Biomedical Engineering Reference
In-Depth Information
6.5.1 Stall Point Potential Well Model
Due to the heterogeneous distribution of charged amino acids, the calculated net
charge of a segment of a
LGa polypeptide chain, Q
AA
¼
P
q
i
in the pore
, can be
positive, negative and zero as a function of the number of residues translocating in a
pore as shown in Fig.
6.5b
. Locations where the net charge or force is zero, we call
stall points. The positive and negative net charge positions in the curve imply
that at these positions during the linear translocation the electrophoretic force,
F
e
b
/
H
eff
, can either oppose or drive the translocation. This can be seen
more clearly in the translocation potential plot in Fig.
6.5c
. The potential profile is
calculated by
¼ Q
AA
C
ð
x
0
H
eff
qðxÞdx:
DUðxÞ¼
(6.5)
The potential profile plot shows that
LGa has two stall points and Hpr has one.
Near the stall points, the peptide chain is likely thermally fluctuating in the potential
well that is formed due to the polypeptide charge sequence. If the potential well is
deep enough, the unfolded protein will be metastably trapped in the nanopore; the
translocation process would still go forward eventually because the total electrical
driving force is downhill. While the unfolded protein is trapped at a stall point, its
escape is thermally activated. The total translocation will therefore be a combina-
tion of electrophoretic and thermally activated processes.
b
6.5.2 The Excluded Volume at Stall Points
Due to the large atomic volume variations between amino acids (up to 3.8 times)
the instantaneous excluded volume profile of an unfolded protein translocation,
L
AA
¼
∑
V
Ai
, is a function of the protein primary sequence as shown in Fig.
6.5d
.
b
LGa has more large volume amino acids,
V
Ai
, compared to HPr,
thus the
calculated excluded volume (
L
AA
) for
LGa is larger compared to HPr. The
measured excluded volume is a time average of the local segment volume.
Since the polypeptide chain dwells in the pore longer at the stall points, the
corresponding excluded volume at the stall points (red circles in Fig.
6.5d
) should
have more weight. Since the purely electrophoretic part of the translocation
should contribute ~1-2
b
s to the translocation time of these small single-domain
proteins, the net translocation time is likely dominated by the sojourns at the stall
points. Thus the experimentally determined excluded volume should correspond
to the stall point volumes.
The experimental data measured in the same ~4 nm diameter pore for
m
LGa
(Fig.
6.5e
) and HPr (Fig.
6.5f
) under denatured conditions (8 M urea) show that
the peak value of the mean
b
LGa than HPr supporting
this analysis. The volumes measured from the peak values of
DI
b
is indeed larger for
b
DI
b
data match
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