Over the past decade, single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) has permitted the mechanical forced unfolding of single proteins. However, the lack of a robust specific attachment of the protein to the surface and AFM tip has been a limiting factor. Here we present a reliable method for mechanically unfolding a broad range of proteins with the AFM. Proteins are covalently and site-specifically bound to a gold surface functionalized with a self-assembled-monolayer presenting maleimide groups. Thus, proteins engineered with a cysteine at one terminus, can be bound to that surface via the S-Maleimide bond from one end. On the other hand, the AFM tip is functionalized with amine-reactive succinimide groups. The latter covalently bind the surface immobilized protein's N-terminus, as the tip comes into contact with the surface during SMFS. Using surface plasmon resonance, mass spectroscopy, and SMFS, we show that this approach prevents non-specific protein-surface, tip-surface, and protein-protein interactions, and therefore allows the controlled unfolding of individual proteins. We further use this method to perform SMFS on the concensus tetratricopeptide repeat (CTPR) protein, with no subjective curve selection. The results revealed that partially folded states are present during the mechanical unfolding of a CTPR protein, contrary to a two-state behavior. Namely, we show the possibility of an unfolding pattern consistent with the sequential unfolding of individual domains. In addition, the force vs. extension curves reveal a constant force plateau as a reproducible feature. These results are consistent with simulations in the context of a 1D Ising model representation of repeat proteins.
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