Download Spring mechanics of α-helical polypeptide

Protein Engineering vol.13 no.11 pp.763–770, 2000
Spring mechanics of α-helical polypeptide
Alimjan Idiris, Mohammad Taufiq Alam and
Atsushi Ikai1
Laboratory of Biodynamics, Faculty of Bioscience and Biotechnology,
Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama
226-8501, Japan
1To whom correspondence should be addressed.
E-mail: [email protected]
To design protein- and polymer-based micro-machineries,
it is important to understand the mechanical properties of
basic structural elements such as the α-helix of polypeptides. We employed the force measurement mode of an
atomic force microscope (AFM) to investigate the spring
mechanics of poly-L-glutamic acid (PGA) in its helical and
randomly coiled states. After covalently anchoring the
polypeptide between a silicon substrate and an AFM tip,
the force required to stretch the polymer was measured.
The results indicated that PGA in its helical conformation
could be stretched almost fully with a continuous increase
in the stretching force, suggesting that it can be used as a
reliable coil-spring in the future design of spring-loaded
molecular machineries.
Keywords: α-helix mechanics/atomic force microscopy/calmodulin/force–extension relationship/poly-L-glutamic acid
Introduction
Knowledge of the energetics of protein stability is central to
understanding the structure–function relationship of protein
molecules and to design nano-mechanical devices based on
protein chemistry. One of the simplest and best studied of
such mechanical elements is the α-helix of polypeptides. The
helical behavior of poly-amino acids in aqueous solutions in
the absence of tertiary interactions has been intensely studied.
A variety of model peptides have been synthesized and
investigated with respect to their tendencies for α-helix formation (Schulz and Schirmer, 1979). Poly-L-glutamic acid (PGA),
among others, has a high tendency to form α-helices in acidic
media. The conformational transition between coil and helix
states has been studied by using a variety of methods, such as
spectroscopic, hydrodynamic, thermodynamic and theoretical
analyses (Doty et al., 1957, 1958; McDiarmid and Doty, 1966;
Olander and Holtzer, 1968; Poland and Scheraga, 1970; Evans
et al., 1995; Muñoz and Serrano, 1995). Each of these studies
was aimed at the estimation of thermodynamic parameters in
a well-controlled solution system to explain best the relative
stability of the helical conformation against conversion to the
coiled state and the sharpness of the transition curve. With the
advent of a variety of methods of single molecular measurement, it is now possible to study the mechanical properties of
helical polymers by, for example, measuring the required force
to stretch the molecule mechanically from its two ends. The
atomic force microscope (AFM), among other tools, has made
it possible to perform such mechanical experiments with single
molecules (Kishino and Yanagida, 1988; Ikai, 1996; Rief et al.,
© Oxford University Press
1997a). The forces within molecules produced by motor
proteins (Finer et al., 1994; Svoboda and Block, 1994) and
the binding forces of receptor–ligand systems (Florin et al.,
1994; Gad et al., 1997) and those between complementary
DNA strands (Essevaz-Roulet et al., 1997) have been measured.
Recently, the forces required to unfold proteins have also been
determined (Mitsui et al., 1996; Kellermayer et al., 1997; Rief
et al., 1997b, 1998; Tskhovrebova et al., 1997; Ikai et al.,
1997; Oberhauser et al., 1998; Carrion-Vazquez et al., 1999;
Fisher et al., 1999; Wang and Ikai, 1999).
In this work, we stretched poly-L-glutamic acid in aqueous
solutions of pH between 8.0 and 3.0, first to study the
mechanical behavior of the polymer throughout the pH range
during its helix–coil transition, and second to learn about the
spring-like behavior of a helical polymer. An interesting finding
was that a helical polypeptide could be stretched remarkably
smoothly with a continuous increase in the force without
reversion to randomly coiled conformations, thus describing
its coil-spring behavior throughout almost full-length stretching
for the first time. A sigmoidal behavior was observed in a plot
of the stretching work against solution pH provided that the
extension of the polymer was within 150–200% of the prestretched length. Mechanical extension of helical polylysine
has recently been performed by Lantz et al. (1999), who
reported stepwise extension of the helical polymer in contrast
to our smooth stretching. Consequently, we present a different
view of the mechanics of helix extension.
