Nanopore Recordings to Quantify Activity-Related Properties of Proteins - Nanopores: Sensing and Fundamental Biological Interactions - page 204

9.1 Introduction

This chapter describes the use of nanopores for quantifying activity-related properties
of proteins. We review recent advances in characterizing the most basic structural
properties of proteins that relate to their activity such as size, charge, and conforma-
tion, as well as functional properties such as binding of ligands to proteins and the
activity of enzymes.
Sensing proteins in nanopores, in most cases, is based on applying an electric
potential difference between the electrolyte solutions separated by a pore-containing
membrane and measuring the fluctuations of ionic current through the nanopore.
Under the appropriate experimental conditions (i.e. size of the nanopore, ionic
strength, and applied electric potential difference), the presence of a single protein
in a nanopore displaces a sufficiently large conducting volume of electrolyte to induce
a detectable change in the resistance (Fig. 9.1 ). The resulting decrease in the current
through the pore is transient and is recorded with high-gain, low-noise amplifiers,
which were originally developed for electrophysiology applications such as patch-
clamp recordings of single ion channels in the membranes of living cells [ 18 , 34 ].
Taking advantage of this simple concept of resistive-pulse sensing, nanopores
provide a unique platform for characterizing the function of proteins. For example,
the magnitude, frequency, and duration of these current fluctuations can provide
information on the volume of the protein, concentration of the protein, and
properties such as conformation, surface charge, bound ligands or enzymatic activ-
ity of the protein in solution. In addition, nanopore sensing can be accomplished with
a simple instrumental setup (Fig. 9.1 ) compared to other single molecule techniques
such as single molecule fluorescence microscopy. Furthermore, nanopore sensing
of proteins is attractive because it is rapid (1-20 min), reports on multiple properties
from individual proteins in the same recording, and may be applied in a label-free
manner. In addition, nanopore sensing can explore the function of proteins in
solution without immobilization of the analyte.
Recently, nanopores with ligands immobilized on the nanopore walls have been
employed as biosensors to detect the presence and concentration of specific proteins.
A recent, excellent review article by Howorka and Siwy outlined these methods [ 20 ].
This chapter will focus, however, on methods that detected single proteins, quantified
the binding of proteins and ligand molecules, or quantified the activity of enzymes
with nanopores.
On a fundamental level, the size, charge, and conformation of a protein influences
its function, and thus we will begin with a discussion on recent research that
employed nanopores to quantify these properties. Next, we will discuss nanopore-
based techniques to measure protein function quantitatively. Discussed in order of
increasing complexity, these parameters include the equilibrium association and
dissociation constants between proteins and ligands, the kinetic on- and off-rates of
ligands binding to proteins, and the catalytic rate constant and Michaelis constant,
which describe enzyme kinetics. Novel strategies to characterize proteins on a single
molecule level rapidly will be useful in a variety of fields ranging from drug