Coiled Coil Probes Capture the Mechanical Unfolding Pathway of a Large Protein

Abstract

Folding behaviors and mechanisms of large, multi-domain proteins remain largely uncharacterized, primarily because of the lack of appropriate research tools. To address these limitations, we develop novel mechanical folding probes 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 probes 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 we use this probe in a large multidomain protein with 10 identical ankyrin repeats and we determine, unequivocally and precisely, the unfolding pathway, its direction and order of sequential unfolding. This development facilitates examination of the folding pathways of large proteins that predominate proteasomes of all organisms but have since eluded study because of technical limitations using traditional techniques.