Biomedical Engineering Reference
In-Depth Information
event duration
t
d
. The average current of all points between the start and stop times
is calculated as
DI
b
. The events with
t
d
that are very short (beyond the time
resolution of the measuring system) and too long are discarded.
6.2 Principles of Protein Translocation
The translocation of proteins through nanopores differs from that of the more
commonly studied polynucleic acids. The physical properties of proteins differ
from those of polynucleic acids in several important ways that directly impact their
nanopore capture, insertion, and translocation. These differences result in changes
in data analysis and interpretation for protein translocation in nanopores.
6.2.1 Protein Capture by Nanopores
A charged molecule near the entrance of a properly biased nanopore will be
captured. Entrance into the pore depends on the molecule's ability to attain a
sterically compatible geometry for translocation during its encounter with the
nanopore opening. For a polynucleotide this typically requires threading one end
of the chain or forming a bend in the chain. Since a polynucleotide is uniformly
charged, bend insertion can occur essentially anywhere in the sequence. By con-
trast, proteins have both pre-formed loops and charges of both sign distributed
along their length suggesting that particular locations of bend insertion will be
preferred. Protein C and N termini can have opposite polarity and therefore may
also exhibit selectivity during the insertion event.
6.2.2 Protein Shape or Geometry During Translocation
In contrast to polynucleotides, proteins most often have a single well-defined
three-dimensional native-state structure. This structure can be partly or completely
disrupted by denaturants, temperature, or the application of electric fields. Tertiary
contacts in proteins can be stabilized by both covalent and non-covalent
interactions. Thus, the structure of the protein during translocation can be globular
(Fig.
6.1a
), looped, or a completely unfolded linear chain and (Fig.
6.1b
) depending
on the conditions during the measurement.
When the protein translocates as a loop or linear chain, only a segment of the
amino acid chain will typically be inside the nanopore (Fig.
6.1b
) and be exposed to
the influence of the electric field therein. The molecular volume of amino acids
varies much more than that of nucleotides. As a result, the magnitude of the current
drop can vary more for proteins and is potentially more sensitive to particular
features in the sequence. Moreover the distribution of charges along the polypeptide
chain is sequence dependent; the net charge inside the pore can fluctuate as a
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