2005-04-04540B
Supplementary Information to “Nanospring behavior of ankyrin repeats”
Gwangrog Lee1, Khadar Abdi2, Yong Jiang1, Peter Michaely3, Vann Bennett2, and Piotr E.
Marszalek1
1
Department of Mechanical Engineering and Materials Science and Center for
Biologically Inspired Materials and Material Systems, Duke University, Durham, NC
27708, USA
2
Howard Hughes Medical Institute and Department of Cell Biology, Duke University
Medical Center, Durham, NC 27708, USA
3
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas,
Texas 75390, USA
1.
Purification and characterization of ankyrin-B polypeptides
DNA encoding human ankyrin-B with ankyrin repeats 1-24 (bp 1-2877, aa 1-959), or 1324 (bp 1279-2877, aa 427-959) both with 100 amino acids of the spectrin-binding
domain, were cloned into a modified pGex6p1 vector (Amhersham Pharmacia)
engineered to contain 7 C-terminal histidine residues. Both constructs were sequenced
and were free of any mutations. Polypeptides were expressed in BL21-Gold (D3) pLysS
(Stratagene) and purified from lysates by sequential affinity chromatography on high
performance Ni-sepharose (Amhersham Pharmacia) and glutathione-sepharose
(Amhersham Pharmacia). Final purification was achieved by cation exchange
1
chromatography on a Mono S HR 5/5 column (Amhersham Pharmacia). GST was
removed in some experiments with GST-precision protease (Amhersham Pharmacia)
followed by adsorption with glutathione-sepharose. Ankyrin-B (repeats 1-24) was
properly folded and monomeric based on circular dichroism spectroscopy and calculation
of molecular weitght from hydrodynamic measurements (see below).
2.
Physical Properties of 24 Ankyrin-B repeats
Methods. Hydrodynamic values were evaluated for the 24 repeat ankyrin-GST using gel
filtration analysis and sedimentation on sucrose gradients. Briefly, gel filtrations were
carried out on a Superose 6 column standardized with catalase (Rs = 5.22 nm), aldolase
(Rs = 4.81 nm), bovine serum albumin (Rs = 3.55 nm), oval bumin (Rs = 3.05 nm), and
ribonuclease A (Rs = 1.64 nm). Sedimentation coefficients were determined through ratezonal sedimentation on gradients of 5-20% sucrose using a SW 50.1 rotor spun at 50K
rpm for 12 hours. The standards used for sedimentation are catalase (11.3 S), aldolase
(7.3 S), bovine serum albumin (4.6 S), and oval bumin (3.5 S).
Results. The molecular weight in solution of the 24 repeat ankyrin-GST was calculated
using values from gel filtration and sedimentation coefficients. Gel filtration analysis
reveals that the protein has a stokes radius of 4.8 nanometers while sedimentation on a
sucrose gradient shows that the protein has a sedimentation coefficient of 7.3 S. This
gives the protein a calculated molecular weight in solution of 140, 074 Da confirming
that the protein is a monomer in solution (Supplementary Table 1).
2
Supplementary Table 1
Structural Properties of the 24 ANK Membrane-Binding Domain GST
Properties
Values
Sedimentation coefficient, S20,w (a)
Partial specific volume, v (b)
Stokes radius, Rs (c)
MW, calculated (d)
MW, actual (e)
Frictional ratio, f/f0 (f)
(a)
(b)
(c)
(d)
7.3 s
0.716 cm3/g
4.8 nm
140, 074 Da
130, 240 Da
1.35
From sucrose gradient sedimentation
Estimated from the amino acid composition
Determined from gel filtration on a calibrated Superose 12 column
MW (molecular weight) Calculated from the equation below
MW = (6NRsS20,w)/ (1-vp20,w)
(Eq. 1)
(e) Calculated from the amino acid sequence
(f) Calculated from the equation below


f/f0 = Rs ( (43MW (v+

The molecular weight in solution of the 24 repeat ankyrin with GST was calculated using
values from gel filtration and sedimentation coefficients. Gel filtration analysis reveals
that the protein has stokes radius of 4.8 nanometers while sedimentation on a sucrose
gradient shows that the protein has a sedimentation coefficient of 7.3 S. This gives the
protein a calculated molecular weight in solution of 140, 074 Da revealing that the
protein is a monomer in solution.
