Nanopore Recordings to Quantify Activity-Related Properties of Proteins - Nanopores: Sensing and Fundamental Biological Interactions - page 210

9.2.3 Determining the Conformation of Proteins
with Nanopores

Protein function depends on correct folding of proteins. Nanopore-based
investigations have started to use translocation time and current pulse amplitudes to
distinguish different states of protein folding. Li and colleagues recently employed
nanopores to distinguish folded, partially unfolded, and completely unfolded proteins
based on their translocation signatures [ 46 ]. Talaga and Li used pores fabricated in
silicon nitride membranes with diameters of 4-8 nm to study unfolding of the protein
bovine LGa) in solutions containing up to 8 M urea (maintaining

the same pH). These authors analyzed the peak amplitudes of the translocation events
in combination with ( 9.1 ) to calculate the volume excluded by the protein. Smaller
excluded volumes and longer t d for unfolded proteins compared to folded proteins
made it possible to distinguish folded, partially folded, and unfolded protein states.
Earlier work by Auvray's group showed that using the biological pore

b -Lactoglobulin ( b

-HL, it is
possible to distinguish between folded and unfolded E. coli maltose binding protein
(MBP) that was denatured by guanidium chloride [ 36 ]. In this work t d was used to
distinguish folded proteins from denatured proteins, as t d was longer for the folded
than the unfolded proteins.
These examples illustrate the potential of nanopores for characterizing proteins
with regard to size, charge, and conformation.

a

9.3 Nanopore Recordings for Characterizing Equilibrium
Binding Constants and Stoichiometries of Binding

The function of most proteins involves binding to other proteins or to small
molecules. Characterizing these interactions is important, for example, in develop-
ing drugs with a high affinity and specificity for a target protein. Table 9.1 lists the
protein-ligand interactions that have been investigated quantitatively using
nanopores to date.

9.3.1 Using Nanopores to Determine the Stoichiometry
of Ligand Binding to Proteins

The stoichiometry of protein-ligand interactions (i.e. the number of ligand molecules
that a protein can bind), is one of the most basic properties of protein-ligand binding.
Two nanopore-based techniques have been employed so far to determine stoichiome-
try of binding.
Uram and coworkers recently used current recordings through nanopores to deter-
mine the volume of virus particles with and without antibodies bound (Fig. 9.3 )[ 50 ].