surface-enhanced 2d ir spectroscopy to probe voltage- and ph …€¦ · surface-enhanced 2d ir...
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Surface-enhanced 2D IR spectroscopy to probe
voltage- and pH – sensitive peptides and ion channels
in a single bilayer
Ion channels play vital roles in biological processes. These channels have been long
recognized for their role in controlling electrically excitable tissues such as muscles and nerves.
However, there is still debate as to the mechanism with which the ions permeate the channel,
specifically if water passes through the channel with the ions or if collisions between the ions are
necessary for transduction. Previous work in the Zanni group has utilized two-dimensional infrared
(2D IR) spectroscopy to determine the method of ion permeation in a model ion channel, KcsA.
2D IR spectroscopy is an excellent technique for this study because vibrational frequencies and
lineshapes are excellent probes of molecular structure and electrostatic environment. Since the
publication of the original report, others have questioned our conclusions and have found different
ways to average the modeled spectra to match our experimental spectra. Therefore, we need to do
more in depth studies of KcsA. We also are working towards a voltage-dependent experiment that
will allow us to study the impact of an applied potential on the protein of interest. In order to do
these potential experiments, I have been working to develop a surface-sensitive 2D IR method
using localized surface plasmon enhancement in order to work with proteins embedded in a single
bilayer. This work has yielded promising results in a model peptide and we are working to
implement this same technique into larger systems.
Research Plan:
Research Objectives
The objective of my research is to determine the mechanism of ion permeation through ion
channels by using two-dimensional infrared (2D IR) spectroscopy, a technique known for its
inherent time and structural resolution. We begin by following up on previous work in the Zanni
group, studying the potassium (K+) channel from Streptomyces lividans (KcsA) in micelles using 13C/18O isotope labels.1,2 However, since we want to probe the effect of an external stimulus on
ion occupancy and general structure, we are moving this work into a single bilayer. This is
necessary in order to facilitate a potential drop across the membrane-bound protein of interest.
With stacked bilayers, the potential required to trigger protein movement increases additively with
each successive layer, leading to unnecessarily dangerous applied potential. With a single bilayer,
the applied potential can be as low as 50 mV.3 However, studying a protein in a single bilayer
yields a very low signal due to the inherently small amount of material present. This can be
remedied by the recent push towards surface-enhanced spectroscopies. Using thin, rough gold as
a source of plasmon enhancement, combined with the inherent non-linear scaling of 2D IR, we
have been able to take spectra of proteins in a bilayer.4,5
Determine the ion occupancy of KcsA – Work by the Zanni group that determined the ion
occupancy of KcsA to predict the mechanism of permeation has recently come under scrutiny.6,7
In short, it was put forth that our data was inconclusive as to the presence or lack of water in the
selectivity filter (a short, highly conserved sequence making up the channel). We need to collect
more spectra of KcsA with different isotope labeling schemes in the filter to better determine ion
occupancy. Work done by the Skinner Group at the University of Chicago has indicated that in
labelling Val76 and Gly77 individually, spectra that are more narrow and red-shifted would be
indicative of the water-free (hard-knock) mechanism and broader, blue-shifted spectra will
indicate water in the channel during transduction (soft-knock mechanism).
Surface-sensitive techniques – In order to be able to observe a spectrum of a single bilayer, while
being able to place a voltage across it, we use a thin, conductive layer of rough gold to function
both as an electrode and a source of plasmon enhancement. Furthermore, by taking advantage of
the ease of sulfur-gold chemistry, we can easily tether a monolayer of the sample to the gold. The
signal enhancement from the gold has been shown to have enhancements of up to 6000x. A further
3x enhancement can be gained by using a reflection geometry for a 3-nm thick thermally
evaporated film of gold.8 However, we have found that the 3-nm film is not macroscopically
conductive. To overcome this, I am working to find a thickness of gold that is conductive while
yielding suitable enhancements in the mid-IR, without lineshape distortions. This could also be
accomplished by adding a non-plasmonic conductive layer under the gold (e.g. indium tin oxide
(ITO) with a small amount of rough, plasmonic gold coated on top.9 We will investigate this
technique by studying the model peptaibol, alamethicin, comparing its static pH and voltage
dependences, as it forms pores in the tethered membrane. We will translate these static experiments
into transient, voltage-jump experiments to gain insight into the dynamics of voltage-gating.