3
3. Circular dichroism
Methods. The 24 repeat ankyrin-GST protein was dialyzed in buffer containing 0.010 M
Na phosphate and 0.1 M NaF pH 7.4. The dialyzed protein at 0.15 mg/ml concentration
was loaded onto a 0.1 millimeter path-length far UV quartz cuvette (Hellma). Circular
dichroism spectra were analyzed at room temperature between wavelengths of 260 and
200 nanometers on an Aviv 62 DS model circular dichroism spectrometer.
Results. Using circular dichroism we show that the 24 repeat ankyrin-GST exhibits a
strong alpha helical profile with double minima at wavelengths of 208 and 220
nanometers (Fig. S1). The signal profile is presented in mean molar ellipticity. The
crystal structure of the 12 repeats in ankyrinR reveals that a single ankyrin repeat is
composed of two anti-parallel alpha helices. As predicted the stack of 24 repeats in our
protein generates a structure with a strong alpha helical profile confirming that our
protein used in AFM measurements has the proper secondary structure fold.
Fig. S1. Circular dichroism spectra shows that the 24
repeat ankyrin -GST exhibits a strong alpha helical profile.
4
4.
AFM imaging
Methods. Imaging was performed with a Nanoscope IIIa MultiModeTM Scanning Probe
Microscope (Veeco Instruments Inc., Santa Barbara, CA) using the Tapping Mode in the
buffer fluid. NP-S probes (Veeco Instruments Inc.) with spring constants of 0.32 N/m
and resonance frequencies of 8-10 kHz were used.
A 30 µl protein solution was
incubated on a freshly-cleaved mica surface at room temperature for 10 min in the same
buffer that was used in the pulling experiments. All images were collected at a scan rate
of 2.0 Hz, with 512x512 pixels, and scan sizes ranging from 200 to 1000 nm.
Results. The atomic structure of 12 ankyrin-R repeats suggested that ankyrin stacks
composed of n>=24 repeats should form a full superhelical turn with putative spring
properties, however such structures have never been observed. We used an AFM to
visualize individual stacks of 24 ankyrin-B repeats. Figure S2 shows a series of six AFM
images revealing the hook-like appearance of ankyrin similar to the shape that was
originally proposed for the extrapolated structure of the ankyrin-R membrane-binding
domain. The molecules’ end-to-end distance, measured from the AFM images (arrows in
Fig. S2) is 12.9±1.2 nm (mean± s.d., n=7) which is close to the ~12 nm determined for
the extrapolated structure. Thus, the AFM images strongly suggest that the engineered
protein, bearing at its terminal a GST module, is correctly folded and does not aggregate.
5
13.7
nm
11.8 nm
12.4
nm
14.7 nm
13.5 nm
12.9 nm
11.3 nm
Fig. 1
Fig. S2. AFM images of individual 24 ankyrin-B repeats. The
images, obtained in solution with the Tapping Mode technique
display an error signal. The arrows indicate the end-to-end
distance of ankyrin.
5.
Principles of Single Molecule Force Spectroscopy by AFM
Figure S3 describes the force-measuring mode of the AFM, used by us and others to
study the elasticity of single biopolymers. The sample, covered by a drop of solution, is
attached to the piezoelectric positioner and moved up to contact with the AFM tip in
order to allow the unbound parts of the molecules to adsorb also to the tip. In this way the
molecules form bridges between the substrate and the tip. Usually, multiple bridges are
formed when the tip penetrates the polymer brush, but by lowering the surface density of
the adsorbed molecules it is possible to greatly limit the number of molecules adsorbed to
the tip. These molecules can be stretched in a controlled manner by moving the substrate
away from the tip and relaxed by moving the substrate back. The stretched molecule
exerts a mechanical force on the cantilever producing its deflection that is a direct
6
measure of the force stretching the molecule (molecule tension). The experimental result
is a force-extension curve (force spectrogram) that reflects the elasticity of a single
molecule and reveals structural changes that may be induced by the external force. This
method relies on the ability of most of
biopolymers to adhere nonspecifically to
x
=  zp -  zc
the AFM tip and frequently allows
F = k c z c
 zc
generating forces exceeding one
1
piezo
nanonewton, before the molecules
detach from the tip or the substrate. The
 zp
2
extension, x, of a molecule being
Fig. S3. A schematic illustrating AFM
measurements of the elasticity of a
single-molecule.
