Lab: Single-Molecule Kinetics — PKA–ATP Binding

In this lab, we will walk through the process of:

Loading and visualizing real single-molecule experimental data from a carbon nanotube sensor.
Building a current histogram and fitting it with a double-Gaussian model to test whether binding is a single-step or multi-step process.
Extracting dwell times from pre-detected switching events and computing first-order rate constants.
Connecting microscopic kinetics to macroscopic thermodynamics ( $K_\text{eq}$ , $\Delta G$ ).

Data source: Coroneus et al., PKA–ATP binding measured via carbon nanotube field-effect transistor.

# Run this cell to import important packages for this lab
import numpy as np
import matplotlib.pyplot as plt
from matplotlib.gridspec import GridSpec
from scipy.optimize import curve_fit

1. Introduction¶

In a typical chemistry experiment, you measure the behaviour of trillions of molecules at once — an ensemble measurement. The result is an average: you learn the mean binding rate, but not what any individual molecule is doing.

In this lab, we analyse data from a single-molecule experiment. A single Protein Kinase A (PKA) enzyme is attached to a carbon nanotube transistor. When an ATP molecule binds to PKA, the device current jumps up; when ATP unbinds, the current drops back down. We can watch these events one at a time.

              ATP binds                ATP unbinds
  State 0 ──────────────▶ State 1 ──────────────▶ State 0
  (low I)     k₀ (s⁻¹)    (high I)    k₁ (s⁻¹)    (low I)

Symbol	Meaning
$I_0$ (State 0)	PKA alone — ATP unbound (low current)
$I_1$ (State 1)	PKA·ATP complex — ATP bound (high current)
$k_0$	Rate constant for leaving State 0 (binding)
$k_1$	Rate constant for leaving State 1 (unbinding)

Our data file covers a 2-second window (153–155 s) sampled at 100 kHz — that’s 200,000 current measurements. The data has been lightly smoothed (20-point moving average), so we can jump straight into analysis.

Question: Why might watching a single molecule reveal information that an ensemble measurement cannot? Think about what happens when you average over trillions of molecules.

Type your answer here, replacing this text.

2. Loading the Data¶

Our data is stored in a TDMS file (a LabVIEW format). We use the nptdms library to read it. The file contains three channels:

CleanSwitch — the smoothed current trace (200,000 samples)
EventState — pre-detected state labels (0 or 1 for each event)
EventDuration — duration of each event in samples

Run the following cell to load the data.

# Run this cell
%pip install nptdms
from nptdms import TdmsFile

DATA_PATH = "Data/Time 153 to 155s State 2 Smooth 20 Supp 15.tdms"
tdms = TdmsFile.read(DATA_PATH)

# Show file structure
for group in tdms.groups():
    print(f"Group: '{group.name}'")
    for ch in group.channels():
        print(f"  Channel: '{ch.name}'  —  {len(ch)} samples")
        for prop in ch.properties:
            print(f"    {prop}: {ch.properties[prop]}")

Now let’s extract the three channels and convert to convenient units.

# Run this cell
group = tdms["Switching analysis"]

current_raw   = group["CleanSwitch"][:]     # Current trace in Amps
event_state   = group["EventState"][:]      # 0 or 1 per event
event_dur     = group["EventDuration"][:]   # Duration in samples

# Physical parameters
sampling_rate = 100_000                     # 100 kHz
dt = 1.0 / sampling_rate                   # 10 µs per sample

# Convert current to nanoamps for readability
current_nA = current_raw * 1e9

# Time axis in milliseconds
time_ms = np.arange(len(current_nA)) * dt * 1000

# Valid events (first 66 per metadata)
n_events = 66
states    = event_state[:n_events].astype(int)
durations = event_dur[:n_events]

print(f"Current trace : {len(current_nA):,} samples  ({time_ms[-1]/1000:.1f} s)")
print(f"Current range : {current_nA.min():.2f} – {current_nA.max():.2f} nA")
print(f"Valid events  : {n_events}")
print(f"  State 0 events: {np.sum(states == 0)}")
print(f"  State 1 events: {np.sum(states == 1)}")

Question: The current trace has 200,000 samples covering 2 seconds. What is the time resolution (time between consecutive samples)? Why might such high time resolution be necessary for this experiment?