Background and Significance
Ion Channels and Pores – Nerves and muscles have been known to be electrically excitable for
over 200 years. However, the movement of ions across the membrane to trigger this excitation was
not discovered until the early twentieth century by Julius Bernstein and built upon by many, such
as Nobel Prize winners Hodgkin, Huxley, and MacKinnon.10–13 Ion channels are so central to cell
physiology and communication, that study of these channels has expanded from monitoring the
ionic flux with voltage- and patch-
clamp experiments to determining
the crystallographic structure of
the membrane-bound proteins
themselves.
A notable feature of ion
channels, which generally control
the flow of Na+, K+, Ca2+, and Cl-
ions is that they have extremely
high ion specificity while
maintaining an ion flux of millions
of ions per second, nearly
matching the diffusion limit.10 The
model potassium channel, KcsA,
has been extensively used in order
to study the mechanism of ion
permeation.14–17 Selection for one
ion over another occurs in the
selectivity filter, a highly
conserved sequence (TVGYG) in
K+ channels composed of four
binding sites bound by five
backbone carbonyls on each of the
monomers forming the tetrameric
channel.11,17 Currently, there are
two competing models for ion
transduction: the hard-knock and
soft-knock (or knock-on) models
as shown in Figure 1. In the hard-
knock model, the potassium ions
are completely desolvated as they
enter the selectivity filter. As a
new K+ ion enters, coulombic forces repel the neighboring K+ ion, causing them to move through
the channel.6,18 In the soft-knock model, the selectivity filter is filled with alternating K+ ions and
water. The new K+ ion enters with an accompanying water, pushing another K+ ion and water out
into the extracellular side of the channel.1,19–23 Experiments, such as x-ray crystallography, single-
channel patch-clamp experiments, and computational modeling, have generally supported the soft-
knock mechanism, though these techniques were not able to give definitive evidence.22–24
However, in recent years, several theoretical groups have proposed the hard-knock model, which
they support by its ability to explain the high ion throughput and selectivity of the channel as the
energy penalty of desolvation greatly favors K+ over Na+.6
Previous work by the Zanni group resulted in a significant advance in the field, yielding
data that could be matched to results from molecular dynamics (MD) simulations. In 2016,
Figure 1. KcsA (two of four monomers shown) with the two proposed
methods of ion permeation A) the soft-knock and B) the hard-knock
with K+ ions in yellow and water molecules in blue. Backbone
carbonyls are shown in the selectivity filter.
Kratochvil and colleagues took 2D IR spectra of the KcsA channel in micelles.1 In order to observe
the environment of the selectivity filter, 13C/18O isotope labels at specific residues were used. The
additional mass of the labels red-shifts the amide I peak corresponding to the labelled amino acid
by approximately 60 cm-1, far enough to distinguish it from the rest of the protein. This method
provides a vibrational probe of the secondary structure and electrostatic environment of a single
residue.25–28 The isotope labels are sensitive to the electrostatics of their environment because
interactions with the partial negative charge on the 18O will effectively increase its mass, causing
a red-shifted peak in the spectrum. In this case, the inward-facing carbonyls that make up the ion
binding sites are sensitive to the electrostatics of the ions and molecules in the selectivity filter.
Placing isotope labels in three locations (Val76, Gly 77, and Gly79) allowed Kratochvil et al. to
observe the ion positions in the channel. In order to see the isotope labels, which were otherwise
masked due to the size of the protein (41 kDa), a non-isotope labeled spectrum was subtracted
from the isotope labelled spectrum. Data from the isotope-labelled experiments of KcsA in
micelles showed good agreement with spectra derived from MD simulation trajectories of the soft-
knock model. A state which included the carbonyl of Val76 flipped out of the selectivity filter was
included to create better agreement with experiment. This state has also been observed in NMR
structural studies of the channel as well as other MD simulations, though not through x-ray
crystallography.29–31
Papers published in the last year have called these results into question.6,7 One showed that
it was possible to generate a spectrum similar to experiment by using a weighted average of states
from the hard-knock model. They argued that the hard-knock model also has a better explanation
to the energetics of ion selectivity. They calculated the difference in energy required to desolvate
the K+ ion is much less than the Na+ ion. This difference explains the rapid K+ ion flow while Na+
ions are blocked. They also argue against the inclusion of the Val76 flipped state as previous work
has indicated that it corresponds to a non-conductive filter. However, in order to fit the hard-knock
model to our data, they have a configuration with four successive K+ ions in the channel account
for 25 percent of the contributing states, which would involve large repulsive forces in the channel.
