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Chapter 8

Single Molecule Protein Unfolding Using a Nanopore

A likely candidate for next-generation protein sensing is solid-state nanopores. The pores developed here are fabricated in a 50 nm thick silicon nitride membrane and a single nanopore is drilled using a focused ion beam or a focused electron beam. The detection method employed is largely based on resistive pulse sensing where analytes are electrokinetically transported through a pore and identified by their unique modulation of ionic current (i.e. an ionic blockade). Since the dimensions of the nanopore are on the same scale as the molecule being sensed, only a single molecule can enter the pore allowing individual protein kinetics to be probed. Traditionally proteins are detected by ensemble averaging which hides important kinetics and sub-populations of molecules that may be important to understanding protein misfolding. In this chapter, it was discovered that the voltage which drives the protein through the pore also has denaturing effects. 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.

Print publication date: 18 Nov 2016
Copyright year: 2017
Print ISBN: 978-1-84973-404-2
PDF eISBN: 978-1-84973-523-0
ePub eISBN: 978-1-78262-958-0