Materials and methods
Synthetic peptide and amino acid analysis
Poly-L-glutamic acid was provided by the Peptide Institute
(Osaka, Japan). The peptide had a Cys residue at the
C-terminus. The nominal degree of polymerization from its
synthetic cycle was 101 including the terminal Cys. The
polymer will be referred to as (Glu)n-Cys, where the subscript
n represents the degree of polymerization. The SH group of
Cys was necessary for single molecule stretching experiments
under AFM when using a chemical cross-linker system. (Glu)nCys was used after further purification by HPLC using a
Superdex 75 gel permeation column (Pharmacia, Uppsala,
Sweden). Amino acid analysis of the purified peptide was
performed by the provider of the peptide. They reported the
molar ratio of glutamic acid to half-cystine as 60. MALDITOF mass spectrometry yielded a peak of m/z 13 100 as the
highest m/z peak. Therefore, the glutamic acid component of
the sample had, respectively, an average and the highest degree
of polymerization of 60 and 100. Commercial poly-L-glutamic
acid (PGA, MW ⫽ 50 000; Sigma-Aldrich, Tokyo, Japan) was
also used to measure circular dichroism (CD).
Protein expression
The plasmid pRCaM (3.8 kb for dimers) was constructed by
inserting a 900 bp fragment of the calmodulin dimer sequence
and cysteine residue codons at two termini, between the
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HindIII and BamHI sites in the multiple cloning region of
pRSETB (Invitrogen, San Diego, CA). It was confirmed by
restriction analysis and DNA sequencing that the plasmid
contained the correct gene sequence of the calmodulin dimer.
An N-terminal (His)6 tag (encoded by the pRSET vector) and
two terminal cysteine residues were fused in frame to the
calmodulin dimer fragment. The plasmid was transformed into
competent BL21 (DE3) cells. The mutant calmodulin dimer
was induced in SOB culture medium augmented with ampicillin
and grown to OD600 ⫽ 0.6, at 37°C, 200 r.p.m., using a
0.1 mM IPTG for 4 h. The expressed (His)6-tagged recombinant
protein was purified from cytosolic supernatants by metal
chelate affinity chromatography on an Ni-NTA agarose column
(Qiagen, Hilden, Germany). The purity of the protein
preparation was ⬎98% as determined by polyacrylamide gel
electrophoresis (PAGE) in the presence of sodium dodecyl
sulfate. The protein was stored in the presence of a 1:100
molar ratio of 1,4-dithiothreitol (DTT) at 4°C. Gel chromatography (with a Superdex-75 HPLC column, Pharmacia) was
performed immediately before AFM force measurement to
remove the reducing reagent.
Circular dichroism (CD) analysis
All circular dichroism (CD) measurements for (Glu)n-Cys and
commercially available poly-L-glutamic acid were carried out
on a J-720WI spectropolarimeter (JASCO, Tokyo, Japan) at
25°C using a fused quartz cell of 1 mm pathlength. All
solutions were prepared with deionized water containing 0.1 M
NaCl and the pH was adjusted immediately before CD measurements by adding a concentrated solution of either HCl or
NaOH. The helicity of the samples was calculated using the
equation α (%) ⫽ –100⫻([θ]222/40 000), where [θ]222 is the
mean residue ellipticity at 222 nm (Adler et al., 1973).
AFM force measurements, cross-linkers and other reagents
A Nanoscope III multiprobe AFM (Digital Instruments, Santa
Barbara, CA) with a J-scanner was used for all the force measurements. Silicon nitride (Si3N4) tips (a sharpened type, abbreviated
to NP-S) were purchased from the same manufacturer. Narrow
cantilevers 200 µm in length and with a nominal spring constant
of 0.06 N/m were used in the measurements. Crystalline silicon
wafers with a (111) surface were purchased from Shin-Etsu
Silicon (Tokyo, Japan). The wafers were cut into square pieces
(1⫻1 cm) before use. The silicon substrates and NP-S tips were
treated with 3-aminopropyltriethoxysilane (APTES) (Shin-Etsu
Chemical, Tokyo, Japan) after cleaning and oxidation (Brzoska
et al., 1992), to ensure that the surface of the silicon substrates
and tips became covered with amino groups. The aminated
substrate surface was further treated with a mixture of the heterobifunctional cross-linker NHS-PEG3400-MAL (abbreviated to
PEG cross-linker, with a theoretical length of 30 nm) and the
monofunctional cross-linker NHS-PEG2000 (Shearwater Polymers, Huntsville, AL). The N-hydroxysuccinimidyl (NHS) and
maleimidyl (MAL) groups on the PEG cross-linker can react
with amino and sulfhydryl groups, respectively. It also contains
polyethylene glycol of MW 3400 as a long spacer. By reacting
with the amino groups on a silanized silicon surface, the crosslinker enables the silicon substrate to be reactive to sulfhydryl
groups. Then (Glu)n-Cys or purified recombinant calmodulin
dimers were anchored to the silanized and functionalized surface
of the silicon wafers through the terminal cysteine residues. The
co-existing monofunctional cross-linker NHS-PEG2000 does
not react with (Glu)n-Cys molecules after its NHS group reacts
with NH2 on the substrate, but it reduces the chance of the
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interaction between immobilized (Glu)n-Cys molecules by
decreasing the density of NHS-PEG3400-MAL on the substrate.