We take this result to indicate that more experimental data is needed in order to constrain the
weights of the individual occupancies and come to a general conclusion. This may include
individual carbonyl isotope labels as detailed in future work below. Finally, some of these models
take into account the presence of a 50-mV potential drop across the bilayer, which was not used
in the previous experiments.18
Surface Enhanced 2D IR spectroscopy – Previous work to study ion channels have relied on
structural information from x-ray crystallography and NMR. X-ray crystallography has atomic
resolution, but is inherently static and the crystallization process can impact structure and ion
occupancy.11,15,29,32 Furthermore, it is difficult to distinguish between internal waters and ions.33
NMR gives both structurally- and time-resolved information but is limited as to the size of
protein.34 The dynamics of ion channels systems have been studied with time resolved current data
from patch-clamp experiments. Patch clamp experiments have good time resolution, but lack
overall structural information. In order to get residue-specific structural information from patch-
clamp experiments, time-intensive point-mutation must be done.32,35 2D IR has the ability to give
residue-specific structural information with inherent femtosecond time resolution and is therefore
an excellent method to study these systems without compromising structural or dynamical
information.4,25,36–38
Briefly, 2D IR measures the third-order response function of the vibrational transitions of
the sample after three light-matter interactions (see the SI for a more details). The vibrations of the
molecule are sensitive to which atoms are vibrating and the chemical environment.4 In proteins, a
commonly probed vibration is the amide I mode, largely composed of the backbone carbonyl
stretch, which absorbs between 1600 and 1680 cm-1.39,40 In our measurements, we observe the
frequencies in this region to determine the secondary structures present in the protein and how they
change. The main secondary structures, α-helix, β-sheet, and random coil, have distinct center
frequencies (1650 cm-1, 1620 cm-1, and 1645 cm-1 respectively) and lineshapes (narrower defined
structures and broad for random coil).4,39 Lineshapes also give insights into inhomogeneous and
homogenous broadening which are indicative of chemical environment. 2D IR is helpful in
distinguishing between these structures because the signal scales with μ4, rather than μ2 in 1D IR.
This increases signal and narrows the lineshape of some peaks while suppressing noise, making it
easier to resolve different secondary structures.4,25
In order to continue our work with membrane-embedded KcsA, we want to work in a single
bilayer. Doing so will allow us to apply external stimuli due to the voltage-divider-like nature of
the membrane as well as simulate a more biomimetic set up.3 2D IR spectroscopy is an odd-ordered
experiment, and is therefore not surface-specific. However, it is possible to generate surface
sensitivity, which is necessary to study the small number of molecule in a single bilayer.41 This is
commonly done by utilizing plasmon enhancement, similar to surface-enhanced Raman
spectroscopy (SERS) and surface-enhanced infrared spectroscopy (SEIRAS). There are several
methods of plasmon enhancement, including nanoarrays of various geometries to create specific
wavelengths of plasmonic resonance in a region of interest.42–46 Our method uses a thin, rough
gold film with small islands of gold on the surface. Between the islands are ‘hot spots’ which are
created when light is highly confined in the gaps, resulting in a higher concentration of the electric
field.41 The peak plasmon frequency is in the visible range, but extends into the mid-IR (vide infra).
For our experiments, this non-resonant enhancement leads to smaller enhancements than a
resonant enhancement method like SERS, but is still on the order of 1000x.8 Thicker gold red-
shifts the plasmon frequency, thus modulating the enhancement. However, thicker gold leads to
lineshape distortions, so a
compromise must be struck
between enhancement and spectral
quality.9
Recent work in the Zanni
and Fayer groups have further
manipulated the spectrometer
geometry to yield further surface
enhancements.8,47 In a BOXCARs
geometry, which is fully non-
collinear, the intensity of the local
oscillator (LO), which is overlaid
with the signal electric field to
produce the heterodyned signal, can
be independently modulated in
order to reduce the background.4
Due to the technical challenges of
collecting purely absorptive spectra
in this geometry, a pump-probe
geometry, which is partially
collinear and self-heterodyned (i.e.
the probe pulse acts as the LO), is popularly employed. However, due to the self-heterodyned
nature of the pump-probe geometry, the LO intensity is tied to the probe intensity and directly
influences the signal strength. To combat this, we took advantage of the fundamental qualities of
different polarizations of light as described in the Fresnel equations. For p-polarized light, there is
Figure 2. Reflectivity of s- and p- polarized light at a CaF2-air
interface, where Brewster’s angle is where the p-polarized light has
zero reflectivity.
an angle (Brewster’s angle) at which when it hits an interface, no light is reflected as shown in
Figure 2. At near-Brewster’s angle reflection geometries, therefore, it is possible to excite the
sample with the probe beam, but attenuate the LO strength such that the background is suppressed,
effectively enhancing the signal in the reflected direction. This was first demonstrated by the Fayer
group, and later applied to biological samples in strongly absorbing solvents in my first year. This
method itself gives a 4x enhancement that can be combined with localized surface plasmons for
enhancements on the scale of 104.8
Research Progress
I have developed a method for surface-enhanced 2D IR spectroscopy using a nanometers-thin,
thermally-evaporated layer of rough gold on a calcium fluoride window to enhance the signal from
a lipid bilayer. I have also demonstrated the ability to not only see signals from a model peptide,
but also changes in the peptide caused by changes in its environment.
