Li, Qing; Scholl, Zackary N.; Marszalek, Piotr E.
Capturing the Mechanical Unfolding Pathway of a Large Protein with Coiled-Coil Probes

The folding behaviors and mechanisms of large multidomain proteins have remained largely uncharacterized, primarily because of the lack of appropriate research methods. To address these limitations, novel mechanical folding probes have been developed that are based on antiparallel coiled-coil polypeptides. Such probes can be conveniently inserted at the DNA level, at different positions within the protein of interest where they minimally disturb the host protein structure. During single-molecule force spectroscopy measurements, the forced unfolding of the probe captures the progress of the unfolding front through the host protein structure. This novel approach allows unfolding pathways of large proteins to be directly identified. As an example, this probe was used in a large multidomain protein with ten identical ankyrin repeats, and the unfolding pathway, its direction, and the order of sequential unfolding were unequivocally and precisely determined. This development facilitates the examination of the folding pathways of large proteins, which are predominant in the proteasomes of all organisms, but have thus far eluded study because of the technical limitations encountered when using traditional techniques.


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