Nanopores: fundamental science

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Lecture 19 Nanopores fundamental science and
biotech prospects
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Outline

• Tethered functional polymers
• under confinement

2. Elementary secondary structures in a nanopore
3. Temperature-responsive pore
Aksimentiev Schulten, 2005
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Song, L., Hobaugh, M.R., Shustak, C., Cheley, S.,
Bayley, H., Gouaux, J. E. 1996, Science
2741869-1865.
- seven subunits -each subunit contributing with
two ?-strands to form a 14-stranded ?-barrel
protein pore - cis entrance is 70 Å above the
bilayer - total of 100 Å - long lumen -average
20 Å in internal diameter -the trans entrance is
close to the bilayer surface
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AFM view
with Jie Yang, U Vermont
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Motivation to work with ?-hemolysin versatility
and robustness
1. Structural It has a known crystal structure
(Song et al., 1996)
2. Biochemical It is a robust membrane
proteinaceous complex stable in SDS preparative
Gels easy to carry out genetic engineering of
the channel.

• 3. Biophysical
• Large single-channel conductance pretty signal
• The channel remains open indefinitely for extreme
  environmental
• conditions (pH, salt, T, ??V)
• Bayley and coworkers, Angew. Chem. Int, January
  2005.
• ?-barrel is intact up to 95 C!

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Single channel electrical recording
DV 40 mV 700 pS 30 pA 1 M NaCl
cis
trans
In this talk various DV, salt,
filtering, timescales etc.
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Single-channel current signature for the
wild-type protein
100 Hz
?V100 mV cKCl 300 mM
WT7
Current (pA)
10 s
Number of Points
Quiet ionic flow
Current (pA)
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COMBINATIONS and PERMUTATIONS
WT7
WT6-(CM-PEG)1
WT5-(CM-PEG)2
WT4-(CM-PEG)3
WT3-(CM-PEG)4
WT2-(CM-PEG)5
WT1-(CM-PEG)6
-(CM-PEG)7
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Pre-formed heteroheptamers with different number
of PEGs
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a
H6S106C-PEG3.4k-biotin1
Current (pA)
1 sec
b
Number of Points
Current (pA)
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3.4 kDa biotinylated PEG
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C. Jeppesen et al., Science 293 (2001) 465-471
T. P. Russell, Science 293 (2001) 446-447
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Experimental design
A
Analyte/cis
B
Analyte/trans
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b
a
WT streptavidin/(trans)
WT streptavidin/(cis)
Current (pA)
Current (pA)
2 s
2 s
c
d
W120A streptavidin/(trans)
W120A streptavidin/(cis)
Current (pA)
Current (pA)
500 ms
10 s
.
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PEG can be trapped out in both sides of the
bilayer (in the same experiment!)
W120A streptavidin/(cistrans)
W120A (cis)/WT streptavidin(trans)
Current (pA)
Current (pA)
10 s
10 s
1. Transient captures/cis 2. Permanent
capture/trans
1. Transient captures/cis 2. Transient
captures/trans

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Stochastic sensing provides kinetic information
ton 1/konA
toff 1/koff
Kd koff / kon
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Quantification of the protein analyte at nM
concentration
0 nM
14.5 nM
1/? (s-1)
Current (pA)
W120A streptavidin (nM)
43.5 nM
Get kon, koff, Kd
Events are detected in the microsecond timescale
10 s

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Detection of an unknown protein - anti-biotin
monoclonal antibody
mAb/(trans)
Current (pA)
5 s
W120A streptavidin(cis)/mAb(trans)
Current (pA)
5 s

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Other applications
Single-polymer dynamics
Design of other nanostructures with movable
polymeric arms
Single-molecule detection of other macromolecules
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Thermodynamics and kinetics of duplex DNA
formation probed at the single molecule level
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A
B
M113
S106
T129
E111
L135
T117
K8
lipid bilayer
cis
trans
polymer
transmembrane pore
constriction
? barrel
vestibule
100 Å

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4 mM OPSS-PEG/trans - Reaction monitored at -40 mV
Apparent reaction rate constant (s-1)
PEG repeat units, N
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What is the polymer concentration within the pore?
PcaHL/csolution
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Subunit combinations permutations
1 mM MePEG-OPSS-1kDa/cis
0
1
2
3
4
5
Current (pA)
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7
modified
unmodified
K8C7
Time (s)
e.g., determination of pore stoichiometry for
other barrel proteins
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Current (pA)
4 s
Current (pA)
2 s
T117C7100mV(n)10kHzOPSS-PEG1k/cis10 mM DTTcis
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3. A targeted built-in temperature-responsive
gating mechanism
(VPGGG)m
? 410-5 atoliters
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Temperature-dependent single-channel activity of
ELP-engineered ?HL pores
A
E101W6
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GS-rich peptide loop engineered ?HL pores
L651W6
20 C
40 C
60 C
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Kinetic model for temperature-dependent
ELP-induced closures
Low conductance
High conductance
open
hydrated (unfolded)
collapsed (folded)
open
T
Low T
High T
- T
Low conductance
High conductance
hydrated (unfolded)
collapsed (folded)
closed
closed
Full current blockade
Small current blockade