Monovalent Streptavidin Research Articles

Improving the Performance of Selective Solid-State Nanopore Sensing Using a Polyhistidine-Tagged Monovalent Streptavidin.

Solid-state (SS-) nanopore sensing has gained tremendous attention in recent years, but it has been constrained by its intrinsic lack of selectivity. To address this, we previously established a novel SS-nanopore assay that produces translocation signals only when a target biotinylated nucleic acid fragment binds to monovalent streptavidin (MS), a protein variant with a single high-affinity biotin-binding domain. While this approach has enabled selective quantification of diverse nucleic acid biomarkers, sensitivity enhancements are needed to improve the detection of low-abundance translational targets. Because the translocation dynamics that determine assay efficacy are largely governed by constituent charge characteristics, we here incorporate a polyhistidine-tagged MS (hMS) to alter the component detectability. We investigate the effects of buffer pH, salt concentration, and SS-nanopore diameter on the performance with the alternate reagent, achieve significant improvements in measurement sensitivity and selectivity, and expand the range of device dimensions viable for the assay. We used this improvement to detect as little as 1 nM miRNA spiked into human plasma. Overall, our findings improve the potential for broader applications of SS-nanopores in the quantitative analyses of molecular biomarkers.

Oligonucleotide-Blocked Streptavidin for Biotinylation Analysis.

Binding between streptavidin, or its homologues, to biotin is one of the most widely exploited biological interactions in the biomedical sciences. Controlling the extent of biotinylation is important for meeting the requirements of the intended design and to preserve the native function of the biotin recipient. Within the protein world, a"trial-and-error" optimization approach toward biotinylation reaction conditions is often necessary due to widely varying properties of proteins. Therefore, product analysis is important. We show here that a oligonucleotide-blocked streptavidin, effectively "monovalent streptavidin", can tag biotin moieties individually and the tagged products visualized via a polyacrylamide gel shift assay to reveal the product distribution, i.e., [protein-(biotin)n] products where n = 1, 2, 3, etc. This is in contrast, and complementary, to current commercially available analytical reagents for biotinylation characterization, which use an absorbance or fluorescence signal to yield the mean number of biotin moieties.

Construction of Orthogonal Modular Proteinaceous Nanovaccine Delivery Vectors Based on mSA-Biotin Binding.

Proteinaceous nanovaccine delivery systems have significantly promoted the development of various high-efficiency vaccines. However, the widely used method of coupling the expression of scaffolds and antigens may result in their structural interference with each other. Monovalent streptavidin (mSA) is a short monomer sequence, which has a strong affinity for biotin. Here, we discuss an orthogonal, modular, and highly versatile self-assembled proteinaceous nanoparticle chassis that facilitates combinations with various antigen cargos by using mSA and biotin to produce nanovaccines. We first improved the yield of these nanoparticles by appending a short sugar chain on their surfaces in a constructed host strain. After confirming the strong ability to induce both Th1- and Th2-mediated immune responses based on the plasma cytokine spectrum from immunized mice, we further verified the binding ability of biotinylated nanoparticles to mSA-antigens. These results demonstrate that our biotinylated nanoparticle chassis could load both protein and polysaccharide antigens containing mSA at a high affinity. Our approach thus offers an attractive technology for combining nanoparticles and antigen cargos to generate various high-performance nanovaccines. In particular, the designed mSA connector (mSA containing glycosylation modification sequences) could couple with polysaccharide antigens, providing a new attractive strategy to prepare nanoscale conjugate vaccines.

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation.

Solid-state nanopores have been used extensively in biomolecular studies involving DNA and proteins. However, the interpretation of signals generated by the translocation of proteins or protein-DNA complexes remains challenging. Here, we investigate the behavior of monovalent streptavidin and the complex it forms with short biotinylated DNA over a range of nanopore sizes, salts, and voltages. We describe a simple geometric model that is broadly applicable and employ it to explain observed variations in conductance blockage and dwell time with experimental conditions. The general approach developed here underscores the value of nanopore-based protein analysis and represents progress toward the interpretation of complex translocation signals.

Strategies for the Site-Specific Decoration of DNA Origami Nanostructures with Functionally Intact Proteins.

