion. Glycerol added to increase viscosity slowed confined diffusion of the tethered beads but did not change excursion. The confined-diffusion coefficients for all tethered beads were smaller than those expected for freely diffusing beads and decreased for shorter tethers. Tethered-particle motion is a sensitive framework for diffusion experiments in which small beads on long leashes most closely resemble freely diffusing, untethered beads.     

Protein engineering of streptavidin for in vivo assembly of streptavidin beads

Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/microg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/mug bead protein, respectively. However, biotin binding of about 61 ng biotin/mug bead protein with an equilibrium dissociation constant (KD) of about 4 x 10(-8)M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.     

Tethering forces of secretory granules measured with optical tweezers

Fusion of a vesicle with its target membrane is preceded by tethering or docking. However, the physical mechanism of vesicle-tethering is unknown. To study this mechanism, we used eosinophil secretory granules, which undergo stimulated homotypic fusion events inside the cell during degranulation. Using a dual optical trap system, we observed tether formation between isolated eosinophil secretory granules. The results show that secretory granules interact stochastically with a target membrane forming physical tethers linking the vesicle and target membrane, rather than via interactions with the cytoskeleton. The necessary components are membrane-associated, and the addition of cytosolic components is not required. Tether-lifetime measurements as a function of applied mechanical force revealed at least three kinetically distinct tethered states. The tethered-state lifetimes of isolated eosinophil granules match the residence times of chromaffin granules at the plasma membrane in intact cells, suggesting that the tethering mechanisms reported here may represent the physiological mechanisms of vesicle-tethering in the cell.     

Surface charge density measurement of a single protein molecule with a controlled orientation by AFM

The spatial distribution of functional groups causes a charge distribution that often has a close relationship with its biofunctions. To understand them of the protein molecules, measurements of the charge distribution under physiological conditions are desired. Atomic force microscopy (AFM) has been utilized to measure the surface charge density by measuring the electric double layer (EDL) force caused by the overlap of the EDLs on the surfaces of the AFM tip and the biomolecule. Here, we demonstrated the surface charge density measurement of a single streptavidin (SA) protein molecule by the three-dimensional force mapping method based on frequency modulation AFM (FM-AFM). The SA has a strong affinity to biotin because of the electrostatic interactions between the molecules. Therefore, the surface charge density measurements of the biotin-binding sites and other surface areas of the molecule have been anticipated. However, the surface charge density of the surfaces other than the biotin-binding side has never been measured. We demonstrate the surface charge density measurement of the top surface of the single SA molecule, which is perpendicular to the biotin-binding sides, with a controlled orientation using DNA origami as a template by FM-AFM in an electrolyte solution. The surface charge density of the top surface of the SA molecule was estimated by fitting the experimental force curves to the Derjaguin-Landau-Verwey-Overbeck theory. We found that the surface charge density of the top surface of the SA molecule is comparable to those reported earlier for the biotin-binding sides of the molecule. We expect that, by using the DNA origami technology, one can control the orientation of a biomolecule attached to the substrate and measure the surface charge density of the specific surface areas of the biomolecule to obtain information that will help us to understand the relationship between their structures and functions.     

Direct force measurements of the streptavidin-biotin interaction

The interaction between streptavidin and its ligand, biotin, were studied by direct force measurements. The complimentary approaches of surface force apparatus (SFA) and atomic force microscopy (AFM) were used to elucidate both long-range and short-range adhesive interactions of the streptavidin biotin interaction. The high spatial resolution of the SFA provided a detailed profile of the intersurface forces of apposing surfaces functionalized with streptavidin and biotin. Measurements obtained by the SFA corresponded to long and intermediate-range forces that are important in determining ligand receptor association. AFM was used to measure the unbinding force of individual streptavidin biotin complexes. These measurements revealed the short-range interactions (i.e. hydrophobic and hydrogen bonding forces) that stabilize the intermolecular bond.     

Balls and chains--a mesoscopic approach to tethered protein domains

Many proteins contain regions of unstructured polypeptide chain that appear to be flexible and to undergo random thermal motion. In some cases the unfolded sequence acts as a flexible tether that restricts the diffusion of a globular protein domain for the purpose of catalysis or self-assembly. In this article, we present a stochastic model for tethered protein domains under various conditions and solve it numerically to deduce the general and dynamic properties of these systems. A critical domain size dependent on the length of the tether is presented, above which a spherical domain tethered to an impenetrable wall by a flexible chain displays a restricted localization between two concentric half-shells. Results suggest that the diffusion of such a spherical domain is effectively reduced in its dimensionality and able to explore the available space with high efficiency. It also becomes clear that the orientation of the ball is not independent of the distance from the tethering point but becomes more constrained as the linking tether is extended. The possible biological significance of these and other results is discussed.     

Atomic force microscopy of long and short double-stranded, single-stranded and triple-stranded nucleic acids.

Atomic force microscopy (AFM, also called scanning force microscopy) is proving to be a useful technique for imaging DNA. Thus it is important to push the limits of AFM imaging in order to explore both what types of DNA can be reliably imaged and identified and also what substrates and methods of sample preparation are suitable. The following advances in AFM of DNA are presented here. (i) DNA molecules as short as 25 bases can be seen by AFM. The short single-stranded DNAs imaged here (25 and 50 bases long) appeared globular in the AFM, perhaps because they are all capable of intramolecular base pairing and because the DNAs were in a Mg(ll) buffer, which facilitates intramolecular cross-bridging. (ii) AFM images in air of short double-stranded DNA molecules, 100-200 bp, gave lengths consistent with A-DNA. (iii) AFM images of poly (A) show both short bent lumpy molecules with an apparent persistence length of 40 nm and long straight molecules with an apparent persistence length of 600 nm. For comparison, the apparent persistence length for double-stranded DNA from phX-174 under the same conditions was 80 nm. (iv) Structures believed to be triple- stranded DNA were seen in samples of poly(dA.poly(dT) and poly (dG).poly(dC). These structures were twice as high as double-stranded DNA and the same width. (v) Entire molecules of lambda DNA, approx. 16 micron long, were imaged clearly in overlapping scans. (vi) Plasmid DNA was imaged on oxidized silicon, although less clearly than on mica.     

Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole‐labeled biotin

In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole‐labeled biotin. BODIPY‐FL‐aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position‐specifically incorporated into Trp120 position of streptavidin by four‐base codon method. On the other hand, carbazole‐labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole‐labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole‐labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole‐labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd.     

Stable,high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (K_d = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (T_m = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67. © 2012 Wiley Periodicals, Inc.     

Investigation of biotin-streptavidin binding interactions using microcantilever sensors

We report the investigation of biotin-streptavidin binding interactions using microcantilever sensors. A symmetric cantilever construction is employed to minimize the effects of thermal drift and the control of surface chemistry on the backside of the cantilever is demonstrated to reduce the effects of non-specific binding interactions on the cantilever. Three structurally different biotin modified cantilever surfaces are used as a model system to study the binding interaction with streptavidin. The cantilever response to the binding of streptavidin on these biotin sensing monolayers is compared. The lowest detection limit of streptavidin using biotin-HPDP is found to be between 1 and 10nM limited by the optical measurement setup. Surface characterization using quartz crystal microbalance (QCM) and high-resolution atomic force microscope (AFM) is used to benchmark the cantilever sensor response. In addition, the QCM and AFM studies reveal that the surface density of bound streptavidin on biotin modified surfaces was low, thereby implying that effects other than steric hindrance are responsible for defining cantilever response.