A single monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPTE) was
created by incubating a solution of the DPTE in ethanol with a 2 nm thick gold-coated window in
order to form a gold-sulfur bond, tethering the head group of the lipid to the window.48 Vesicles
of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) were allowed to fuse to the monolayer
of DPTE. The window was washed thoroughly to leave a single bilayer behind. Spectra of both
the DPTE and the mixed-lipid bilayer show the success of this technique seen in Figure 3. The
peaks arise from the ester carbonyls which link the head group to the tails.49 The presence of
multiple peaks is likely due to the difference in the two tails of POPC and interactions with the
gold. Furthermore, it is important to note that there are no substantial signals arising in the amide
I region, which is vital in being able to take peptide spectra without background correction.
To determine if we could observe a peptide embedded in the membrane, alamethicin (alm),
a 20-residue model peptaibol, was chosen to test our experimental methods. Alm associates with
the membrane, and has both α- and 310-helical character.50,51al A change of voltage or pH causes
alm to form bundles and insert itself into a membrane, initiating its antimicrobial activity.48,50,52–55
The alm was deposited onto the membrane at a 10:1 lipid:peptide ratio. The sample was dried then
rehydrated with D2O at a neutral or basic pH. 2D IR spectra of this sample were then taken
demonstrating the sensitivity of our methods as seen in figure 4. These spectra are consistent with
previous linear IR and SFG spectra taken of alm. We attribute the 1670 cm-1 peak to the α-helical
half of the peptide. The 1635 cm-1 peak arises from the 310-helix, consistent with both SFG spectra
of alm and 2D IR spectra of 310-helices.52,56
Figure 3. Representative 2D IR spectra of the ester carbonyl region for A) A monolayer of DPTE and B) a
single bilayer of DPTE and fused POPC vesicles both tethered to 2-nm of thermally evaporated gold on a
CaF2 window in D2O.
A B
Pu
mp
Fre
qu
ency
(cm
-1)
Pu
mp
Fre
qu
ency
(cm
-1)
Probe Frequency (cm-1
) Probe Frequency (cm
-1)
The differences between the pH 6.8
and the pH 12 spectra demonstrate our
sensitivity to the change of environment that
the amide backbone is experiencing. There
are evident shifts between the relative
intensities of the 1670 cm-1 and the 1630 cm-
1 peaks. This can be explained by a change in
angle as the peptide inserts into the membrane
or due to peptide-peptide interations.48,52,57,58
In this system, membrane-peptide
interactions that are created can also
influence the amide I frequency as the
bundles are formed. We can also consider
possible changes in secondary structure. For
example, the alm is destabilized when it
forms a 310-helix, causing it to insert into the
membrane in order to stabilize the structure.
Most importantly, these results gave us the
confidence to move beyond small peptides.
Being able to detect these structural changes
with the small amount of sample is
encouraging as we move to larger systems
where we will need to detect changes in a
single isotope label.
In order to test our abilities to run alm
as a voltage-gated experiment, several
modifications were needed. First, the thin
gold-coated substrates were not
macroscopically conductive, despite being
thicker than the percolation threshold
(thickness needed for conductivity). This
required us to determine what thickness of
gold yielded a good signal without lineshape distortions previously observed when adding a large
amount of plasmonic material to a CaF2 window.9 Thicknesses of gold between 2 and 10 nm of
gold were tested for both conductivity and signal ‘quality’ with alamethicin. Conductance was
determined by measuring the resistance across approximately one centimeter of the coated window
with a multimeter. The standard of reasonable conductance of the electrode was taken to be less
than 50-kΩ of resistance, as previously noted.9 It was determined that 8 nm of thermally evaporated
gold was the minimum thickness that resulted in an appropriate resistance, and spectra were taken
using 10 nm and 8 nm windows.
Figure 4. Representative 2D IR spectra of the amide I
region for A) alamethicin at pH 6.8 B) alamethicin at pH
12 both deposited on a single DPTE/POPC bialyer
tethered to 2-nm of thermally evaporated gold on a CaF2
window in D2O.