DNA origami structures provide flexible scaffolds for the organization of single biomolecules with nanometer precision. While they find increasing use for a variety of biological applications, the functionalization with proteins at defined stoichiometry, high yield, and under preservation of protein function remains challenging. In this study, we applied single molecule fluorescence microscopy in combination with a cell biological functional assay to systematically evaluate different strategies for the site-specific decoration of DNA origami structures, focusing on efficiency, stoichiometry, and protein functionality. Using an activating ligand of the T-cell receptor (TCR) as the protein of interest, we found that two commonly used methodologies underperformed with regard to stoichiometry and protein functionality. While strategies employing tetravalent wildtype streptavidin for coupling of a biotinylated TCR-ligand yielded mixed populations of DNA origami structures featuring up to three proteins, the use of divalent (dSAv) or DNA-conjugated monovalent streptavidin (mSAv) allowed for site-specific attachment of a single biotinylated TCR-ligand. The most straightforward decoration strategy, via covalent DNA conjugation, resulted in a 3-fold decrease in ligand potency, likely due to charge-mediated impairment of protein function. Replacing DNA with charge-neutral peptide nucleic acid (PNA) in a ligand conjugate emerged as the coupling strategy with the best overall performance in our study, as it produced the highest yield with no multivalent DNA origami structures and fully retained protein functionality. With our study we aim to provide guidelines for the stoichiometrically defined, site-specific functionalization of DNA origami structures with proteins of choice serving a wide range of biological applications.

Lipid Bilayer Strengthens the Side Chain Interaction Network of a Membrane Protein

The lipid bilayer in a cell membrane serves as a natural solvent for membrane proteins. The physical principle of how the lipid bilayer mediates the folding, stability, and cooperativity between residues to facilitate the functions of membrane proteins in cells is not well understood. Here, we employed steric trapping as a tool to study membrane protein folding in a native lipid environment. This method couples spontaneous denaturation of a doubly biotinylated protein to the competitive binding of bulky monovalent streptavidin.

Switchable reinforced streptavidin.

The complex of the small molecule biotin and the homotetrameric protein streptavidin is key to a broad range of biotechnological applications. Therefore, the behavior of this extraordinarily high-affinity interaction under mechanical force is intensively studied by single-molecule force spectroscopy. Recently, steered molecular dynamics simulations have identified a low force pathway for the dissociation of biotin from streptavidin, which involves partial unfolding of the N-terminal β-sheet structure of monovalent streptavidin's functional subunit. Based on these results, we now introduced two mutations (T18C,A33C) in the functional subunit of monovalent streptavidin to establish a switchable connection (disulfide bridge) between the first two β-strands to prevent this unfolding. In atomic force microscopy-based single-molecule force spectroscopy experiments, we observed unbinding forces of about 350 pN (at a force-loading rate of 10 nN s-1) for pulling a single biotin out of an N-terminally anchored monovalent streptavidin binding pocket - about 1.5-fold higher compared with what has been reported for N-terminal force loading of native monovalent streptavidin. Upon addition of a reducing agent, the unbinding forces dropped back to 200 pN, as the disulfide bridge was destroyed. Switching from reducing to oxidizing buffer conditions, the inverse effect was observed. Our work illustrates how the mechanics of a receptor-ligand system can be tuned by engineering the receptor protein far off the ligand-binding pocket.

Optimizing Nucleic Acid Biomarker Detection in a Solid-State Nanopore Through Probabilistic Modeling

We have established a solid-state (SS-) nanopore assay with high specificity for nucleic acid targets, enabling applications like epigenetic screening, microRNA analysis, and pathogen detection . Despite its proven specificity and sensitivity, translational biomarker detection at clinically relevant concentrations is hindered by very low event rates and long acquisition times. It has been noted that most small-molecule translocations are not detectable due to low signal to noise ratio or fast timescales. Here, we describe the probability that a given translocation will produce a detectable event by considering tunable system parameters, and use it to show that these key experimental variables can influence the measured event rate in a predictable fashion. This model elucidates the dependencies of nanopore/analyte interactions and suggests methods by which measurement sensitivity can be optimized. Event probability can be determined experimentally by comparing expected (Rexp) to observed (Robs) event rates for dsDNA constructs of various lengths bound to monovalent streptavidin. Rexp can be derived analytically from the Nernst-Planck equation and Knudsen diffusion coefficient, and Robs measured as a function of fundamental experimental parameters, including the driving potential, target concentration, nanopore radius, and data acquisition rate. Using these data to generate probability distributions, we show that Robs can be predicted for arbitrary experimental conditions from first principles. With this approach, we are able to better understand and even influence the sensitivity of our SS-nanopore assay. The explicit use of the probability distribution provides insight into the mechanisms that govern event generation, and our approach may be expanded to take into account any fundamental system parameter. Tuning system parameters according to the underlying probability distribution will enable optimized detection of low-abundance or challenging nucleic acid biomarker targets.

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.

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.

Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State Nanopores.

