Detection of streptavidin-biotin intermediate metastable states at the single-molecule level using high temporal-resolution atomic force microscopy

Abstract

Although the streptavidin-biotin intermolecular bond has been extensively used in many applications due to its high binding affinity, its exact nature and interaction mechanism have not been well understood. Several reports from previous studies gave a wide range of results in terms of the system's energy potential landscape because of bypassing some short-lived states in the detection process. We employed a quasi-static process of slowly loading force onto the bond (loading rate = 20 pN s^-1) to minimize the force-induced disruption and to provide a chance to explore the system in near-equilibrium. Therein, by utilizing a fast sampling rate for the detection of force by atomic force microscopy (20 μs per data point), several transient states of the system were clearly resolved in our force spectroscopy measurements. These key strategies allow the determination of the states' relative positions and free energy levels along the pulling reaction coordinate for the illustration of an energy landscape of the system.

Fig. 1. Histogram of observed rupture forces at non-equilibrium unbinding of the streptavidin–biotin system. The inset shows a typical force–distance curve, which contain the rupturing event of the streptavidin–biotin bond.

Fig. 2. Streptavidin–biotin bond dynamics in near-equilibrium probed at a slow loading rate. (a) Force plotted as a function of time. (b) Magnified force–time plot of black box in (a) and a histogram of force values observed in the force–time plot. Transient events were spotted (green arrows as representatives) suggesting the presence of intermediate metastable states.

Fig. 3. Histograms of each segmented region. The region presented in Fig. 2b was divided into 10 segments, which was optimized by minimizing peak-fitting error. The dashed lines correspond to the mean value of the force peak obtained though multipeak fitting and the shaded region represents the standard deviation. The mean values of force peak 8 and 1 are interpreted as the equivalent force for the lowest bound and unbound states, respectively as suggested by eqn (1). It should be noted that all states were not observed in every segment. A total of 8 states were observed with high reproducibility (ESI†) presented in Table 1 and by the broken lines in this figure.

Fig. 4. Schematic model of an energy potential landscape for SMFS experiments (ref. 14 and 37).

Fig. 5. Method to evaluate lifetime of transient states though sliding window method. (a) Force (red, left axis) and std. dev. (green, right axis) plotted against the same time series. (b) Zoomed region (box in (a)) highlighting the peak distance as the lifetime of the state and employing a threshold to capture specific events.

Fig. 6. Illustration of energy potential landscape using the results obtained in this study. This shows the location of the various states (lowest bound, metastable, and unbound) with the corresponding bond free energy (+markers). The blue dashed lines represent the locations of the energy barriers revealed in previous studies. The presence of the other energy barriers is implied by the adjacent energy minima. Note: the curvature of the energy states and the barrier locations other than the already reported values are arbitrary just to show a simplified illustration of the potential landscape.