stretched in the AFM is defined as its
end-to-end distance and is determined
from the separation between the AFM tip and the substrate as shown in Fig. S3. This
separation is calculated as the difference between the travel of the piezo from the
reference position, ZP, and the bending of the cantilever ZC; x= ZP - ZC, while the
force experienced by the molecule, and measured by the bending of the cantilever is
calculated as F=kC ZC , where kC, is the spring constant of the cantilever (needs to be
calibrated). The zero extension is at the reference position of the piezo where the
substrate contacts the cantilever as evidenced by a dramatic change in the slope of the
force extension curve that becomes vertical (e.g. see Fig. S4b; S5a,b and Fig. 1b, main
text). The uncertainty of zero extension is of the order of 1-2 nm, because of adhesive
interactions between the tip and the substrate (stretching trace) and “the cantilever
jumping to contact” phenomenon (relaxing trace). The zero force is defined by the
7
photodiode signal that is produced by the light reflected off of the relaxed cantilever (no
bending, ZC=0).
600
AFM recordings on multiple vs
single intact ankyrin molecules
Figure S4a shows an example of an AFM
stretch-release recording that is frequently
Force / pN
6.
500
a
400
300
200
100
0
obtained on heptahistitine-tagged 24
0
ankyrin-B repeats attached to the substrate
presence of imidazole, which abolished
Force / pN
an AFM recording obtained in the
100
150
200
250
Extension / nm
600
bearing the metal chelate N-nitrilotriacetic acid (NTA) and Fig. S4b shows
50
b
400
200
0
the binding of ankyrin to the substrate.
0
20
40
60
80
100 120
Extension / nm
With no imidazole in the buffer (Fig. S4a),
Fig. S4. a, A typical force-extension
pattern obtained in AFM stretching
measurements on ankyrin in the
presence of strong adhesive interactions
and multi-molecular bridges between the
tip and the substrate. b, A typical
recording obtained in the presence of
0.5 M imidazole shows that the binding
of ankyrin to NTA is abolished.
multiple and irregularly spaced force
peaks reporting the rupture of adhesive
interactions between the AFM tip and the
substrate and the detachment and/or
unfolding of several ankyrin molecules
create complex patterns that vary from one recording to another and are very difficult to
interpret. In ~5 % of all the events the adhesive interactions are however minimal and
occasionally one ankyrin molecule happens to adsorb to the tip upon contacting the
8
substrate (Fig. S5a,b; Fig. 1b-e, main text). This is quite possible because the surface
density of ankyrin molecules in these experiments is quite low (cf. Fig. S2, AFM images
obtained under similar protein concentration conditions). Such infrequent events produce
much simpler force curves than the curve
Force / pN
shown in Fig. S4a. These rare force curves
display similar patterns between various
recordings and this consistency between the
300
a
200
100
0
recordings (see Fig. S5 a,b and Fig. 1b-e,
0
main text) allows one to assume that they
10 20 30 40 50
Extension / nm
have been obtained on single molecules.
400
Force / pN
When selecting recordings to
analyze it is necessary to develop a set of
consistent rules that will help to minimize
b
300
200
100
0
-100
the number of mistaken cases where more
-2
than one molecule has been stretched at a
0
2
4
6
8
10
Extension / nm
time. It is also necessary to exercise an
Fig. S5. Examples of singlemolecule force-extension recordings
obtained on ankyrin-B repeats. a,
The force curve obtained on 12
ankyrin repeats + GST. b, The force
curve obtained on 24 repeats with
no GST. Stretching force curve
(blue), relaxing force curve (red)
approach free of a bias that could result in
selecting only those recordings that fit
one’s expectations. In the specific case of
ankyrin we did not know what kind of force
extension curves we should expect as there is no relevant experimental data in the
literature. Therefore we did not have any pre-existing bias in selecting the data. We were
struck by the observation that the recordings with minimal adhesion showed a very
9
pronounced linear part at the beginning of the stretch that we have never observed for
other molecules (Fig. S5a,b; Fig. 1b-e, main text). This was a unique, reproducible and
consistent feature that one needs in single molecule force spectroscopy for selecting the
data. Our additional criteria for selecting single-molecule recordings included: (a) a
requirement that the length of the straighten molecule should not exceed 30 nm which
eliminates recordings obtained on unraveled molecules, and (b) that the linear part should
be followed by a single force peak which (c) should occur at the extension <=30 nm, as
multiple force peaks would suggest multiple molecules.