A
B
A new sample cell was also needed to perform these experiments to allow for the
connection of electronics to the sample. To this end, I designed and 3D printed several iterations
of a spectro-electrochemical cell (Figure 5a). The cell is made of plastic and is therefore safer to
use than our conductive metal sample cells. We use conductive aluminum tape to both connect the
gold (working electrode) to the potentiometer and act as a counter electrode (Figure 5b). Since we
are focused on placing a constant potential over a membrane, we are not currently concerned about
having a reference electrode. Due to evaporation issues in losing solution over the course of
averaging, a flow cell design was also implemented so that liquid could be replenished over the
course of the experiment.
Some promising voltage-dependent data were taken with a 10 nm gold-coated window, but
were not replicable. This was due to generally a large negative offset in signal that washed out the
peptide signal, perhaps due to the presence of hot carriers. This signal was not seen in a control
experiment at a negative waiting time (t2) where the probe pulse arrives before the pump pulse,
indicating that it is not scatter. Other experimental set ups attempted included 8 nm of gold in both
a transmission and reflection geometry, 10 microns of gold in a reflection geometry, and 10 nm of
palladium topped with 2 nm of gold. In the successful preliminary experiment, spectra of the alm
on the lipid bilayer in a KCl/D2O buffer were taken at 0 and ±900 mV. The 0-mV spectrum was
subtracted from each the -900- and +900-mV spectra in order to both eliminate the non-resonant
response of the thick gold and act as a background subtraction shown in figure 6. This yielded
difference spectra qualitatively similar to the basic pH spectrum.
Figure 5. A) Designed spectro-electrochemical flow cell for the voltage-dependent 2D IR studies. The slot
visible at the top of the window is for inserting the conductive aluminum tape. B) Proposed electrochemical
experiment set up with alamethicin on a DPTE/POPC fused bilayer tethered to a thin gold film on a CaF2
window. Space between the two CaF2 windows is created by a 56-μm spacer.
A B
Due to
aforementioned
difficulties, we
are attempting to
create a more
reproducible
window as an
electrode.
Towards this
end, our
collaborator in
the Arnold group
is beginning to
make substrates
with a 5 nm layer
of spin-coated
then annealed
indium tin oxide
(ITO) under 3
nm of thermally
evaporated gold.
This may work
better due to the lack of plasmon from the ITO so we are able to use the thickness of the gold we
found to be best for the plasmonic enhancement of monolayers. We are still observing a negative
response from the material, but have been able to subtract it out to see an alm signal by a peptide-
free spectrum from a peptide spectrum.
While working
on this new
experimental set up, we
have also been
collecting surface-
enhanced spectra of
KcsA to clarify the
mechanism of ion
permeation. As before,
we prepared these
samples by creating a
monolayer of DPTE on
a calcium fluoride
window coated with 3
nm of gold. Buffered
KcsA in vesicles were
deposited, allowed to
form into a bilayer, then
gently washed to
eliminate any stacked
bilayers. They were then
exchanged with D2O 50
times to eliminate any
Figure 7. Representative 2D IR spectra of
the amide I region for A) unlabeled KcsA
B) Val76, Gly 77, and Gly79 labelled
KcsA C) a zoom of the unlabeled spectrum
subtracted from the labeled spectrum to
leave just the isotope labels. The two
samples were deposited on the same DPTE
coated window with a 3-nm gold thermally
evaporated film, hydrated in D2O with a 12
μm spacer.
A
B
B
C
Figure 6. Spectra of alamethicin in a tethered
lipid bilayer on 10 nm thermally evaporated
Au at A) 0V B) -900mV and C) the 0V
spectrum subtracted from the -900 mV
spectrum, yielding qualitative similarities to
the basic pH spectrum.
Pu
mp
Fre
qu
ency
(cm
-1)
Pu
mp
Fre
qu
ency
(cm
-1)
Pu
mp
Fre
qu
ency
(cm
-1)
Pu
mp
Fre
qu
ency
(cm
-1)
Pu
mp
Fre
qu
ency
(cm
-1)
Pu
mp
Fre
qu
ency
(cm
-1)
Probe Frequency (cm-1
) Probe Frequency (cm-1
)
Probe Frequency (cm-1
)
Probe Frequency (cm-1
) Probe Frequency (cm-1
)
Probe Frequency (cm-1
)
A B
C
H2O from appearing in the amide I region as described in previously. The spots are then rehydrated
in D2O. Preliminary spectra have shown consistency with the work of previous work in the Zanni
group as shown in figure 7. Furthermore, we are able to see isotope labels by using a subtraction
scheme where an unlabeled spectrum is subtracted from an isotope labelled spectrum by
normalizing to the α-helical peak.1 This allows the bulk of the amide I region to be eliminated,
making the isotope labels visible. We are now prepared to take spectra of the newly labelled KcsA
as detailed in my future plans.
