Biomedical Engineering Reference
In-Depth Information
This result demonstrated that a nanopore measurement is sensitive to the
electrical charge of proteins and can be used to probe the pI of an unknown native
state protein in an aqueous solution close to their native conditions.
6.5 Linear Amino Acid Chain Translocation
The electrophoretic force on an unfolded protein will tend to stretch it out during
translocation. If the chain length
l
m
is longer than the pore length
H
eff
, only a
segment of the chain is occupying the pore as illustrated in Figs.
6.1b
and
6.5a
. The
electrophoretic translocation kinetics of a driven linear amino acid chain is
expected to depend on the details of the primary sequence of charged amino
acids rather than the total net charge as is the case for a globular proteins
(Fig.
6.1a
) or polynucleic acids. Using bovine
LGa)
and a histidine-containing phosphocarrier protein (HPr) as model proteins [
35
], we
now discuss the consequences of unfolded
b
-lactoglobulin variant a (
b
LGa and HPr translocation in silicon
nitride nanopores. In addition, we discuss and compare the difference between
folded and unfolded protein translocation kinetics.
b
Fig. 6.5 (a) Schematic of linear translocation for
GLa as predicted
by treating the ionization of the individual residues as independent at pH 7.0 as a function of AA
number. (c) The electrostatic contribution to the potential energy of
b
GLa. (b) The net charge of
b
GLa and Hpr as a function of
AA through the nanopore. This potential is for the C terminus entering first. (d) The calculated
excluded partial volume
b
GLa and Hpr proteins as a function of number of amino
acids (AA) translocated. The slopes on the
left
and
right sides
show the insertion and exit of the
linear chain. The
circles
mark the location of stall points (charge zero) during the translocation.
Event Scatter Plot and marginal distributions of Sojourn Times and Calibrated Excluded Volume
for
L
profile for
bb
GLa and (e) HPr (f) in a small-diameter nanopore (D
p
~4 nm) under denaturing conditions
(8 M urea). The open pore current was the same (I
o
¼
3.2 nA) during these measurements.
b
GLa
(PDB file 2AKQ, 73,549.60 kDa) has 162 amino acids and it has a charge of
8
e
at pH 7. HPr
(PDB file 1POH, 9121.54 Da) has 85 aa and it has a charge of
2e at pH 7. The pore effective
thickness
H
eff
¼
20 nm was used for all calculations
b
Search WWH ::
Custom Search