7.
Additional evidence that force-extension curves of ankyrin were measured on
individual molecules.
In single molecule force spectroscopy there is rarely an absolute certainty that a given
measurement has been performed on one molecule. However, all the recordings that we
considered as obtained from single and intact ankyrin stacks revealed the same unique
and persistent features suggesting that they indeed were obtained on individual stacks.
Additional evidence in support of our claim is shown in Figure S6 (which originated from
Fig. 1d, main text) and in Fig. 2b and c (main text). In Fig. S6a the stretch recording
captured the linear part, which was followed by the rising force curve, which culminated
in the unfolding force peak. The relaxing trace (Fig. S6b) captured a number of small
force peaks that we hypothesize correspond to the refolding of individual repeats from
the stack. In Fig. S6c we superimposed this refolding trace on our template unfolding
recording (Fig. 2a, main text). Both traces overlap reasonably well suggesting that they
indeed report the unfolding and refolding behavior of very similar structures, namely
individual ankyrin repeats. Thus, we argue that since the relaxing trace is indicative of the
10
mechanical refolding behavior of individual ankyrin repeats then the stretching trace
must have been obtained on a single ankyrin stack. Therefore, the linear elasticity comes
from a single ankyrin stack. However, the strongest and most direct evidence that the
features captured in our AFM recordings on ankyrin, including its unusual linear
elasticity, indeed represents the properties of individual molecules is shown in Fig. 2b
and c of the main text. The force-extension curves displayed in these figures were each
captured in single pull measurements. They clearly reveal the linear elasticity of the stack
followed by its breakdown at a high force, which in turn is followed by the unraveling of
individual repeats as evidenced by a series of regularly spaced small force peaks.
Significantly, these small force peaks overlap well with the small force peaks of our
template unfolding recording (Fig. 2a) strongly suggesting that all these force peaks
represent the unfolding of similar structures, namely individual ankyrin repeats. The fact
that all three characteristic events were captured in a single pull experiment very strongly
supports our conjecture that in these and other measurements we indeed stretched single
ankyrin molecules.
11
100
c
250
10
20
b
Refolding
40
30
20
0
-20
60
Force / pN
300
0
0
Force / pN
a
Stretching
Force / pN
Force / pN
200
200
150
40
20
0
100
100
120
140
160
Extension / nm
50
0
-50
0
10
20
30
0
20 40 60 80 100 120 140
Extension / nm
Extension / nm
Fig. S6. Stretching and refolding traces from Fig. 1d, main text, at a higher
magnification. a, Stretching plot. b,Refolding trace. c, Refolding trace
superimposed on the template unfolding trace from Fig. 2a (main text).
8.
Refolding of individual ankyrin repeats and refolding of the stack
8a. Refolding of ankyrin repeats
When investigating the unfolding/refolding behavior of ankyrin repeats, we used
another criterion for selecting recordings, which we believed were obtained on single
ankyrin molecules. Namely, we looked for a fingerprint of small equally spaced force
peaks that appeared to be very consistent between various recordings. Here we relaxed
our requirement that the force curves must show the linear part, as most of these
measurements were carried out on molecules that were already partially unfolded in the
process of picking them up with the AFM tip for force spectroscopy measurements. Here
we used a strategy that is a common practice in single-molecule force spectroscopy,
12
allowing, with a much higher frequency to single out one molecule from a number of
molecules that initially adsorbed to the tip, by a careful manipulation of the
piezo/substrate position relative to the AFM cantilever. This forces other molecules to
detach, until only one molecule stays attached to the tip for repeated measurements.