The silanized tips were treated with the homobifunctional
cross-linker disuccinimidyl suberate (DSS) (Pierce, Rockford,
IL) for stretching of (Glu)n-Cys and treated with the
heterobifunctional cross-linker N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or succinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP, LC for long chain) (Pierce)
for calmodulin force measurement, since the pyridyldithio moiety of SPDP (or LC-SPDP) reacts with free SH groups and there
is a high probability of forming a disulfide bond with SH groups
on the free end of anchored calmodulin dimers. Similarly, the
free end of DSS on the tip was expected to react with the Nterminal amino group of (Glu)n-Cys anchored on the substrate.
When the extension of polyethylene glycol part of the PEG
cross-linker was measured, the substrate was functionalized with
APTES that reacts with the NHS group of the cross-linker
whereas the tip was with 3⬘-mercaptopropyltriethoxysilane that
reacts with the MAL group. Each coated substrate with a polypeptide or a protein layer was mounted on the AFM sample stage
after rinsing and a liquid cell was constructed over it. Force–
extension experiments were done first, contacting the substrate
and tip briefly and then gradually increasing the distance between
them. Formation of covalent cross-links on both sides of the
polymer chain was confirmed by downward deflection of the
AFM cantilever. To establish an experimental standard, the chain
was pulled to and beyond its full length (i.e. rupturing covalent
cross-links). Cyclic increases and decreases in distance between
the substrate and the tip were also performed within the full
length of the chain by limiting the displacement range of the
AFM piezo scanner. All experiments were performed at room
temperature. A schematic representation of the AFM tip and
silicon substrate modification and the procedure for sandwiching
(Glu)n-Cys between the tip and substrate are illustrated in Figure
1. The bottom illustration of Figure 1 represents a schematic
view of how we applied a similar method described above to
extend calmodulin, an α-helical protein, in its dimeric form. The
presence of peptides on the silicon surface was confirmed by
ESCA analysis performed with an AXIS-ULTRA (Shimadzu,
Tokyo, Japan). Deconvolution of ESCA peaks around 399–400
and 285–289 eV revealed the presence of organic nitrogens and
carbons, respectively.
Worm-like chain (WLC) model
To model the force versus extension characteristics of an
unfolded polypeptide chain, a theoretical extension curve of
an ideal random coil, the WLC model was used (Bustamante
et al., 1994), with the equation
F(L)⫽ (kBT/p)[0.25/(1 – L/L0)2 – 0.25 ⫹ L/L0]
where the persistence length p describes the polymer stiffness,
kB is Boltzmann’s constant, L and L0 are the stretched length
and contour length of the chain, respectively, and T is the
absolute temperature. A value of 0.37 nm was used for p from
the known dimensions of polypeptide chains (Schulz and
Schirmer, 1979).
Results
The result of CD measurements confirmed that (Glu)nCys formed α-helical and random coil structures in acidic
(pH ⬍4.0) and in neutral (pH ⬎5.0) media, respectively, and
showed a helix–coil transition between pH 5.0 and 4.0
(Figure 2) in a similar manner to commercial PGA. It exhibited
Spring mechanics of α-helical polypeptide
Fig. 1. Schematic representation of the modified AFM tip and silicon substrate and the procedure for stretching a (Glu)n-Cys molecule: (1) modification of the
silanized tips with the homobifunctional cross-linker DSS (the surface of tip is covered with DSS molecules under experimental conditions, but only one DSS
molecule is shown here for simplicity); (2) modification of the silanized silicon substrate with NHS-PEG3400-MAL and NHS-PEG2000; (3) immobilization
of (Glu)n-Cys to the modified silicon substrate; (4) sandwiching of an immobilized (Glu)n-Cys molecule between the AFM tip and substrate. The bottom
illustration is an artist’s view of calmodulin dimer stretching under an AFM emphasizing the α-helical secondary structure of the protein.