Type your answer here, replacing this text.

3. Visualizing the Switching Trace¶

Before doing any analysis, let’s look at the data. We’ll plot the current trace at three different zoom levels.

# Run this cell
fig, axes = plt.subplots(3, 1, figsize=(12, 9))

# (a) Full 2-second trace
axes[0].plot(time_ms, current_nA, lw=0.3, color='steelblue')
axes[0].set_title("(a)  Full 2-second trace")
axes[0].set_ylabel("Current (nA)")
axes[0].set_xlim(time_ms[0], time_ms[-1])

# (b) First 200 ms
mask_b = time_ms <= 200
axes[1].plot(time_ms[mask_b], current_nA[mask_b], lw=0.4, color='steelblue')
axes[1].set_title("(b)  First 200 ms — individual events become visible")
axes[1].set_ylabel("Current (nA)")
axes[1].set_xlim(0, 200)

# (c) First 50 ms
mask_c = time_ms <= 50
axes[2].plot(time_ms[mask_c], current_nA[mask_c], lw=0.6, color='steelblue')
axes[2].set_title("(c)  First 50 ms — clear two-level switching")
axes[2].set_ylabel("Current (nA)")
axes[2].set_xlabel("Time (ms)")
axes[2].set_xlim(0, 50)

for ax in axes:
    ax.set_ylim(current_nA.min() - 0.5, current_nA.max() + 0.5)
    ax.grid(True, alpha=0.3)

fig.tight_layout()
plt.show()

Question: Describe what you see. How many distinct current levels are there? Do the switching events appear periodic (regular) or random? What does this tell you about the underlying process?

Type your answer here, replacing this text.

4. Current Distribution and the Gaussian Test¶

This is the central analysis of the lab. We will:

Histogram every current sample in the 2-second trace.
Fit the distribution with a double Gaussian.
Extract the width $\sigma$ of each peak.

Why does this matter?¶

The current fluctuates around each level because of thermal noise — random jostling of atoms and electrons at room temperature. Statistical mechanics (via the Central Limit Theorem) tells us that if the molecular process is a single-step reaction, these thermal fluctuations produce a Gaussian distribution:

I \sim \mathcal{N}(\mu,\,\sigma^2)

(1)

If instead there are hidden intermediate states (a multi-step process), the distribution deviates from Gaussian — it becomes broader, skewed, or develops shoulders.

The Gaussian test: Narrow, symmetric Gaussians → single-step process. Non-Gaussian tails → evidence for hidden intermediate steps.

The PKA–ATP system has already been characterised as single-step, so we expect clean Gaussians. Let’s verify.

4a. Building the Histogram¶

Run the cell below to build a histogram of all 200,000 current values.

# Run this cell
n_bins = 400
counts, bin_edges = np.histogram(current_nA, bins=n_bins)
bin_centres = 0.5 * (bin_edges[:-1] + bin_edges[1:])

fig, ax = plt.subplots(figsize=(10, 4))
ax.bar(bin_centres, counts, width=np.diff(bin_edges)[0],
       color='steelblue', alpha=0.7, edgecolor='none')
ax.set_xlabel("Current (nA)")
ax.set_ylabel("Number of samples")
ax.set_title("Current histogram — all 200,000 samples")
ax.grid(True, alpha=0.3)
plt.tight_layout()
plt.show()

You should see two sharp peaks — one for each conductance state. Because the data was smoothed (20-point filter), the noise around each level is very small, making the peaks appear as narrow spikes. The Gaussian shape becomes visible when we zoom in.