However, in this procedure the molecule of interest typically becomes at least partially
unfolded. These molecules would not qualify as intact stacks for tertiary-structure
elasticity measurements but they still produce consistent results in the unfolding/refolding
measurements.
13
1000
a
800
Force / pN
Force / pN
1000
600
400
200
0
c
800
600
400
200
0
0
20
40
60
80
0
Extension / nm
40
60
80
Extension / nm
150
1000
b
800
Force / pN
Force / pN
20
600
400
200
d
125
100
75
50
25
0
0
0
20
40
60
0
80
20
40
60
80
Extension / nm
Extension / nm
Fig. S7. Individual ankyrin molecules are isolated for repeated single-molecule
unfolding/refolding measurements by rupturing of the adhesion bond and a
controlled detachment of shorter ankyrin fragments.
Figure S7 shows a sequence of AFM recordings which were obtained using this
approach. The first recording revealed a strong adhesive interaction and several ankyrin
molecules between the AFM tip and the substrate (Fig. S7a, blue trace). However, by
carefully manipulating the position of the substrate relative to the cantilever tip, which is
achieved by moving the piezo actuator away from the AFM cantilever in small steps, it
was possible to rupture the adhesion bond and to detach most of the attached ankyrin
molecules (Fig. S7b) so eventually only one molecule remained attached between the tip
and the substrate (Fig. S7c). Isolated in this way, a single ankyrin fragment was stretched
from its initial extension of 8.6 nm, at a force of 115 pN, to the extension of 25.5 nm
14
where it broke down at a force of 916 pN (Fig. S7c, blue trace). After this event the
molecule remained attached to the tip as evidenced by the rest of the stretching trace
(blue) and the relaxing trace (red). This breakdown released ~40 nm of the polypeptide
chain which is equivalent to the unfolding of ~ 3 ankyrin repeats. When the relaxing
measurement finished and the piezo returned to its starting position, it was separated from
the cantilever by 17 nm. The unfolded molecule was long enough (L > 60 nm) to be fully
relaxed at the end of this measurement (F=0 pN). The next recording (Fig. S7d) revealed
small force peaks during the stretching (blue trace) and also during the relaxing of the
molecule (red trace). These force peaks suggest the unfolding and refolding of the same
three repeats that have been released from the stack during the major unfolding event
captured in Fig. S7c. The recording shown in Fig. S7d is actually the same as shown in
the main text as Fig. 3a, and is the first in the series of stress-release cycles, which
captured the refolding of individual ankyrin repeats. We note that in the series of
measurements following the recording shown in Fig. S7c (Fig. 3a and later records, main
text), the cantilever was always separated from the substrate by a minimum of 17 nm.
This separation prevented other ankyrin molecules from adsorbing to the tip, which could
interfere with the measurements on the original molecule of interest. We note that most
of the unfolding/refolding results on single ankyrin molecules shown in this work were
obtained by isolating an individual molecule as described above. It is also important to
realize that in this procedure the molecule of interest is subjected to increasing forces and
at some point it unfolds (as in Fig. S7c). In such a case, the subsequent recordings are
carried out not on an “intact” molecule, but on the molecule that already experienced a
single or multiple unfolding/refolding events. As we show the measurements on such a
15
partially unfolded molecule can be very informative, but this molecule would not qualify
for the measurements aimed at determining the linear elasticity where the stack needs to
be intact. Therefore, when studying the elasticity of intact ankyrin stacks (Fig. 1 main
text) we could not use this approach but instead had to perform hundreds of trials to catch
single molecules in the first pull.
8b. Reattachment of unfolded ankyrin repeats to the stack
A very intriguing result of the experiment shown in Fig. 3c, main text, is the possibility
that the unfolded repeats may have reattached to the stack. To seriously consider this
hypothesis we have to eliminate some simpler alternative explanations, for example, that
the observed event resulted from the unrecognized additional separation of the substrate
relative to the cantilever, which could have been triggered by a drift or a creep of the
piezo actuator. Such an event would further pre-stretch the molecule and would shift the
force curve towards shorter extensions and higher forces. Our piezoelectric stages that
control the position of the substrate are equipped with high resolution position sensors (a
strain gauge on one AFM instrument and a capacitive sensor on another AFM) that have
the resolution of the displacement measurement in the z direction of the order of 0.1nm.
