Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates

Abstract

It is difficult to determine the structural stability of the individual subunits or protomers of many proteins in the cell that exist in an oligomeric or complexed state. In this study, we used single-molecule force spectroscopy on seven subunits of covalently linked cochaperonin GroES (ESC7) to evaluate the structural stability of the subunit. A modified form of ESC7 was immobilized on a mica surface. The force-extension profile obtained from the mechanical unfolding of this ESC7 showed a distinctive sawtooth pattern that is typical for multimodular proteins. When analyzed according to the worm-like chain model, the contour lengths calculated from the peaks in the profile suggested that linkedGroES subunits unfold in distinct steps after the oligomeric ring structure of ESC7 is disrupted. The evidence that structured subunits of ESC7 withstand external force to some extent even after the perturbation of the oligomeric ring structure suggests that a stable monomeric intermediate is an important component of the equilibrium unfolding reaction of GroES.Keywords: force spectroscopy; mechanical unfolding intermediates; protein subunit stability; covalently linked oligomeric protein; GroES; cpn10Abbreviations: AFM, atomic force microscopy; CH-ESC7, ESC7 that has two-cysteine residues and six-histidine residues extending from the N terminus and C terminus, respectively; ESC7, engineered form of cochaperonin GroES with covalently linked subunits; Gdn-HCl, guanidine hydrochloride; PBS, phosphate saline buffer.

Prying Open Single GroES Ring Complexes by Force Reveals Cooperativity across Domains

“…The relatively low mechanical strength of GroES subunits accords with its mixed secondary structure and the arrangement of these structural elements relative to the pulling direction, as observed previously for other proteins with mixed a/b and a þ b structures, such as MBP (24,25), DHFR (26,27), and lysozyme (28,29). However, the unfolding values reported here are significantly smaller than thẽ 50 pN reported previously (30) for sc(GroES) 7 . This probably reflects the fact that the different constructs used required different methods of surface attachment, leading to differences in the ability to identify bona fide unfolding events.…”