4b. Fitting the Double Gaussian¶

We model the histogram as the sum of two Gaussians:

f(I) = A_0 \exp\!\left(-\frac{(I - \mu_0)^2}{2\sigma_0^2}\right) + A_1 \exp\!\left(-\frac{(I - \mu_1)^2}{2\sigma_1^2}\right)

(2)

where $\mu_0, \mu_1$ are the peak centres, $\sigma_0, \sigma_1$ are the widths, and $A_0, A_1$ are the amplitudes.

# Run this cell — define the model and estimate initial parameters

def gaussian(x, A, mu, sigma):
    """Single Gaussian peak."""
    return A * np.exp(-0.5 * ((x - mu) / sigma)**2)

def double_gaussian(x, A0, mu0, sigma0, A1, mu1, sigma1):
    """Sum of two Gaussians."""
    return gaussian(x, A0, mu0, sigma0) + gaussian(x, A1, mu1, sigma1)

# --- Initial parameter estimates ---
# Split data at the midpoint and find the mode of each half.
I_mid = (current_nA.min() + current_nA.max()) / 2.0
samples_lo = current_nA[current_nA < I_mid]
samples_hi = current_nA[current_nA >= I_mid]

# Peak centres from sub-histograms
counts_lo, edges_lo = np.histogram(samples_lo, bins=100)
centres_lo = 0.5 * (edges_lo[:-1] + edges_lo[1:])
mu0_est = centres_lo[np.argmax(counts_lo)]

counts_hi, edges_hi = np.histogram(samples_hi, bins=100)
centres_hi = 0.5 * (edges_hi[:-1] + edges_hi[1:])
mu1_est = centres_hi[np.argmax(counts_hi)]

# Peak amplitudes from main histogram
A0_est = counts[np.argmin(np.abs(bin_centres - mu0_est))]
A1_est = counts[np.argmin(np.abs(bin_centres - mu1_est))]

# Sigma from samples near each peak
near0 = samples_lo[np.abs(samples_lo - mu0_est) < 1.0]
near1 = samples_hi[np.abs(samples_hi - mu1_est) < 1.0]
sig0_est = max(np.std(near0) if len(near0) > 10 else 0.1, 0.01)
sig1_est = max(np.std(near1) if len(near1) > 10 else 0.1, 0.01)

p0 = [A0_est, mu0_est, sig0_est, A1_est, mu1_est, sig1_est]

print("Initial estimates:")
print(f"  State 0:  A₀ = {A0_est:.0f},  μ₀ = {mu0_est:.2f} nA,  σ₀ = {sig0_est:.4f} nA")
print(f"  State 1:  A₁ = {A1_est:.0f},  μ₁ = {mu1_est:.2f} nA,  σ₁ = {sig1_est:.4f} nA")

# Run this cell — perform the fit
bounds_lo = [0, mu0_est - 2, 0.001, 0, mu1_est - 2, 0.001]
bounds_hi = [np.inf, mu0_est + 2, 5.0, np.inf, mu1_est + 2, 5.0]

# Ensure starting values are within bounds
p0[2] = np.clip(p0[2], 0.002, 4.9)
p0[5] = np.clip(p0[5], 0.002, 4.9)

popt, pcov = curve_fit(double_gaussian, bin_centres, counts,
                       p0=p0, bounds=(bounds_lo, bounds_hi), maxfev=20000)

A0, mu0, sigma0, A1, mu1, sigma1 = popt
perr = np.sqrt(np.diag(pcov))

print("=" * 50)
print("     DOUBLE-GAUSSIAN FIT RESULTS")
print("=" * 50)
print(f"  State 0 (ATP unbound)")
print(f"    μ₀  = {mu0:.3f} ± {perr[1]:.4f} nA")
print(f"    σ₀  = {sigma0:.4f} ± {perr[2]:.4f} nA  ◀ Gaussian width")
print(f"    A₀  = {A0:.0f}")
print()
print(f"  State 1 (ATP bound)")
print(f"    μ₁  = {mu1:.3f} ± {perr[4]:.4f} nA")
print(f"    σ₁  = {sigma1:.4f} ± {perr[5]:.4f} nA  ◀ Gaussian width")
print(f"    A₁  = {A1:.0f}")
print()
print(f"  Current separation: Δμ = {abs(mu1 - mu0):.2f} nA")
print(f"  Signal-to-noise: Δμ / (σ₀ + σ₁) = {abs(mu1-mu0)/(sigma0+sigma1):.1f}")
print("=" * 50)