In Fig. S8a-d we re-plotted the recordings shown in the main text (Fig. 3a-d). Now,
instead of the molecule extension we show the relative position of the piezo stage for
each force curve and tabulate the starting and ending position of the piezo for each
stretch-release cycle. There are small differences between the starting and ending
positions of the piezo of less than 1.5 nm caused by piezo hysteresis and even smaller
16
700
a
Force / pN
Force / pN
150
100
50
c
600
500
400
300
200
100
0
0
0
20
40
60
80
0
Piezo position / nm
40
60
80
Piezo position / nm
150
b
Force / pN
Force / pN
150
20
100
50
d
100
50
0
0
0
20
40
60
0
80
20
40
60
80
Piezo position / nm
Piezo position / nm
Record
number
Starting piezo position
(blue trace / nm)
Ending piezo position
(red trace / nm)
a
17.7
18.93
b
17.86
19.16
c
17.94
19.1
d
17.97
19.11
Fig. S8. Unfolding/refolding force curves of ankyrin originally shown in Figure 3ad (main text) re-plotted with the molecule’s extension replaced by the relative
position of the piezo actuator. Table shows the starting and ending position of the
piezo for each measurement shown in a-d.
differences between subsequent starting positions, of less than 0.3 nm (apparently the
piezo creep at the end of the relaxing travel reduced the effect of the hysteresis). Thus,
during these and other measurements the piezo stage was extremely stable and the
starting positions of the sample in the recordings shown in Fig. S8a-d were practically
identical. Therefore we have to reject an explanation of the observed effect as caused by
17
700
700
a
500
600
400
400
300
300
200
200
100
100
0
0
0
600
10 20 30 40 50 60 70 80
e
200
150
100
50
0
400
400
-50
300
300
200
200
100
100
0
0
0
10 20 30 40 50 60 70 80
300
0
100
100
c
50
50
0
0
-50
-50
50
100
150
Extension / nm
10 20 30 40 50 60 70 80
f
Force / pN
0
g
250
500
500
Force / pN
700
b
300
0
10 20 30 40 50 60 70 80
700
600
d
500
Force / pN
600
h
250
200
150
100
50
0
0
10 20 30 40 50 60 70 80
-50
0
10 20 30 40 50 60 70 80
Starting piezo
position
(blue trace / nm)
Ending piezo
position
(red trace / nm)
c
28.69
29.37
d
31.08
32.61
e
31.36
32.3
f
31.02
32.11
50
100
150
Extension / nm
Extension / nm
Record
number
0
Fig. S9. Another example of individual
ankyrin repeats detaching, unfolding,
refolding and reattaching to the stack.
In g and h we superimposed the
unfolding and refolding traces from f
onto our template unfolding recording.
Table shows the starting and ending
position of the piezo during that series
of measurements.
some piezo artifacts. We conclude that the force curve shown in Fig. S8c and in Fig. 3c
(main text) indeed represents the elasticity of the stack to which two out of the three
unfolded repeats have reattached and a further extension of this molecule was not
possible by unfolding of these repeats.
Another example of the reattachment behavior of the unfolded ankyrin repeats is
shown in Fig. S9. A single ankyrin molecule was isolated for these force spectroscopy
measurements following the procedure described above (Fig. S7). Figure S9b shows the
force extension curve of an ankyrin fragment that unfolded and broke down in two events,
a minor peak corresponds likely to the unfolding of a single repeat and a major force peak
18
corresponds to the unfolding of at least two repeats, based on the amount of the
polypeptide chain released in these events. The relaxing trace (red curve) reveals a series
of small force peaks that report the refolding of these previously unfolded repeats. In Fig.
