Nanopore-Based Measurements of Protein Size, Fluctuations, and Conformational Changes

TLDR

Proteins are structurally dynamic macromolecules, and nanopores provide an efficient solution‑based platform to probe their native conformational properties. The method uses an electric field to drive proteins through a nanopore slightly larger than the molecule, while high‑bandwidth ion‑current recordings capture each transit. Measurements show that mean current blockade amplitude scales with radius of gyration, amplitude‑distribution shapes reflect protein fluctuations from simulations, fluctuation magnitude correlates with α‑helix/β‑sheet ratio, and the technique resolves two calcium‑induced states of calmodulin.

Abstract

Proteins are structurally dynamic macromolecules, and it is challenging to quantify the conformational properties of their native state in solution. Nanopores can be efficient tools to study proteins in a solution environment. In this method, an electric field induces electrophoretic and/or electro-osmotic transport of protein molecules through a nanopore slightly larger than the protein molecule. High-bandwidth ion current measurement is used to detect the transit of each protein molecule. First, our measurements reveal a correlation between the mean current blockade amplitude and the radius of gyration for each protein. Next, we find a correlation between the shape of the current signal amplitude distributions and the protein fluctuation as obtained from molecular dynamics simulations. Further, the magnitude of the structural fluctuations, as probed by experiments and simulations, correlates with the ratio of α-helix to β-sheet content. We highlight the resolution of our measurements by resolving two states of calmodulin, a canonical protein that undergoes a conformational change in response to calcium binding.