Force Spectroscopy: AFM Measurements of the Streptavidin-Biotin Interaction

This section describes methods for acquiring force measurements between purified proteins immobilized on apposing surfaces. Numerous methods exist for immobilizing ligand to a variety of different substrates. Some commonly used ligands for affinity chromatography (e.g., biotin or D-mannose) can be found already attached to agarose beads from vendors such as Sigma ( St. Louis, MO) and Pierce ( Rockford, IL). As a substrate for the force measurements, the agarose beads provide several attractive features. The agarose matrix has low affinity for most proteins, keeping non-specific interaction at a minimum. Proteins can be readily coupled to activated agarose beads. Moreover, the elastic agarose substrate will conform to the shape of the cantilever tip, thus increasing the contact surface area and creating a higher probability for receptor-ligand interactions (Moy et al., 1994). Ligand-receptor binding is also greatly enhanced by attaching receptors to molecular tethers (e.g., dextran and polyethyleneglycol) that allow for a wider range of lateral motion (Hinterdorfer et al., 1996; Rief et al., 1997b). In addition, tethers can provide latitude for proper reorientation of the molecule during stretching so that the external force being applied to the molecule is perpendicular to the surface.

Here, we will use the streptavidin-biotin system to illustrate how single molecule AFM force measurements of ligand-receptor interactions are acquired. First, a streptavidin-functionalized cantilever is generated as described elsewhere. The following is a protocol that we use for the preparation of biotinylated agarose beads. Note that the biotinylated agarose beads are plated on a streptavidin-coated Petri dish to minimize the movement of the beads during the AFM measurements.

MATERIALS

35 mm plastic petri dishes, streptavidin (Sigma), phosphate-buffered saline (pH 7.4), biotinylated agarose beads (Sigma).

METHODS

• To prepare streptavidin-coated dishes for immobilizing biotinylated agarose beads:

• Place a 100 µl drop of streptavidin (0.05 mg/ml in sodium bicarbonate, pH 8.3) on the bottom of a 35 mm plastic petri dish.

• Incubate overnight at 37°C in a humidified incubator.

• Rinse the dish three times in PBS just before adding beads.

• Add 100 µl of biotinylated agarose beads to 1.5 ml PBS.

• Centrifuge beads at 10,000 g for 10 seconds and remove supernatant.

• Repeat wash twice.

• After removing supernatant from last wash, resuspend beads in 0.01% BSA in PBS.

• Add 100 µl of washed beads to the streptavidin-coated plate. Beads should adhere to the dish almost immediately.

The figure below presents a representative AFM force measurement acquired using a streptavidin functionalized AFM cantilever and a biotinylated agarose bead. The measurement consists of an approach trace and a retract trace. During the approach trace, expansion of the piezoelectric translator lowers the cantilever onto the agarose bead and presses the AFM tip into the elastic bead. Surface contact is registered by an upward deflection of the cantilever and allows for the formation of streptavidin-biotin complexes. The number of complexes formed depends on density of biotin and streptavidin on the bead and cantilever, respectively, the duration of cantilever-bead contact and the applied force that presses the cantilever against the elastic agarose bead. Typically, an applied force of one nanonewton (nN) results in the formation of several complexes. The sawtooth shape of the retract trace reveals that unbinding of multiple biotin-streptavidin complexes does not necessarily occur simultaneously. The retract trace showed multiple transitions in force that can be attributed to the breakage of one or more streptavidin-biotin bonds. Interactions between the AFM tip and the substrate can be reduced to a single streptavidin-biotin linkage by the addition of either soluble biotin or streptavidin. Under conditions where the frequency of adhesion is low, there is a high probability that the force transition at surface separation corresponds to the unbinding force of the single streptavidin-biotin complex (Merkel et al., 1999). The addition of either free streptavidin or free biotin can be used to demonstrate the specificity of the streptavidin-biotin interaction.

5-AFM Measurements on Living Cells