4c. Visualizing the Fit¶

The full-range histogram shows two sharp spikes because the smoothed data has very little noise. To see the actual Gaussian bell-curve shapes, the inset panels zoom in on each peak individually (cf. Figure 2b/2c insets in the original paper).

# Run this cell — paper-style figure with inset zoom panels
fig, (ax_trace, ax_hist) = plt.subplots(
    1, 2, figsize=(14, 6), sharey=True,
    gridspec_kw={'width_ratios': [3, 1.2], 'wspace': 0.03}
)

# --- Left: time trace ---
mask_zoom = time_ms <= 200
ax_trace.plot(time_ms[mask_zoom], current_nA[mask_zoom], lw=0.4, color='steelblue')
ax_trace.axhline(mu0, color='#2196F3', ls='--', lw=1, alpha=0.5)
ax_trace.axhline(mu1, color='#F44336', ls='--', lw=1, alpha=0.5)
ax_trace.set_xlabel("Time (ms)")
ax_trace.set_ylabel("Current (nA)")
ax_trace.set_title("(a) Current trace (0–200 ms)")
ax_trace.set_xlim(0, 200)
ax_trace.grid(True, alpha=0.3)

# --- Right: sideways histogram (full range) ---
n_fine = 800
counts_fine, edges_fine = np.histogram(current_nA, bins=n_fine)
centres_fine = 0.5 * (edges_fine[:-1] + edges_fine[1:])
scale = n_fine / n_bins

ax_hist.barh(centres_fine, counts_fine, height=np.diff(edges_fine)[0],
             color='grey', edgecolor='none', alpha=0.6)
ax_hist.set_xlabel("Counts")
ax_hist.tick_params(labelleft=False)
ax_hist.set_title("(b) Distribution")
ax_hist.grid(True, alpha=0.3)

# --- Inset: zoom State 0 (bottom) ---
pad0 = max(10 * sigma0, 0.05)
samples_near0 = current_nA[(current_nA > mu0 - pad0) & (current_nA < mu0 + pad0)]
c0_local, e0_local = np.histogram(samples_near0, bins=60)
ctr0_local = 0.5 * (e0_local[:-1] + e0_local[1:])

inset0 = ax_hist.inset_axes([0.4, 0.03, 0.55, 0.25])
inset0.barh(ctr0_local, c0_local, height=np.diff(e0_local)[0],
            color='#BBDEFB', edgecolor='none')
y0 = np.linspace(mu0 - pad0, mu0 + pad0, 500)
local_scale0 = len(samples_near0) * np.diff(e0_local)[0] / (sigma0 * np.sqrt(2 * np.pi))
inset0.plot(gaussian(y0, local_scale0, mu0, sigma0), y0, '#2196F3', lw=2)
inset0.set_title(f'σ₀ = {sigma0:.4f} nA', fontsize=8, color='#2196F3', pad=2)
inset0.tick_params(labelsize=6)
inset0.set_ylim(mu0 - pad0, mu0 + pad0)

