Nanopores: a versatile tool to study protein dynamics

Abstract

Proteins are the active workhorses in our body. These biomolecules perform all vital cellular functions from DNA replication and general biosynthesis to metabolic signaling and environmental sensing. While static 3D structures are now readily available, observing the functional cycle of proteins – involving conformational changes and interactions – remains very challenging, e.g., due to ensemble averaging. However, time-resolved information is crucial to gain a mechanistic understanding of protein function. Single-molecule techniques such as FRET and force spectroscopies provide answers but can be limited by the required labelling, a narrow time bandwidth, and more. Here, we describe electrical nanopore detection as a tool for probing protein dynamics. With a time bandwidth ranging from microseconds to hours, nanopore experiments cover an exceptionally wide range of timescales that is very relevant for protein function. First, we discuss the working principle of label-free nanopore experiments, various pore designs, instrumentation, and the characteristics of nanopore signals. In the second part, we review a few nanopore experiments that solved research questions in protein science, and we compare nanopores to other single-molecule techniques. We hope to make electrical nanopore sensing more accessible to the biochemical community, and to inspire new creative solutions to resolve a variety of protein dynamics – one molecule at a time.

FIB-milled plasmonic nanoapertures allow for long trapping times of individual proteins

“…However, such traps require fluorescence labeling of the protein, stochastic diffusion of the protein into the traps, and very low salt concentrations that are well below physiological concentrations. Newer techniques, such as the NEOtrap, which traps a protein using an induced electroosmotic current rather than an electrophoretic force, are able to very significantly increase the trapping times ( Schmid and Dekker, 2021 ), but the use of an ionic sensing method prevents parallelization for which electrical isolation of each sensor is needed. Various groups have proposed other different techniques, most notably, the Anti-Brownian Electrokinetic Traps (ABEL) that tracks the molecule of interest by applying a real-time electrokinetic feedback to compensate for the drift from Brownian motion ( Cohen and Moerner, 2006 ; Cohen and Moemer, 2005 ; Fields and Cohen, 2011 ).…”

Using wide biological pores to cap and contain the COVID-19 spike protein

“…There are many pore-forming proteins in nature that are toxins and function by puncturing the cell membrane to cause cell death [14 ]. On the other hand such an intrinsically malign property has been turned to advantage in vitro (and in silico) by using these pores as analytical tools in a wide range of applications, including sequencing/identification of biomolecules (DNA, protein, oligomers, peptides) [15,16], analysis of species [17], protein conformation and folding [18 ], and protein dynamics [19].…”

In situsolid-state nanopore fabrication

“…being developed for commercial applications such as DNA sequencing, [6][7][8] protein analysis, 9,10 and analyte detection in ultra-dilute samples. [11][12][13] As such, single-molecule biosensing devices hold promise for future applications such as early stage diagnostics and personalised medicine.…”