S9c, the piezo was moved away from the cantilever so their separation increased to 30
nm to prevent other ankyrin molecules from adsorbing to the tip. The stretch-release
cycle performed from the new starting position of the piezo revealed now two small force
peaks separated by ~11 nm followed by the force curve that was separated by ~22 nm
from the second peak (as judged by the WLC fits). In the relaxing trace (red) we also
recorded two force peaks suggesting that the three repeats unfolded in the stretching
cycle refolded. Fig. S9d reveals a remarkable event, very similar to the one shown
already in Fig. S8c and in Fig. 3c (main text). However, in this case all three repeats seem
to reattach to the stack and were able to endure the force higher than 500 pN, as
evidenced by the relaxing trace that accurately followed the stretching trace. However, in
the next measurement, shown in Fig. S9e, the force curve reached the peak of 550 pN and
the stack broke down at a force of ~500 pN, at the beginning of the relaxing trace (see the
event marked by a dashed green circle). Even though the piezo already started its
returning travel and the molecule length should be steadily decreasing, the breakdown of
the stack released enough polypeptide chain so the molecule’s length actually increased,
and almost completely relaxed the cantilever. The detached repeats immediately refolded
as evidenced by the set of small force peaks captured by the rest of the relaxing trace (red
curve). The next recording (Fig. S9f) shows that detached and refolded repeats can be
again unfolded and again spontaneously refold. In Fig. S9g an S9h we superimposed the
forward and backward traces from Fig. S9f on top of our template unfolding recording
19
(Fig. 2a, main text) and the good overlap between these totally independent recordings
strongly suggest that individual (and tandem) ankyrin repeats unfolded and refolded. The
table lists the actual piezo positions for the critical recordings (c, d, e, f) including the
recording that captured the reattachment event. The table shows no significant piezo drift
or creep that could cause the observed effects.
9.
Comments on the mechanical stability of ankyrin stacks and ankyrin repeats
Our measurements suggest that relatively little force (< =60 pN) is required to unfold an
individual ankyrin repeat, once it is broken off of the stack (cf. Fig. S7-S9 and Figs. 2, 3;
main text). However, the mechanical stability of the intact stack seems to be much higher
as evidenced by the recordings shown in Fig. 1 (main text), where the intact stacks
detached from the tip at forces as high as 500 pN, but they did not unravel. In addition,
the observation of the refolding of the stack by the reattachment of the previously
detached repeats also suggests that the stack is mechanically very strong. This is because,
the refolded stacks were able to sustain forces in excess of 500 pN (Fig. S8c, S9d,e).
This strong stability of the stack that surpasses the stability of other mechanical proteins
studied so far such as titin or fibronectin is extremely interesting and warrants further
studies. We think that the strength of the stack is determined by unusually strong
interfacial interactions between the neighboring repeats, a notion that is consistent with
the recent observation by Mello & Barrick (Mello, C.C. & Barrick, D. An experimentally
determined protein folding energy landscape. PNAS 101, 14102-14107 (2004)) who
measured a very favorable interfacial energy between the neighboring repeats in the
ankyrin stack and postulated a cooperative behavior of the repeats. In our AFM study we
20
also observed such cooperativity in that we recorded simultaneous unfolding of two or
more repeats (Fig. 2a, 3ab) and a frequent
refolding of tandem repeats.
150
V = 0.012 nm/ms
10.
Force / pN
120
Speed dependence of the
unfolding/refolding of ankyrin repeats
a
90
60
30
0
-30
0
In Fig. S10 we show a short ankyrin fragment
measurements aimed at testing the effect of
30
40
50
V = 0.042 nm/ms
120
Force / pN
stack and were subjected to stretch-release
20
Extension / nm
150
composed of 3 repeats that detached from the
10
60
b
90
60
30
0
-30
varying the extension speed on their
0
20
30
40
50
unfolding and refolding traces overlapped well
and the magnitude of the unfolding forces did
Force / pN
V = 0.2 nm/ms
120
intermediate extension speeds (Fig. S10a, b) the
However, at the highest speed (Fig. S10c) the
magnitude of the second unfolding force peak
increased over two times, while the refolding
force peaks remained unchanged indicating the
robust refolding kinetics.
21
c
90
60
30
0
-30
0
not decrease significantly at the lowest speed.
60
Extension / nm
150
unfolding/refolding behavior. At low and
10
10
20
30
40
50
Extension / nm
Fig. S10. The effect of
increasing the stretching
rate on the
unfolding/refolding
behavior of a short ankyrin
fragment. Stretching (blue
trace); refolding (red trace).
In c, the gray trace is the
same as the stretching trace
in a.
60