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Fig. 2. pH dependence on the degree of helicity of (Glu)n-Cys based on
the results of CD spectroscopy. The helicity was calculated by measuring
the mean residue ellipticity at 222 nm in 0.1 M NaCl solution at different
pHs.
~70% helicity at pH 艋4.0 and almost all of PGA molecules
were in the random coil state at pH ⬎6.0. Ellipticity was not
observed at pH ⬍4.0, because the solubility of PGA decreased
sharply under such conditions. We also measured the CD
spectrum of PGA in the presence of 5% PEG to see the effect
of the PEG part of the cross-linker on the helix formation of
(Glu)n-Cys. The result indicated a slight shift of the transition
curve to a higher pH region compared with the curve in
Figure 2, but the sharpness of the curve was unchanged within
experimental error.
From CD spectroscopy, subsequent force measurements
were conducted between pH 3.0 and 8.0. A rate of extension
(pulling rate) beween 0.1 and 0.2 nm/ms was employed.
(Glu)n-Cys was estimated to have a maximum theoretical
elongation length of 37 nm when n ⫽ 100 and a minimum
length of 15 nm (i.e. complete α-helix with a distance of
0.15 nm between the neighboring residues along the helical
axis). Typically, one end of the peptide was cross-linked to
the silicon substrate and the other end was cross-linked to the
tip on the AFM cantilever. As the sample stage was retracted,
the peptide was stretched between the tip and substrate and
the force applied to the peptide was recorded as the deflection
of the cantilever. To ensure that, in most cases, only one
polymer chain became covalently sandwiched between the
substrate and the tip during their contact time, the success rate
of cross-link formation was kept to ⬍5% of the total contact
numbers by adjusting the concentration of (Glu)n-Cys in the
immobilization solution.
A first series of stretching experiments were performed
using cross-linkers without polyethylene glycol arms. Figures
3A and B show the force–extension (F–E) curves and distribution of extended chain length in such experiments. Parts of
the F–E curves with negative slopes do not contain actual data
but rather correspond to jumps in cantilever movement. Large
peaks at the beginning of the F–E curves (Figure 3A) revealed
a strong adhesion between the sample and either the substrate
or tip. Such adhesive interactions are thought to obscure the
true mechanics of polypeptide stretching and should therefore
usually be avoided. However, in this particular case, an
adequate estimate of the polymer length at its full extension
was obtained. From the data in Figure 3B, a mean extension
length with a standard deviation of 30 ⫾ 3 nm (n ⫽ 52) was
calculated. The mean length corresponded to a theoretical
stretched length of ~80 amino acid residues. Considering the
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Fig. 3. (A) F–E curves of (Glu)n-Cys at pH 8.0 in the absence of PEG
cross-linker. A large adhesion force appeared when stretching (Glu)n-Cys
without PEG cross-linker. (B) Frequency distribution of the extension length
of (Glu)n-Cys. Mean extension length is 30 ⫾ 3 nm (n ⫽ 52).
number average degree of polymerization of 60 as determined
from amino acid analysis, we concluded that our cross-linking
conditions were favorable towards longer than average chains.
Since the cross-linking site of a sample chain to the tip may
or may not be closely located to the corresponding site on the
substrate, we must make an allowance for this discrepancy.
If the vertical and horizontal distances between the two
cross-linking points when the tip is in contact with the substrate
are v and h, the relationship between the actual length of a
fully stretched peptide, L, and the observed stretch length in
the F–E curve, S, is
L2 ⫽ (S ⫹ v)2 ⫹ h2
Although it was not possible to estimate either v or h from
our experimental design, S remains ⬎0.9L when v and h are
⬍0.1L. Moreover, if v ⫽ 0, h can be as large as 0.43L, whereas
if h ⫽ 0, v must be ⬍0.1L for S to be 艌0.9L. Therefore, a
vertical discrepancy of the two cross-linking points yields a
considerably more serious error in estimation of L from S than
does a horizontal one. If we assume that a randomly coiled
(Glu)n-Cys can be approximated by a Gaussian chain with
fixed bond angles but with free internal rotations, the expected
average of its end-to-end distance is ~3.7 nm, which is equal
to 0.1L. In this study, polymer chains were found to be strongly
adsorbed to the substrate before they were stretched, meaning
that v was much smaller than 0.1L. Therefore, the observed
total chain length at the final rupture point of the covalent
bonds would have been measured at ⬍10% error.
