The I27RS8 Protein as a Reference Protein for Force Extension Technical Brief
Sheldon E. Broedel, Jr., Ph. D.
Athena Environmental Sciences, Inc., Baltimore , MD
Atomic force microscopy (AFM) is a remarkably simple instrument. Originally
used for measuring surface contours of individual molecules, it has provided
important information about the structure of proteins. In the force measuring
mode, AFM is capable of measuring forces down to piconewtons and can resolve
force changes caused by the displacement of its probe by a fraction of a nanometer.
Single molecule measurements are routinely done (for reviews see references 2, 3).
Since measurements can be made on many individual molecules in a short period of
time, a statistical evaluation of the measurements can be performed. Perhaps the
most powerful aspect of AFM is that the measurements can be done in an aqueous
environment, allowing for the study of biological material under conditions that
resemble those in vivo. The folding and unforlding of the protein can be studied
in the presence of substrate, product, and co-factors. The temperature can be
controlled as can the buffer conditions, i.e., ionic strength, pH, solvents, etc.
The mechanical unraveling of proteins using atomic force microscopy is a
powerful tool for the study of protein structure. Single molecules stretched
between the AFM probe and stage result in force-extension recording which provide
information about the stability and kinetics of protein and protein domain unfolding.
AFM in a force extension mode offers several advantages over thermal or chemical
denaturation to the study of protein structure. Until recently, the only way
to measure the stability of a protein was to change its physical (e.g., with
heat or high pressure) or chemical environment (e.g., with guanidinium chloride
or urea, acidic or alkaline pH) and monitor the loss of protein conformation,
using spectroscopic techniques from which folding free energies could be obtained.
AFM allows the measurement of folding reactions at the single molecule level
and therefore can uncover rare unfolding events that are not observed by using
traditional bulk methods, in which measured parameters are averaged over many
molecules. It allows a direct measurement of the mechanical stability of the fold.
The process of mechanical unfolding is therefore defined as a function of extension
(here the reaction coordinate is well defined, i.e., X-ray, crystallography),
force spectroscopy is dynamic and it works in real time. AFM works in aqueous
solution, so crystal formation is not required. Thus, force extension using AFM
promises to elucidate the dynamic structural properties of a wide variety of proteins
at the single molecule level and provide an important complement to other structural
and dynamic techniques (e.g., X-ray, crystallography, NMR, spectroscopy).
When working with a protein with unknown unfolding properties, it is helpful
to employ a reference protein as a means of calibrating the AFM instrumentation.
The I27RS8 engineered polyprotein is ideal for this purpose. I27
RS8 is composed of eight direct repeats of the Ig module 27 of the human
titin protein. The construction and characterization of the polyprotein has been
previously described by Dr. Julio Fernandez and his colleagues.4 In this paper,
the properties of the proein are reviewed in the context of using it as a reference
in AFM analysis.
Force Extension Properties of I27RS8
Forced unfolding of the polyprotein
The force-extension relationships for the I27RS8 polyprotein were
measured using a single axis atomic force microscope. Single proteins were fully
extended by retracting the sample holder away from the AFM tip. Stretching the I27RS8
polyprotein resulted in a force extension curve with peaks that varied randomly
in amplitude with an average value of ~200pN (Fig. 1). A histogram of force peaks
(unfolding force) measured from 266 unfolding events revealed an asymmetrical
distribution of events that had a maximum at ~200pN (Fig. 1C). Hence, the only
variations in the force required to unfold were stochastical, consistent with the
idea that during stretching, the tandem I27 modules unfold independently from each
other. The force extension curves of I27RS8 are well described by
the worm-like chain model (WLC) which predicts the entropic restoring force
generated upon the extension of a polymer (Fig. 1B, thin lines). Fits of the WLC
to the force-extension curves of I27RS8 gave a persistence length of
p = 0.39 ± 0.07nm (n=10). The persistence length is a measure of the distance
over which the polymer retains memory of a direction. It is significant that the
persistence length is of the size of a single amino acid. Consecutive peaks were
fitted by the WLC with the same persistence length and a contour length increment
of Δlc = 28.4 ± 0.3nm (Fig. 1B, thin lines; n=16). The increase
in contour length of a module upon unfolding is 28.4nm, which is approximately
equal to the expected length of 89 fully extended amino acids minus the folded
length of the module ([89 x 0.38nm] - 4nm = 29.8nm).
Figure 1. Force-extension relationships for
recombinant poly-I27 measured with AFM techniques: A) Sawtooth pattern of unfolding observed with the I27LG11
polyprotein. B) Stretching of single I27RS8 polyproteins gave
force-extension curves with a sawtooth pattern with equally spaced force peaks. The
sawtooth pattern is well described by the WLC equation (B, bottom trace continuous
lines). C) Unfolding force frequency histogram for I27RS8. The
filled circles correspond to a Monte Carlo simulation of the mean unfolding forces
(n = 1,000) of eight domains placed in series using an unfolding rate constant,
ku0, of 3.3 x 10-3s-1,
an unfolding distance, Δxu of 0.25nm and a V = 0.6nm/ms.