# --- Inset: zoom State 1 (top, lowered to avoid bar) ---
pad1 = max(10 * sigma1, 0.05)
samples_near1 = current_nA[(current_nA > mu1 - pad1) & (current_nA < mu1 + pad1)]
c1_local, e1_local = np.histogram(samples_near1, bins=60)
ctr1_local = 0.5 * (e1_local[:-1] + e1_local[1:])

inset1 = ax_hist.inset_axes([0.4, 0.55, 0.55, 0.25])
inset1.barh(ctr1_local, c1_local, height=np.diff(e1_local)[0],
            color='#FFCDD2', edgecolor='none')
y1 = np.linspace(mu1 - pad1, mu1 + pad1, 500)
local_scale1 = len(samples_near1) * np.diff(e1_local)[0] / (sigma1 * np.sqrt(2 * np.pi))
inset1.plot(gaussian(y1, local_scale1, mu1, sigma1), y1, '#F44336', lw=2)
inset1.set_title(f'σ₁ = {sigma1:.4f} nA', fontsize=8, color='#F44336', pad=2)
inset1.tick_params(labelsize=6)
inset1.set_ylim(mu1 - pad1, mu1 + pad1)

fig.suptitle("PKA–ATP Single-Molecule Switching — cf. Figure 2b/2c", fontsize=13)
fig.tight_layout()
plt.show()

4d. Interpreting the Results¶

Both peaks are well-described by Gaussians. The widths ( $\sigma_0$ and $\sigma_1$ ) are extremely small compared to the peak separation (~8 nA), giving an excellent signal-to-noise ratio.

Key result: The Gaussian shape of both peaks confirms that PKA–ATP binding and unbinding are each single-step processes with no hidden intermediates. This is a direct application of the Central Limit Theorem: many small, independent thermal fluctuations produce a Gaussian distribution. Any deviation would signal a multi-step mechanism.

Question: The data was smoothed with a 20-point moving average, which reduced the noise (and therefore $\sigma$ ). If we had used the raw (unsmoothed) data, would you expect $\sigma$ to be larger or smaller? Would the Gaussian test still work? Why or why not?

Type your answer here, replacing this text.

5. Dwell Time Analysis and Rate Constants¶

Every time the molecule switches state, it stays in the new state for a random length of time — the dwell time $\tau$ . For a first-order (single-step, memoryless) process, dwell times follow an exponential distribution:

P(\tau) = k\, e^{-k\tau}

(3)

where $k$ is the rate constant for leaving that state. The mean dwell time is simply $\langle\tau\rangle = 1/k$ .

The pre-analysed event list in our data file gives us the state identity and duration of each event.

Run the cell below to extract dwell times and compute statistics.

# Run this cell
dwell_ms_0 = durations[states == 0] / sampling_rate * 1000  # ms
dwell_ms_1 = durations[states == 1] / sampling_rate * 1000  # ms

print(f"State 0 (unbound) — {len(dwell_ms_0)} events")
print(f"  Mean dwell time  : {np.mean(dwell_ms_0):.2f} ms")
print(f"  Median           : {np.median(dwell_ms_0):.2f} ms")
print(f"  Std dev          : {np.std(dwell_ms_0):.2f} ms")
print(f"  Range            : {np.min(dwell_ms_0):.2f} – {np.max(dwell_ms_0):.2f} ms")
print()
print(f"State 1 (bound) — {len(dwell_ms_1)} events")
print(f"  Mean dwell time  : {np.mean(dwell_ms_1):.2f} ms")
print(f"  Median           : {np.median(dwell_ms_1):.2f} ms")
print(f"  Std dev          : {np.std(dwell_ms_1):.2f} ms")
print(f"  Range            : {np.min(dwell_ms_1):.2f} – {np.max(dwell_ms_1):.2f} ms")

Question: For an exponential distribution, the mean and the standard deviation are both equal to $1/k$ . Compare the mean and std dev for each state. Are they approximately equal? What does this tell you about whether the process is truly first-order?

Type your answer here, replacing this text.

5a. Rate Constants¶

TO-DO: Use the mean dwell times to calculate rate constants. Recall that $k = 1/\langle\tau\rangle$ . Be careful with units — dwell times are in milliseconds, but rate constants should be in $\text{s}^{-1}$ .