Nanopore sensors show great potential for rapid, single-molecule determination of DNA sequence information. Here, we develop an ionic current-based method for determining the positions of short sequence motifs in double-stranded DNA molecules with solid-state nanopores. Using the DNA-methyltransferase M.TaqI and a biotinylated S-adenosyl-l-methionine cofactor analogue we create covalently attached biotin labels at 5′-TCGA-3′ sequence motifs. Monovalent streptavidin is then added to bind to the biotinylated sites giving rise to additional current blockade signals when the DNA passes through a conical quartz nanopore. We determine the relationship between translocation time and position along the DNA contour and find a minimum resolvable distance between two labeled sites of ∼200 bp. We then characterize a variety of DNA molecules by determining the positions of bound streptavidin and show that two short genomes can be simultaneously detected in a mixture. Our method provides a simple, generic single-molecule detection platform enabling DNA characterization in an electrical format suited for portable devices for potential diagnostic applications.

Direct Observation of Single Biopolymer Folding and Unfolding Process by Solid-State Nanopore

Biomolecular conformation and their transition play a crucial role in various in vivo or in vitro system. The most of the practical techniques for resolving the secondary structures of biomolecules could provide quite precise structural information for their solid-state or steady state, even at atomic resolution. For example, Cryo-EM determines high-resolution structures for the frozen-hydrated specimens of biomolecules. polymers, but it is still challenging to resolve the dynamic process of multiple functional conformational states for biomolecules at single-molecular scale. Here, we direct observed DNA folding and unfolding process in real-time by using sub-5 nm solid-state nanopores. In our experiments, a single-stranded DNA adhered to single monovalent streptavidin could be reversibly trapped in a solid-state nanopore. Then, the fluctuations of the blockade current could be recorded, which reveals the dynamic structural transitions among DNA secondary structures. For example, after trapping the cytosine-rich DNA strains in slightly alkaline solution, the formation of multiple unstable and semi-folded i-motif structures could be observed. More important, well time-resolved transitions between these structures could be obtained. When using slightly acidic solution, the stable structures with stable blockade current could be found. With this new approach, we can directly observe the dynamic conformational change of biomolecules at single-molecular scale, which would be of great help for resolving single molecule interactions, designing single-molecule machine and understanding the working process of biomolecular in biological system.

The Crystal Structure of Monovalent Streptavidin.

The strong interaction between streptavidin (SA) and biotin is widely utilized in biotechnological applications. A SA variant, monovalent SA, was developed with a single and high affinity biotin-binding site within the intact tetramer. However, its structural characterization remains undetermined. Here, we seek to determine the crystal structure of monovalent SA at 1.7-Å resolution. We show that, in contrast to its ‘close-state’ in the only wild-type subunit, the L3,4 loops of three Dead SA subunits are free from crystal packing and remain in an ‘open state’, stabilized by a consistent H-bonding network involving S52. This H-bonding network also applies to the previously reported open state of the wild-type apo-SA. These results suggest that specific substitutions (N23A/S27D/S45A) at biotin-binding sites stabilize the open state of SA L3,4 loop, thereby further reducing biotin-binding affinity. The general features of the ‘open state’ SA among different SA variants may facilitate its rational design. The structural information of monovalent SA will be valuable for its applications across a wide range of biotechnological areas.

Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.

Membrane proteins are assembled through balanced interactions among protein, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements in membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping which couples unfolding of a doubly-biotinylated protein to binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to an intramembrane protease GlpG of Escherichia coli, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain the stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.

General Steric Trapping Strategy Reveals an Intricate Cooperativity Network in the Intramembrane Protease GlpG under Native Conditions

Membrane proteins are assembled through balanced interactions among protein, lipids and water. Studying their folding while maintaining the native lipid environment has been a necessary but challenging. Here we present a set of methods for analyzing key elements in membrane protein folding, including thermodynamic stability, compactness of the unfolded state and unfolding cooperativity under native conditions. The methods are based on steric trapping which couples unfolding of a doubly-biotinylated protein to binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying those methods to an intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions for maintaining the stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.

A Novel Virus-Like Particle Based Vaccine Platform Displaying the Placental Malaria Antigen VAR2CSA.

Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA) and is responsible for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP) based vaccines (e.g., the licensed human papillomavirus vaccines) have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTagTM) can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA)-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1st and 2nd immunization; however, this difference was not statistically significant after 3rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of parasites to CSA. This study demonstrates that the described Avi-L1 VLP-platform may serve as a versatile system for facilitating optimal VLP-display of large and complex vaccine antigens.