AFM measurements of the unfolding rate constant
In order to simulate a chain of modules placed under force we have used Monte
Carlo techniques and compiled the force required to unfold a domain during the simulated
extensions. The force applied to the modules was calculated from the WLC model of
elasticity and varied depending on the contour length and the total extension of
the protein.6,7 The unfolding and refolding of the modules was assumed to be a simple two
state reaction with rate constants that depend exponentially on the applied force.6, 7
We found that an unfolding rate at zero force of ku0 = 3.3 x 10-4s-1
and an unfolding distance Δxu = 0.25nm was required in order to fit the
distribution of unfolding forces measured from the I27 polyprotein (Fig. 1B).
An independent estimate is obtained by fitting a Monte Carlo simulation to the
dependency of the average force of unfolding on the rate of pulling by the AFM.
Experiments where the polyprotein was pulled at rates varying over the range 0.01 - 10nm/ms
are shown in Figure 2. The average force of unfolding was shown to depend on the pulling
rate. This dependency was reproduced by a Monte Carlo simulation using ku0 = 3.3 x 10-4s-1
and Δxu = 0.25nm. Thus, by two independent AFM experiments, we found that the
transition state for unfolding was reached after an extension of only 2.5Å and at
an unfolding rate constant at 0 force of 3.3 x 10-4s-1.
Figure 2. Stretching
single I27RS8 proteins at different pulling speeds.
A): i) 0.05nm/ms, ii) 0.5nm/ms, and iii) 5nm/ms. The average unfolding forces (continuous
lines are 116pN, 224pN, and 272pN, respectively. B) Dependence of the unfolding forces
on the pulling speed. Each symbol (squares) is the average of (from left to right)
16, 19, 16, 266, 21, 21, 9 data points. The solid line corresponds to the result of a
Monte Carlo simulation. The best fit was obtained with ku0 =
3.3 x 10-4s-1 and a Δxu = 0.25nm.
Estimation of the folding rate constant
A double pulse experiment designed to measure the folding rate of the I27RS8
polyprotein is illustrated in Figure 3. A first extension of the polyprotein allows counting
the available folded domains. After reaching the extended state, the protein was
relaxed to its initial length. After a variable time period the protein was stretched
again and a variable number of force peaks were observed. We interpret these results
as an indication that some of the domains unfolded in the first extension of the protein
had spontaneously refolded upon relaxation. The number of refolded domains counted this way
was found to depend exponentially on the amount of time that the protein remained relaxed
(Fig. 3). For a simple two-state model for the folding reaction, when a protein module
is relaxed to its original length and there is no applied force, then kf(F) =
kf0>>ku(F) = ku0 ~0, resulting in
a simple first order folding reaction where the folding probability is given by Pf
(t) = 1-e-tk0f. This function describes well the folding data of Figure 3B with
kf0 = 1.2s-1.
Figure 3. A) Unfolding and refolding cycles of an I27RS8 protein probed with a
double-pulse protocol (inset). The protein is first stretched to count the number of domains
that unfold, Ntotal, (a-c, top traces), and then it is relaxed to its initial
length. A second extension after a delay, Δt, measures the number of refolded domains,
Nrefolded (a-c, bottom traces). B) Plot of the refolded fraction, Nrefolded/Ntotal
versus Δt. The solid line is a fit of the data (squares) to the function, Pf(t) = 1 -
e-tk0f, where kf0 = 1.2s-1.
The filled circles correspond to a Monte Carlo simulation of a two state folding/unfolding
kinetic model using a folding rate constant, kf0 = 1.2s-1.
Using I27RS8 as a Reference Protein in Force Extension Measurements
The consistency of measurements with the I27RS8 polyprotein makes it an ideal
reference protein for calibrating and controlling force extension experiments. Because the
polyprotein is designed with a homogenous eight domain structure, it permits the measurement
of extensions across a single molecule. Further, the polyprotein has proved to be robust
allowing measurements to be made on multiple molecules in a single session as well as across
multiple experiments. For example, the distance between the eight force peaks of 64 individual
molecules was 24 ± 0.34nm (CV of 1.4%).4 In eight separate experiments on a
total of 266 molecules, the force peaks were measured at 204 ± 26 pN (CV of 12.7%).4
The protein is currently available from Athena Enzyme Systems (Baltimore, MD, Cat. No. 0304)
in a ready-to-use format under the trade name I27O. The protein is prepared for extension
measurements by diluting the stock protein solution to a concentration of 100µg/mL in PBS
pH 7.3 buffer and applying the sample to freshly evaporated gold coverslips. The coverslips
are then attached to the piezoelectric positioner stage. I27RS8 was engineered
with an amino-terminal hex-His peptide sequence and carboxy-terminal di-Cys. This configuration
is hypothesized to allow the protein molecules to attach to the coverslip at the carboxy-terminal
leaving the amino-terminus free to attach to the silicon nitride cantilever tip, when the tip is
brought in close proximity to the absorbed protein. It is this model which is believed
to account for the high frequency (>50%) of observing all eight domain extensions
after each retraction as well as the ability to perform extension and retraction
experiments on a single molecule.
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