Future Plans
Finish work with alamethicin for publication – In the next few months we will finish work on the
alm project. Once we have replicable voltage data, we will write a paper on the static voltage
technique. I simply need to finalize the flow cell set up and to find a suitable combination of
conductive materials to form the electrode on the CaF2 window. I will then take the voltage data,
compare the results both to our own pH-modulated data, and SFG data from other groups. This
will pave the way for more complex systems by serving as a strong proof of concept experiment.
KcsA mechanism validation – As we finish the work with alamethicin, we will transition to KcsA.
We want to add additional constraints to the existing models by adding data from new isotope
labelling schemes in the selectivity filter. With our surface-enhanced method, we believe that we
will be able to observe single isotope labels, rather than the multi-label scheme employed in
previous work. We can then observe Val76 and Gly77 individually in samples prepared with native
protein ligation by our collaborators as detailed in the SI. These sites are of significant interest
after modeling work done by the group of Jim Skinner at the University of Chicago (private
correspondence). They modeled both the hard- and soft-knock models including both the flipped
and unflipped states of Val76. They then took the trajectories and used them to generate weighted
2D IR spectra of each individual label as explained in the SI. The models yielded readily
distinguishable spectra. In both cases, the soft-knock mechanism yielded an inhomogenously
broadened features between 1570 and 1590 cm-1, while the hard knock mechanism had narrower
peaks with maxima between 1550 and 1570 cm-1. The differences in peak width is especially
apparent in the case of Gly77, and examples of the different types of broadening are showing in
figure S4. Experiments with the suggested labels will definitively clarify the mechanism for KcsA,
which serves as the model for K+ channels. Furthermore, our data benefits computational models
that can use this data to further constrain averaged spectra.
Many models of the mechanism of K+ permeation through KcsA has included a 50 mV
drop across the bilayer.18 Using the static voltage methods I am working to develop, we plan to
repeat our KcsA experiments with an applied voltage. This will allow us to rectify any differences
between our experimental results and competing models by providing data using the parameters
of the simulated data. Again, this is a case of more data contributing both to a better understanding
of biological processes and also providing data with which to test simulations and models.
Transient Voltage-Jump experiment – Finally, I will work to develop a 2D IR voltage-jump (V-
jump) experiment. The end goal is to be able to bridge the transient ion current data currently
collected from patch clamp experiments and the static structural data gleaned from
crystallography, taking
advantage of the
structural and temporal
resolution of 2D IR
spectroscopy. This will
entail creating a new
pulsed experiment
where we will create a
voltage using an
arbitrary wave
generator (AWG)
triggered off of the
laser. Using a function
generator, we are able
to control both the amplitude and duration of the applied potential. We are writing a code to collect
2D IR spectra at different voltage waiting times (Tv) after the laser pulse (Figure 8), similar to
temperature-jump experiments. This will allow us to observe the structural dynamics of voltage-
gating in real time.
There are two processes that we plan to observe using this method. First, we will be able
to observe the ion permeation through the channel by using isotope labels in the selectivity filter
of an ion channel and again comparing to models. This will allow us to compare the mechanism
we inferred through our static experiments with what is happening in real time. Second, we will
be able to observe allosteric processes that occur in the more complex voltage-gated K+ channel,
KvAP. KvAP uses an arginine-rich S4 helix to sense a potential drop.59–62 The change in voltage
elicits a yet-to-be-determined physical result in the paddle. There are a few existing theories: the
sliding helix (or canonical) model where the S4 helix moves out of the membrane and turns, the
paddle model where the angle of the voltage sensing domain changes and the twisted S4 model
where a kink in the center of the helix causes the two helical segments to move separately.63 Using
the V-jump technique, we will be able to observe the movement of the voltage paddle. By
comparing these results with modeled spectra, we will then be able to determine the correct
mechanism of gating. We could, also, determine if the movement of the voltage paddle is the rate
determining step, or if there is a currently unknown structural rearrangement between the paddle
moving and the pore opening that determines the response time of the opening of the ion channel.
This can easily be extracted by determining if the movement of the paddle is synchronous with the
pore opening (making it the rate determining step) or before (indicating an unknown middle step).
This is only the start of what could be asked by the V-jump experiment.
Figure 8. Proposed pulse sequence for the transient voltage experiments. After zero
potential, a potential will quickly be applied. Then at a delay time, tv a 2D IR
spectrum will be taken. After averaging the 2D IR spectrum at the first value of tv,
the delay time will change, allowing for spectra taken at several points after the
applied voltage in order to measure the structural response to the potential.
