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

Abstract

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.

Fig 1. Possible pulling geometries for SA of different valencies.

(a) Crystal structure of mSA (pdb identification code 5TO2 [15], overlaid with 1MK5 [16] 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.

Fig 2. SDS-PAGE of mSA, tSA and commercial SA from streptomyces avidinii (sSA).

(a) Overview of differently treated SAs with and without addition of biotinylated GFP on a stain-free polyacrylamide gel. Overlay of images taken with UV light excitation (blue) and illumination with a blue LED source (green). Parts of this image are inverted and shown in detail (b-d UV-excitation; e: GFP-channel): (b) Denatured SA samples (5 min at 95°C). Decomposition into monomers (14 kDa) is visible. His-tagged subunits appear larger. sSA subunits are smeared out. (c) Untreated SA samples which maintain tertiary structure. (d,e) Addition of biotinylated GFP to untreated SA samples. Valencies of SAs are visible as different numbers of GFPs are bound. The lowest band in (d) corresponds to Sfp Synthase (26 kDa).

Fig 3. Isothermal titration calorimetry of biotin and SAs of different valency.

The binding of biotin to different SAs was measured with isothermal titration calorimetry. The binding stoichiometry of mSA and biotin was determined as N = 0.95 (blue circles). The measured binding stoichiometry of the engineered tetravalent version (green diamonds) N = 4.31 is in good agreement with the value of commercial SA isolated from Streptomyces avidinii (black squares) N = 4.29. Within the limits of the measurement’s accuracy, the binding enthalpies of the different SAs are the same (ΔH = -26 kcal/mol for monovalent, ΔH = -25 kcal/mol for tetravalent and ΔH = -26 kcal/mol for commercial SA), confirming that the N-terminal modifications do not interfere with the binding of biotin.

Fig 4. Investigation of the mechanical stability of the biotin:mSA binding with a well-defined pulling geometry.

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 (S2 Appendix).

Fig 5. Overlay of force-extension curves and transformation into contour length space.

(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.

Fig 6. Unfolding forces of ddFLN4 and unbinding forces of biotin and mSA for different pulling velocities.

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.

Fig 7. Bell-Evans plot of unfolding and unbinding forces.

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 Fig 6 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.