Single molecule mechanical probing of the SNARE complex by atomic force microscope

We used atomic force microscope (AFM) to perform single molecule study of the SNARE complex. Novel method (Ni2+-H6) was used to attach the proteins on the AFM cantilever and the glass coverslip. The rupture force and extension of the interactions were measured. The result shows that extension plays an important role in the interaction. 12 nm extension of the ternary complex is consistent with the previous X-ray crystallography results. Based on single molecule reaction rate theory, we calculated the lifetime and barrier width of the complex. 2.1 s lifetime of the ternary complex compared with 0.16 s of the binary confirms that the ternary complex is a much more solid structure. Constant force experiment is another independent approach to the bond information. The lifetime and barrier width from constant force measurements are in good agreement with the regular force loading rate experiments. Constant force technique can also identify the strong bond of the complex by analyzing the response curve of the feedback piezo. The result is also consistent with the X-ray structure. The binding free energy of the SNARE complex was calculated by two independent methods. First, we used Jarzynski's non-equilibrium thermodynamics theory to calculate the binding energy. This is the first time this theory is applied to the single molecule study with AFM. Second, we calculated the binding free energy through the single molecule reaction rate theory. Lifetimes of the complex were measured at different temperatures. The binding energy can be obtained from these different lifetimes. The results of non-equilibrium thermodynamics and single molecule reaction rate theory are reasonably comparable, which shows the self-consistence of our measurements. As a practical application, we developed a sensor which can detect Botulinum toxin type B (BoNT-B) rapidly. The technique is general and can be broadly used in different fields. The single molecule study of the SNARE complex provides insight into the bond information of the complex and greatly enhances our understanding of the mechanisms of the SNARE membrane fusion. These general techniques can also be used to investigate other protein systems.