Coiled-Coil Probes Identified the Unfolding Pathway of Yeast Phosphoglycerate Kinase

Abstract

Large multi-domain proteins which have complicated reacting interfaces and dominate the proteomes of life remain mysterious regarding to their folding behaviors and mechanisms. Comprehensive studies towards the understanding of the fundamentals of protein folding mechanisms tend to bias small single-domain monomeric proteins, possibly due to their simplicity and reversible refolding capabilities. Yet the understanding of protein folding behaviors acquired from studying small single-domain proteins are not adequate to generate an accurate extrapolation for predicting the folding mechanisms of proteins that contain more than one domain. Here we present the characterization and interpretation of the unfolding pathway of a model large multi-domain protein yeast phosphoglycerate kinase (PGK) using single-molecule force spectroscopy (SMFS) and insertion of the antiparallel coiled-coil (CC) polypeptide probe we developed previously. When placed into different loop regions inside of yeast PGK, the CC probe affected different portion of the unfolding profile. Statistical comparison between the force-extension (FE) curves of multiple CC inserted yeast PGK's and the wildtype yeast PGK provided insights into figuring out the effects of conformational changes in domains and across the interfaces between domains on the unfolding pathway of the entire yeast PGK protein. Together with coarse-grain simulation modeling of the unfolding pathway, the sequence of the unfolding events occurred along the unfolding pathway of yeast PGK is precisely determined.