To obtain the F–E curve for polypeptide stretching without
adsorption, we used a PEG cross-linker that was expected to
Spring mechanics of α-helical polypeptide
Fig. 5. Histogram of the unfolding work required to stretch a single
(Glu)n-Cys chain from a distinct state to the fully stretched conformation
under different pH conditions. The energy was calculated from the F–E
curves shown in Figure 4B. The unit of work is given in kJ/mol here and in
Figure 6. A work of 1000 kJ/mol corresponds to 1.66⫻10–18 J/molecule,
which is equivalent to 1.66 nN nm.
Fig. 4. Comparison of mean F–E curves of (Glu)n-Cys under different
pH conditions. (A) Before subtracting PEG extension. The curve under
PEG represents the mean extension curve of PEG cross-linker. Here and
below, thin curves between thick lines labeled pH 8.0 and 3.0 are the
F–E curves obtained at intermediate pHs of 7.0, 6.0, 5.0 and 4.0,
respectively, in order. (B) After subtracting PEG extension from the total
extension in (A). The data emphasize the pH dependence of unfolding
force. WLC ⫽ worm-like chain. All of the force curves were measured at a
pulling rate of 0.1–0.2 nm/ms.
free the polypeptide from adhesive interaction with the substrate. Consequently, the F–E curves obtained were devoid of
peaks in their initial regions, supporting our strategy to decrease
unwanted adhesive interactions. Figure 4A shows mean F–E
curves of (Glu)n-Cys (n ⫽ 40) obtained at different pHs and
normalized to an extension length of 60 nm (the mean
extension obtained in Figure 3B plus the mean extension
length of the PEG cross-linker alone obtained in a separate
experiment, n ⫽ 50). To construct such normalized curves,
we collected F–E curves with maximum extensions between
55 and 65 nm and adjusted the total extension of all curves to
60 nm. They formed a collection of highly overlapping curves
and a mean curve is shown in Figure 4. It must be pointed
out that, although the use of PEG cross-linker eliminated
unwanted adsorptions from F–E curves, estimation of the
stretch length for the (Glu)n-Cys part became less accurate.
Consequently, the length-dependent numerical parameters
obtained from the mean F–E curves in the following sections
involves at least ⫾15% errors due to this inaccuracy.
To obtain the F–E relationship of (Glu)n-Cys alone, the
corresponding relationship of the PEG cross-linker must be
subtracted. As shown in Figure 4A, the unfolding force
gradually increased with decreasing solution pH from 8.0 to
3.0, whereas in separate experiments with the PEG crosslinker alone, there was no detectable dependence of the F–E
relationship on solution pH (data not shown). Therefore, an
averaged and pH-independent F–E curve of the PEG crosslinker was used to subtract the contribution of the cross-linker
from the combined extension curves (Figure 4B) and the result
clearly shows that much larger force was required to stretch
(Glu)n-Cys at pH 3.0 than at 8.0 throughout extension. Force
curves resulting from samples of intermediate pHs lie between
those from the high and low extremity pHs. The initial region
of the curve obtained at pH 8.0 is similar to that of the WLC
model but deviates significantly toward the end of extension,
suggesting that there were extra segmental interactions.
An approximate estimate of the Young’s modulus of helical
PGA may be obtained from the average slope of the F–E
curve at pH 3 in Figure 4B, which is ~0.03 nN/nm in the
region of 5–10 nm extension. Assuming that the length of
helical (Glu)n-Cys is ~10 nm (n ⫽ 80, helicity ⫽ 80%) and
the radius of the α-helix is 0.2 nm, a value of 3GPa can be
obtained for the Young’s modulus. This is not an unreasonable
value because it is in agreement, within a factor of two, with
the result of a recent theoretical calculation by Gang Bao of
Georgia Institute of Technology (personal communication),
who estimated the Young’s modulus of poly-L-alanine helix in
water as 5 GPa.
