Single molecule unfolding and stretching of protein domains inside a solid-state nanopore by electric field

• Published in: Scientific reports Vol. 3; no. 1; p. 1638
• Main Authors: Freedman, Kevin J., Haq, S. Raza, Edel, Joshua B., Jemth, Per, Kim, Min Jun
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 10-04-2013; Nature Publishing Group
• Subjects: 631/57/2272/2273; 631/57/2272/2274; 631/57/2282; 631/61/350/1058; Data processing; Denaturation; Electric fields; Humanities and Social Sciences; Microscopes; Models, Molecular; multidisciplinary; Nanopores; Protein Conformation; Protein Denaturation; Protein folding; Protein Interaction Domains and Motifs; Protein Stability - drug effects; Protein Unfolding - drug effects; Proteins; Proteins - chemistry; Science; Urea; Urea - pharmacology
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep01638

Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 10
6
 V/m and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein.