Reconsideration of dynamic force spectroscopy analysis of streptavidin-biotin interactions

Abstract

To understand and design molecular functions on the basis of molecular recognition processes, the microscopic probing of the energy landscapes of individual interactions in a molecular complex and their dependence on the surrounding conditions is of great importance. Dynamic force spectroscopy (DFS) is a technique that enables us to study the interaction between molecules at the single-molecule level. However, the obtained results differ among previous studies, which is considered to be caused by the differences in the measurement conditions. We have developed an atomic force microscopy technique that enables the precise analysis of molecular interactions on the basis of DFS. After verifying the performance of this technique, we carried out measurements to determine the landscapes of streptavidin-biotin interactions. The obtained results showed good agreement with theoretical predictions. Lifetimes were also well analyzed. Using a combination of cross-linkers and the atomic force microscope that we developed, site-selective measurement was carried out, and the steps involved in bonding due to microscopic interactions are discussed using the results obtained by site-selective analysis.

Keywords: atomic force microscopy; dynamic force spectroscopy; site-selective analysis; streptavidin-biotin interaction.

Figure 1.

Structure of streptavidin-biotin complex (reproduced from [28,29]).

Figure 2.

Schematic illustrations of the apex of a modified cantilever (a) and the three types of cross-linker for site-selective analysis; streptavidin is fixed to a SAM of (b) 1,10-decanedithiol/1-octanethiol (1/100 ratio) solution on a Au-coated substrate via a streptavidin-maleimide structure (SM condition), (c) 8-amino, 1-octanethiol molecule on a Au-coated substrate via a biotin-PEG-COO-NHS molecule (B-PEG condition) and (d) HS-(PEG)-SH/1-octanethiol (1/100 ratio) mixed solution on a Au-coated substrate via a streptavidin-maleimide structure (SM-PEG condition).

Figure 3.

Tapping-mode AFM images of a sample substrate: (a) bare Au, (b) with 1,10-decanedithiol/1-octanethiol SAM, (c) magnified image of an etch pit in (b), (d) substrate immersed in 10 mg/l streptavidin-maleimide solution and (e) line profile of streptavidin-maleimide molecules. The spring constant of the cantilever used for all measurements was 2 N/m.

Figure 4.

Schematic diagram representing the method of DFS analysis.

Figure 5.

(a) and (b): Two different forms of the typical force curve obtained using our system [15,16] as functions of (a) distance and (b) time. (c) and (d) show the data obtained for a direct interaction between the cantilever and the substrate, where no stretching of the PEG molecule is observed. (e) shows a typical force curve obtained for the B-PEG condition. (f) Schematic diagram of the approach and retract cycle.

Figure 6.

Schematic illustration of the force measurement and a typical force curve obtained at a high loading rate of 10⁴ pN/s to analyze the effect of the viscous drag on the cantilever.

Figure 7.

Change in binding rate caused by the introduction of free biotin molecules. The loading rate was set at 2.25 × 10² pN/s for all measurements. In the measurements, the ratios of the number of detected streptavidin-biotin bonds to the total number of measurements were (a) 8.1%, (b) 4.5% and (c) 2.2%.

Figure 8.

Typical histograms of the rupture forces obtained for the (a–c) SM and (d–f) B-PEG conditions in PBS (pH 7.4) at various loading rates.

Figure 9.

Relationships between the modal rupture force and the logarithm of the loading rate obtained for (a) SM (Figure 2(b)), (b) B-PEG [Figure 2(c)] and (c) SM-PEG (Figure 2(d)) conditions in 0.01 M PBS (pH 7.4). The barrier positions obtained from these results are shown in the figure. A similar experimental result obtained in 0.05 M sodium nitrate solution under the SM condition is also shown. The result obtained for the 0.01 M PBS (pH 7.4) solution shown in (b) is also indicated for comparison (blue dashed line).

Figure 10.

Schematic illustration of the site-selective analysis using two types of cross-linker. (a) B-PEG condition and (b) SM condition.

Figure 11.

Schematic illustrations of the rupture types for (a) B-PEG and (b) SM-PEG conditions.

Figure 12.

Schematics of the bonding structures of streptavidin-biotin complex.