# TO-DO: Calculate rate constants from mean dwell times
tau0 = ...  # mean dwell time for State 0 in ms
tau1 = ...  # mean dwell time for State 1 in ms

k0 = ...    # binding rate in s⁻¹ (hint: 1000 / tau0)
k1 = ...    # unbinding rate in s⁻¹

print(f"τ₀ = {tau0:.2f} ms  →  k₀ = {k0:.1f} s⁻¹  (binding)")
print(f"τ₁ = {tau1:.2f} ms  →  k₁ = {k1:.1f} s⁻¹  (unbinding)")

5b. Dwell Time Distributions¶

Run the cell below to visualise the dwell time distributions. On a log scale, an exponential distribution appears as a straight line.

# Run this cell
fig, axes = plt.subplots(2, 2, figsize=(12, 8))

bins0 = np.linspace(0, np.max(dwell_ms_0) * 1.1, 15)
bins1 = np.linspace(0, np.max(dwell_ms_1) * 1.1, 15)

# (a) State 0 — linear
axes[0, 0].hist(dwell_ms_0, bins=bins0, color='#2196F3', edgecolor='white', alpha=0.85)
axes[0, 0].axvline(tau0, color='navy', ls='--', lw=2, label=f'⟨τ₀⟩ = {tau0:.2f} ms')
axes[0, 0].set_title("(a) State 0 dwell times (linear)")
axes[0, 0].set_xlabel("Dwell time (ms)")
axes[0, 0].set_ylabel("Count")
axes[0, 0].legend()
axes[0, 0].grid(True, alpha=0.3)

# (b) State 1 — linear
axes[0, 1].hist(dwell_ms_1, bins=bins1, color='#F44336', edgecolor='white', alpha=0.85)
axes[0, 1].axvline(tau1, color='darkred', ls='--', lw=2, label=f'⟨τ₁⟩ = {tau1:.2f} ms')
axes[0, 1].set_title("(b) State 1 dwell times (linear)")
axes[0, 1].set_xlabel("Dwell time (ms)")
axes[0, 1].set_ylabel("Count")
axes[0, 1].legend()
axes[0, 1].grid(True, alpha=0.3)

# (c) State 0 — log scale
axes[1, 0].hist(dwell_ms_0, bins=bins0, color='#2196F3', edgecolor='white', alpha=0.85)
axes[1, 0].set_yscale('log')
axes[1, 0].set_title("(c) State 0 dwell times (log scale)")
axes[1, 0].set_xlabel("Dwell time (ms)")
axes[1, 0].set_ylabel("Count (log)")
axes[1, 0].set_ylim(bottom=0.5)
axes[1, 0].grid(True, alpha=0.3)

# (d) State 1 — log scale
axes[1, 1].hist(dwell_ms_1, bins=bins1, color='#F44336', edgecolor='white', alpha=0.85)
axes[1, 1].set_yscale('log')
axes[1, 1].set_title("(d) State 1 dwell times (log scale)")
axes[1, 1].set_xlabel("Dwell time (ms)")
axes[1, 1].set_ylabel("Count (log)")
axes[1, 1].set_ylim(bottom=0.5)
axes[1, 1].grid(True, alpha=0.3)

fig.tight_layout()
plt.show()

Question: Look at the log-scale plots (c and d). Do they look roughly like straight lines? With only ~33 events per state, we have limited statistics — but is the overall shape consistent with an exponential distribution?

Type your answer here, replacing this text.

6. From Kinetics to Thermodynamics¶

Single-molecule rate constants connect directly to macroscopic thermodynamics.

Equilibrium constant:

K_\text{eq} = \frac{k_0}{k_1}

(4)

This ratio tells us how much time the molecule spends bound vs. unbound.

Free energy difference:

\Delta G = -RT \ln K_\text{eq}

(5)

where $R = 8.314\;\text{J mol}^{-1}\text{K}^{-1}$ and $T = 298\;\text{K}$ .

