Biomedical Engineering Reference
In-Depth Information
driving force. The distribution of translocation times depends strongly on protein
sequence, applied voltage
, as well as
H
eff
.
In summary, our studies of single protein translocation in solid-state nanopores
have shown that:
c
1. A nanopore experiment can measure a protein's size, electrical charge, and
conformation state.
2. If a native state protein has enough disulfide bonds (e.g. BSA has 17) to keep
the protein intact inside a nanopore in spite of the high electric field strength, the
protein translocates through the nanopore like a simple charged particle.
3. If a protein is completely unfolded as an amino acid chain, the translocation
kinetics is highly sequence dependent.
4. It is possible to distinguish proteins based on their nanopore translocation signal
profile in their native and unfolded state as functions of pH (charge state) and
applied voltage (driving force).
5. Furthermore, based on our analysis, an advantage of the nanopore experiment is
that it has the potential to distinguish proteins with single or multi-site mutants.
We describe this possibility in details below.
6.8 Future Trends
The sensitivity of the ionic current signal
DI
b
(
t
) to the charge and volume of specific
segments of the polypeptide chain present inside the nanopore at the stall points
suggests that unfolded protein translocation could ultimately provide enough contrast
to routinely distinguish different proteins in complicated mixtures. The compatibility
of nanopores with microfluidics and their ability to obtain data from zeptomole
samples suggest that these approaches could be used to screen single cell or subcellular
samples. To illustrate these ideas we report some proof-of-concept calculations.
6.8.1 Nanopore Protein Mixture Screening
Using staphylococcal nuclease (SNase) as a model protein, we predict the nanopore
translocation profiles of several SNase mutants and envision the results from a set of
nanopore measurement as illustrated in Fig.
6.8
.
Staphylococcal nuclease (SNase, Fig.
6.8a
) Consists of a polypeptide chain of
149 amino acid residues without disulfide binds [
41
,
42
]. SNase has been used as a
model protein to study protein folding and unfolding. More than 500 mutants have
been made and characterized in order to study the sequence dependence of its
structure and function.
Varying Protein charge Sequence allows control of the magnitude of specific
barriers in the translocation profile and can provide insight into the translocation
time distribution. Fig.
6.8b
illustrates how protein charge sequence changes alter
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