| In single molecule force spectroscopy, a ligand-containing tip is approached towards the receptors on the probe surface, which possibly leads to formation of a receptor-ligand bond. The tip is subsequently retracted until the bond breaks at a certain force (unbinding force). Varying the dynamics of the experiment yields information about the binding pocket, binding energy barriers, and kinetic reaction rates. The attachment of human rhino virus 2 (HRV2) to the cell surface, the first step in infection, was characterized at the single molecule level. Sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra. This suggests that multiple receptors bound to the virus in a timely manner. Unbinding forces required to detach the virus from the cell membrane increased within a time frame of several 100 ms. The number of receptors involved in virus binding was determined and estimates for on-rate, off-rate, and equilibrium binding constant of the interaction between HRV2 and plasma membrane anchored receptors were obtained. Furthermore, we show that accurate free energy values of membrane protein unfolding can be obtained from single molecule force measurements. By applying a statistical theorem developed by Jarzynski, we derived equilibrium unfolding free energies of from unfolding force data acquired at different force loading-rates and temperatures. The reversible free energies were force loading-rate independent and remained unchanged within the temperature range from 18 to 42° C, indicating that indeed equilibrium values were obtained. Finally, we present a method for the localization of specific binding sites and epitopes with nm positional accuracy. A magnetically driven AFM tip containing a ligand covalently bound via a tether molecule is oscillated at a few nm amplitude, during scanning along the surface. In this way, topography and recognition images on membranes and cell surfaces were obtained simultaneously. |
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