Distinguishing Protein Domains and Folded States with Unfoldase-Mediated Nanopore Analysis

Abstract

We have recently shown that the protein unfoldase ClpX can be used to controllably unfold and translocate proteins through an α-hemolysin pore, thereby allowing sequence-dependent features of engineered proteins to be detected [1]. Here we now describe the extension of this technique to the analysis of hetero-multi-domain proteins. The signals generated during the enzymatic unfolding and translocation of these systematically engineered protein constructs were analyzed to correlate domain identity and folded state, in addition to unfolding intermediates, to specific current states. These results demonstrate the utility of this technique to linearly interrogate and distinguish between the individual domains of complex proteins by revealing information on sequence, size, and structural stability at the single-molecule level.1. Nivala, J., Marks, D.B. & Akeson, M. Unfoldase-mediated protein translocation through an α-hemolysin nanopore. Nat. Biotechnol. 31 247-50 (2013).View Large Image | View Hi-Res Image | Download PowerPoint Slide