A novel approach to make homogeneous protease-stable monovalent streptavidin

The interaction between the tetramer streptavidin and biotin is recognized as one of the strongest non-covalent associations. Owing to the tight and specific binding, the streptavidin-biotin system has been used widely for bimolecular labeling, purification, immobilization, and even for targeted delivery of therapeutics drugs. Here, we report a novel approach to make homogeneous monovalent tetramer streptavidin. The purified monovalent protein showed both thermal stability and protease stability. Unexpectedly, we found that two proteases, Proteinase K (PK) and Subtilisin (SU), can efficiently remove the His8-tag from the wild-type subunit without affecting the tetramer architecture of monovalent streptavidin, thus making it more homogeneous. In addition, crystallization was performed to assure the homogeneity of the monovalent protein prepared. Overall, monovalent streptavidin shows increased homogeneity and will likely be valuable for many future applications in a wide range of research areas.

Design and characterization of a membrane protein unfolding platform in lipid bilayers.

Accurate measurement of membrane protein stability—and particularly how it may vary as a result of disease-phenotypic mutations—ideally requires a denaturant that can unfold a membrane-embedded structure while leaving the solubilizing environment unaffected. The steric trap method fulfills this requirement by using monovalent streptavidin (mSA) molecules to unfold membrane proteins engineered with two spatially close biotin tags. Here we adapted this method to an 87-residue helix-loop-helix (hairpin) construct derived from helices 3 and 4 in the transmembrane domain of the human cystic fibrosis transmembrane conductance regulator (CFTR), wherein helix-helix tertiary interactions are anticipated to confer a portion of construct stability. The wild type CFTR TM3/4 hairpin construct was modified with two accessible biotin tags for mSA-induced unfolding, along with two helix-terminal pyrene labels to monitor loss of inter-helical contacts by pyrene excimer fluorescence. A series of eight constructs with biotin tags at varying distances from the helix-terminal pyrene labels were expressed, purified and labeled appropriately; all constructs exhibited largely helical circular dichroism spectra. We found that addition of mSA to an optimized construct in lipid vesicles led to a complete and reversible loss in pyrene excimer fluorescence and mSA binding, and hence hairpin unfolding—results further supported by SDS-PAGE visualization of mSA bound and unbound species. While some dimeric/oligomeric populations persist that may affect quantitation of the unfolding step, our characterization of the design yields a promising prototype of a future platform for the systematic study of membrane protein folding in a lipid bilayer environment.

Developing a Universal Steric Trapping Strategy for Studying Folding and Stability of Helical Membrane Proteins

“Steric trapping” is a method that links binding of monovalent streptavidin (mSA) to unfolding of a biotinylated protein (MP). It allowed the measurements of high affinity protein–protein interactions and thermodynamic stability of polytopic helical MPs in a native environment, which had been difficult to achieve using more conventional methods. In the current steric trapping framework, a conformation-sensitive chromophore or an enzymatic activity in a target MP is required for monitoring mSA-induced unfolding. This target-specific approach is a limiting factor that hinders its application to various MP systems, i.e. MPs in a misfolded conformation, MPs with an assembly role, or MPs without convenient unfolding readout. To further advance the steric trapping strategy for more general application, we have developed novel tripartite probes possessing a thiol-reactive group, a biotin group and a fluorescent or paramagnetic group for sensitization of unfolding or binding of mSA. We applied the new strategy to investigating the stability and unfolding mechanism of an intramembrane protease GlpG. By combining FRET between fluorescently labeled GlpG and quencher-labeled mSA as a measure of mSA binding and the proteolytic activity of GlpG as a measure of unfolding, we proved the thermodynamic coupling between binding and unfolding, and determined the thermodynamic stability and unfolding rate of GlpG in a native micellar environment. While the stabilities were similar independent of the location of biotin pairs, the unfolding was 20 times faster when the biotin pair was placed near the proteolytic active site. This result suggests a subdomain-organization of the helical bundle architecture of GlpG. Steric trapping may serve as a useful tool for elucidating the local versus global flexibility of helical MPs.

Label-Free Free-Solution Single-Molecule Protein–Small Molecule Interaction Observed by Double-Nanohole Plasmonic Trapping

The interaction of proteins with small molecules is fundamental to their function in living organisms, and it is widely studied in drug development. Here we use the double nanohole optical trapping technique to observe real-time label-free free-solution single-molecule dynamics of three complexes: biotin–streptavidin, biotin–monovalent streptavidin, and acetylsalicylic acid–cyclooxygenase 2. Radically different behavior is seen between the protein with and without the small molecule binding. This detection platform is scalable, inexpensive, and highly sensitive, which may transform drug discovery based on protein–small molecule interactions.