Supporting Information:
2D IR spectroscopy: Two-dimensional infrared spectroscopy measures the third-order response
function of a sample as a result of three light-matter interactions exciting the vibrational transitions
of a molecule. The methods of collecting the spectra vary, but all use an identical pulse sequence
(figure S1) where three pulses of
light interact with a sample to create
a signal. The signal can then be
heterodyned by either with the final
probe pulse or with a separate local
oscillator. The first pulse puts the
sample in a coherence state and it
stays there during the coherence
time, t1. The second pulse then
brings it into a population state that
evolves during the population time, t2. Finally, the third pulse will cause a coherence between the
ground and first excited state or the first and second excited states, stimulating a combined ground
state bleach and stimulated emission or excited state absorption signals respectively.4
The direction of this signal depends on the phase matching geometry utilized. The Zanni
group uses a pump-probe geometry in which the first two pump pulses are collinear and spatially
offset from the third, pump, pulse resulting in a signal that is emitted in the same direction as the
probe. This set up is convenient for a few reasons. First, the signal is self-heterodyned in that we
use the probe pulse as our local oscillator to extract the phase information from the sample.
Secondly, the rephasing and non-rephasing signals are emitted in the same direction, allowing us
to cut down on data collection time and yielding purely absorptive data directly.64,65
The two pump pulses are created by using a germanium acousto-optical amplifier (Ge-
AOM). Briefly, this is used as a pulse shaper to turn our pump pulse into two, phase-controlled
pump pulses separated by different time delays. The incoming pulse is first Fourier transformed
from the time to frequency domain with a grating. An acoustic wave, or mask, is applied to the Ge
crystal, acting as a diffraction grating in the frequency domain for the incoming light. After the
Ge-AOM, a second grating is used to return the pulse to the time-domain, yielding the double
pump pulse.25,64 The Ge-AOM can be modulated on a shot-to-shot basis.66 We generate our spectra
in the time-frequency domain such that the pump axis is generated by scanning the delay t1 and
performing a Fourier transform, while the probe axis is collected by the dispersed frequencies on
an MCT array.
The interference between the electric fields of the signal and the local oscillator contains
the information in this experimental set up. The signal in the frequency domain is
𝑆 ∝ ∫ |(𝐸𝐿𝑂(𝜔) + 𝐸𝑠𝑖𝑔(3)(𝜔))|
2
𝑑𝜔 ≈ 𝐸𝐿𝑂2 (𝜔) + 𝐸𝑠𝑖𝑔
(3)2(𝜔) + 2𝐸𝐿𝑂(𝜔)𝐸𝑠𝑖𝑔
(3)(𝜔)
where 𝐸𝐿𝑂2 (𝜔) is the intensity of the pump pulse (local oscillator), 𝐸𝑠𝑖𝑔
(3)2(𝜔) is the intensity of the
signal, and the 2𝐸𝐿𝑂(𝜔)𝐸𝑠𝑖𝑔(3)(𝜔) cross term is the signal of interest. We are able to isolate this
cross term again by using the pulse shaper. Every other pulse takes a spectrum as described, while
the intermittent pulse turns ‘off’ the pump by using the Ge-AOM as a chopper. As a result, we
have spectra that are just the local oscillator by itself. We can then subtract off the local oscillator
as is done is absorption spectroscopy where the signal is approximately
𝑆 = log (𝑝𝑢𝑚𝑝 𝑜𝑛
𝑝𝑢𝑚𝑝 𝑜𝑓𝑓) ≈ log (
𝐸𝐿𝑂2 (𝜔)𝐸𝑠𝑖𝑔
(3)2(𝜔) + 2𝐸𝐿𝑂(𝜔)𝐸𝑠𝑖𝑔
(3)(𝜔)
𝐸𝐿𝑂2 (𝜔)
) ≈ 2𝐸𝐿𝑂(𝜔)𝐸𝑠𝑖𝑔
(3)(𝜔)
𝐸𝐿𝑂2 (𝜔)
Figure S1. Pulse sequence for a 2D IR experiment.
Eq. S1
Eq. S2
The ability to modulate the pump on and off is clearly very useful in data collection. In total, the
use of the pulse shaper aids in spectra collection, allows us to reduce pump scatter through phase
cycling, and allows for faster data collection.4
Sample Preparation: Alamethicin derived from Trichoderma viride purchased from Sigma-Aldrich
was diluted to a concentration of 2 mg/mL in methanol for storage purposes. Lipids, 1-palmitoyl-
2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-Dipalmitoyl-sn-Glycero-3-
Phosphothioethanol (DPTE) (Figure S2) both in chloroform at a concentration of 10 mg/mL were
purchased from Avanti Polar Lipids Inc. Calcium fluoride (CaF2) windows (25 mm x 2 mm) were
purchased from Crystran.
