Discriminating Residue Substitutions in a Single Protein Molecule Using a Sub-nanopore

It
is now possible to create, in a thin inorganic membrane, a single,
sub-nanometer-diameter pore (i.e., a sub-nanopore)
about the size of an amino acid residue. To explore the prospects
for sequencing protein with it, measurements of the force and current
were performed as two denatured histones, which differed by four amino
acid residue substitutions, were impelled systematically through the
sub-nanopore one at a time using an atomic force microscope. The force
measurements revealed that once the denatured protein, stabilized
by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore,
a disproportionately large force was required to pull it back. This
was interpreted to mean that the SDS was cleaved from the protein
during the translocation. The force measurements also exposed a dichotomy
in the translocation kinetics: either the molecule slid nearly frictionlessly
through the pore or it slipped-and-stuck. When it slid frictionlessly,
regardless of whether the molecule was pulled N-terminus or C-terminus
first through the pore, regular patterns were observed intermittently
in the force and blockade current fluctuations that corresponded to
the distance between stretched residues. Furthermore, the amplitude
of the fluctuations in the current blockade were correlated with the
occluded volume associated with the amino acid residues in the pore.
Finally, a comparison of the patterns in the current fluctuations
associated with the two practically identical histones supported the
conclusion that a sub-nanopore was sensitive enough to discriminate
amino acid substitutions in the sequence of a single protein
molecule by measuring volumes of 0.1 nm3 per read.