The Unbinding of Molecular Complexes: Force Curves

Qualification of adhesive properties by means of AFM is usually carried out in the force spectroscopy mode, where force curves are recorded. The force curve is the dependence between a cantilever deflection (that is converted into force) and a relative sample (or scanner) position, which can be transformed to a tip-sample distance. The interaction forces can be obtained from the analysis of the retraction part of the force curve recorded during the AFM cantilever withdrawal from the surface.

In cases, when individual complexes are ruptured with the participation of cell surface, the unbinding can proceed through three most common scenarios. Ideally, only the rupture of a single complex occurs (Fig. 5.16). Then, the resulting force curve contains the characteristic “jump" that can be parameterized by two quantities: the unbinding force F and the rupture length L.

When two (or more) single complexes of the same type are simultaneously ruptured (Fig. 5.17a), the character of the force curves changes, depending on how many complexes are formed within the contact area between the ligand-modified tip and the receptor-covered surface and what the mechanism of the multiple unbinding is (two simplest cases are cooperative and uncooperative one). For cooperative unbinding, multiple molecular complexes break simultaneously, which manifests in the larger unbinding force (being the product of a force F obtained for the single complex and the number of complexes n ruptured at the same time) and in the similar length L as observed for the single molecular complex. The resulting force curve has a similar shape with larger unbinding force (Fig. 5.17b). In such case, the length histogram would have only one peak at the most probable rupture length L but in the force histogram, multiple maxima are expected at positions of F, 2F, ..., nF values. When molecular complexes act uncooperatively, the rupture proceeds sequentially and the detachment force has a similar value as that of a single molecular complex. The force curve will show a sawtooth pattern with unbinding events of the similar rupture length L and the unbinding force Fvalues (Fig. 5.17c).

(a) Illustration of a single molecular complex unbinding

Figure 5.16 (a) Illustration of a single molecular complex unbinding.

(b) An ideal force curve characteristic for the interaction between a single pair of ligand and receptor molecules (red line—approach part, black line—cantilever withdrawing denotes the curve recorded during molecular complex unbinding). Reprinted with permission from [2].

These considered possible shapes of force curves are also valid for experiments with the use of isolated proteins where one protein is attached to the AFM probe and the other, complementary one, is immobilized on the substrate surface. The embedding of receptors in a plasma membrane can often influence the unbinding process by the induction of the membrane deformation that may manifest in the broadening of the rupture length range but the unbinding force remains unchanged (unless any unrecognized non-specific interaction is present in the analyzed data). Thus, the unbinding events obtained for a given receptor, embedded in the cell membrane, produce the maximum in the force histogram and a broad distribution of the rupture lengths with or without distinguished peaks. In this case, the mean value of the rupture length will be shifted to higher values.

Illustration of simultaneous unbinding of two molecular complexes

Figure 5.17 Illustration of simultaneous unbinding of two molecular complexes (a) showing characteristic force curves for (b) cooperative and (c) uncooperative unbinding (red line—approach, black line—unbinding curves. Reprinted with permission from [2].

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