Gold films (Au% > 99.99%) were deposited via thermal evaporation to varying thicknesses
at the rate of 0.1 Å/s at a pressure of 8 x 10-7 torr onto the CaF2 window. Films of indium tin oxide
(ITO) and gold were created first by sputter-coating the window with 5 nm of ITO and annealing
at 350 °C for 1.5 hours. The gold was then deposited to the desired thickness as described. They
layered metal (Pd/Au) samples were created by first thermally evaporating the palladium, then
adding the gold on top separately.
A 100-μL aliquot of DPTE in
chloroform (10 mg/mL) and 250-μL
aliquot of POPC in chloroform (10
mg/mL) were dried down under N2 and
lyophilized overnight to remove
remaining solvent. DPTE was rehydrated
in 10 mL of spectroscopic-grade ethanol.
The coated window was then submerged
in the DPTE/ethanol solution overnight.
The window was then rinsed with ethanol
and allowed to dry. For alamethicin
samples, POPC vesicles were prepared by
rehydrating the POPC in 100 mM NaCl buffer and applying five freeze-thaw cycles in isopropanol
and dry ice and lukewarm water respectively. The solution was then placed in a bath sonicator for
10 minutes to create approximately 50-nm diameter vesicles. Window was then submerged in the
POPC vesicle solution overnight to allow for vesicle rupture. The window was then washed with
deionized water. Alamethicin was prepared by diluting 5.2 μL of the stock solution in 50 μL of
deionized water. This was then distributed over half of the lipid-coated CaF2 window. Assuming
total surface coverage of lipid, this should create a 20:1 lipid:peptide ratio. The window was dried
under N2 and then rehydrated with D2O or phosphate buffer (pH 6, 12) in D2O and a second
window was added separated with a 56 μm spacer.
For the KcsA samples, the DPTE monolayer was created in the same fashion. KcsA (1.5
mg/mL) in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (0.25% w/v) and 50 mM Tris,
150 mM KCl, and 1 mM DTT buffer (pH 7.5), both those that were unlabeled and labelled, were
deposited in separated 0.2-μL spots on the same window, and gently rinsed to remove excess
sample. The spots were then exchanged 50 times with D2O by adding 0.2 μL at a time and allowing
it to dry under N2. The spots were then rehydrated with 0.2 μL of D2O and placed between two
CaF2 windows with a 12-μm spacer.
2D IR data acquisition: A Coherent Libra regenerative amplifier centered at 800 nm (3.4 W, 50 fs)
pump an optical parametric amplifier (OPA) (Light Conversion TOPAS). The output signal and
idler pulses are then mixed in a AgGaS2 difference frequency generation (DFG) crystal to generate
the mid-IR light centered at 6 μm with pulses of <100 fs with energies around 18 μJ. The mid-IR
Figure S2. The structures of the lipids
A
B
is then split with a 90/10 CaF2 beam splitter into the pump and probe lines. The pump line is
directed through an acousto-optical modulator (AOM) which shapes the pulses as previously
described. The polarization (s- or p-) of both the pump and probe lines are modulated by successive
half-wave plates and polarizers. A 7.5-cm focal length parabolic mirror is used to focus the
spatially and temporally overlapped pulses at the sample. Due to the pump-probe geometry used,
the probe/signal beam is then directed into a monochromator to be dispersed onto a 32-pixel MCT
array.
MD Simulations:
The route from MD simulation to spectra is outlined briefly here.4,67 Spectra are derived from MD
simulations by taking the frequency trajectory of the vibration of interest, in our case the isotope-
labelled backbone carbonyl, and from the trajectory determine the distribution of frequencies. This
distribution will yield a frequency fluctuation correlation function, which can be used to determine
the lineshape function. The lineshape function is key in creating the response function. Then, the
response functions for the rephasing and non-rephasing signals can be treated like collected time-
domain data, and Fourier transformed and summed to create the purely absorptive spectra. Because
2D IR spectra take an ensemble average of the sample of interest, it is necessary to take into
account the different ion occupancies of selectivity filter. This was done by taking the trajectories
of each of the possible ion occupancies for both models individually and determine the resulting
spectra. These spectra were then combined in a weighted average to create the final modeled
spectra.
Figure S4. Modeled spectra with A) homogenous broadening B) partially inhomogeneous broadening C)
inhomogeneous broadening.
A B C
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