State occupancy (fraction of time in each state):

f_1 = \frac{k_0}{k_0 + k_1}, \qquad f_0 = 1 - f_1

(6)

TO-DO: Use your rate constants to calculate $K_\text{eq}$ , $\Delta G$ , and the state occupancies. Then compare the occupancies from the kinetic formula to the observed occupancies from total dwell times.

# TO-DO: Thermodynamic analysis
R = 8.314   # J / (mol·K)
T = 298.0   # K

K_eq = ...       # k0 / k1
DeltaG = ...     # -R * T * np.log(K_eq), in J/mol
DeltaG_kJ = ...  # convert to kJ/mol

# State occupancy from rate constants
f1_kinetic = ...  # k0 / (k0 + k1)
f0_kinetic = ...  # 1 - f1_kinetic

# State occupancy from observed total dwell times
total_samples_0 = np.sum(durations[states == 0])
total_samples_1 = np.sum(durations[states == 1])
f0_obs = total_samples_0 / (total_samples_0 + total_samples_1)
f1_obs = total_samples_1 / (total_samples_0 + total_samples_1)

print("=" * 50)
print("        THERMODYNAMICS")
print("=" * 50)
print(f"  K_eq  = {K_eq:.2f}")
print(f"  ΔG    = {DeltaG_kJ:.2f} kJ/mol")
print()
print(f"  State occupancy (from kinetics):")
print(f"    f₀ (unbound) = {f0_kinetic*100:.1f}%")
print(f"    f₁ (bound)   = {f1_kinetic*100:.1f}%")
print()
print(f"  State occupancy (observed):")
print(f"    f₀ (unbound) = {f0_obs*100:.1f}%")
print(f"    f₁ (bound)   = {f1_obs*100:.1f}%")
print("=" * 50)

Question: Do the kinetic and observed occupancies agree? If $K_\text{eq} > 1$ , which state is thermodynamically favoured — bound or unbound? Is $\Delta G$ positive or negative, and what does that mean physically?

Type your answer here, replacing this text.

Question: We extracted a macroscopic quantity ( $\Delta G$ ) from watching a single molecule. A bulk experiment on trillions of PKA molecules would also yield a $\Delta G$ . Should the two values agree? Why or why not?

Type your answer here, replacing this text.

7. Summary¶

TO-DO: Fill in the summary table below with your results from Sections 4–6.

Quantity	Symbol	Units
State 0 current	$\mu_0$	nA
State 1 current	$\mu_1$	nA
State 0 Gaussian width	$\sigma_0$	nA
State 1 Gaussian width	$\sigma_1$	nA
State 0 mean dwell time	$\tau_0$	ms
State 1 mean dwell time	$\tau_1$	ms
Binding rate	$k_0$	s $^{-1}$
Unbinding rate	$k_1$	s $^{-1}$
Equilibrium constant	$K_\text{eq}$	—
Free energy	$\Delta G$	kJ/mol

Discussion Questions¶

The Gaussian test showed clean Gaussians for both peaks. What would you expect the current distribution to look like if ATP binding were a two-step process with a short-lived intermediate?
The switching events are random — no two dwell times are the same. Why can’t thermal fluctuations “average out” to produce a constant dwell time? (Hint: think about the memoryless property.)
If the experiment were cooled from 298 K to 77 K (liquid nitrogen), how would you expect $\sigma$ to change? What about the rate constants $k_0$ and $k_1$ ?
We observed ~66 events in 2 seconds. If you wanted to measure the rate constants with 10× better precision, approximately how much more data would you need? (Hint: standard error scales as $1/\sqrt{N}$ .)

References¶

Coroneus et al., single-molecule dynamics of Protein Kinase A — see Figure 2b/2c.
Thermal energy at 298 K: $k_B T \approx 4.11 \; \text{pN} \cdot \text{nm} = 2.48 \; \text{kJ/mol}$ .

Congratulations! You’ve finished the Single-Molecule Kinetics Lab.¶

Developed by: Jacob Quisumbing, in collaboration with Prof. John Coroneus.