By integrating the curves shown in Figure 4B from 0 to
35 nm in extension, the work required to unfold the polymer
chain in different conformations to its full length was estimated
and plotted in Figure 5. The results reveal that more work is
required for the chain extension at low pH relative to high
pH, but there seems to be no abrupt increase in the work
between pH 5 and 4 where a sharp change was observed in
CD measurement (Figure 2). When integration was carried out
from zero to intermediate extension lengths and the resulting
work was plotted as a function of pH as shown in Figure 6,
more insight was obtained as to the helical nature of (Glu)nCys. The result in Figure 6 reveals that while the extension is
⬍15–20 nm, there is a sigmoidal change in the work of
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A.Idiris et al.
Fig. 6. Unfolding work of stretching a single (Glu)n-Cys chain to limited
extension lengths obtained in a similar manner as explained in Figure 5.
Fig. 7. Unfolding–refolding curves observed in two cycles for samples in
0.1 M NaCl solution, pH 3.0 (A and B) and pH 7.0 (C and D). The
unfolding–refolding cycle could often be repeated over 200 times for a
single chain before mechanical drifts of AFM ruptured the cross-linking
system. The unfolding and refolding force was higher for samples at
pH 3.0 than at pH 7.0.
extension, but it becomes obscure as the extension length is
increased further. We interpret the result as indicating that the
helical nature of the chain persists up to 150–200% extension
but at higher extension the difference between coil and helix
becomes obscure as far as the work of extension is concerned.
To investigate the reversibility of (Glu)n-Cys stretching,
additional stretching experiments were carried out on
stretching–retraction cycles by keeping the maximum
stretching distance less than the maximum extension length of
the chain. As shown in Figure 7, this allowed repeatable
unfolding and refolding of F–E curves as many as 200 times
for a single molecule without breaking covalent bonds. The
cyclical unfolding–refolding results are almost indistinguishable from the one-time stretching results in Figure 3. Therefore,
quantitatively, the results are highly reproducible and reliably
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represented as the F–E curve of a single polypeptide without
adsorptive interactions. Here, similarly to the previously discussed data shown in Figure 4A, it was also confirmed that the
unfolding force was higher at lower pH and vice versa.
To explore whether there is a dependence of the stretching
force on the pulling rate, unfolding experiments were conducted
at different scan speeds. At pH 7.0 and 8.0, stretching force
was independent of pulling rate for rates between 0.2 and
1.2 nm/ms (data not shown). At pH 3.0 and 4.0, however,
there was a slight increase in force as the pulling rate was
increased (data not shown).
F–E measurement was then extended to engineered
calmodulin dimers (see the bottom illustration of Figure 1). A
dimer of calmodulin has 296 amino acid residues. Cysteine
residues were added to the ends of the dimers and two
more residues (Glu and Phe) were inserted between the two
monomers, bringing the total number of amino acid residues
to 300. The linear contour length of the dimer was therefore
expected to be 110 nm, assuming that the contour length of
one residue is 0.37 nm. Considering that the extended length
of the PEG cross-linker is 30 nm, the maximum stretch should
be about 140 nm. From the CD measurement, the engineered
dimer of calmodulin exhibited 50% helicity in the presence of
Ca2⫹, indicating that the genetically engineered calmodulin
dimer maintained a level of α-helical conformation comparable
to that of the native calmodulin (Klee, 1977; Watterson et al.,
1980; Babu et al., 1985; Chattopadhyaya et al., 1992; Falke
et al., 1994). Stretching experiments of the engineered
calmodulin dimer in 50 mM Tris–HCl buffer (pH 7.5) with
5 mM CaCl2, resulted in the F–E curves shown in Figure 8A
(the contributions of the PEG cross-linker have been subtracted). In the F–E curves in Figure 8A, a distinct small force
peak of 0.4 nN was observed at 40–60 nm and multiple force
peaks of 0.6–1.0 nN were observed at 85–105 nm of extension
before final rupturing. Since the shapes of the above F–E
curves between the peaks were similar to that of the F–E curve
of (Glu)n-Cys at pH 3.0, such parts were interpreted as
representing stretching of helical parts of the molecule. The
force peaks mentioned above could be attributed to the
sequential breakdown of tertiary conformations in the molecule,
but detailed interpretation must await more precise measurement of the force curves of calmodulin stretching.
To confirm that the observed F–E curves were mainly the
result of the unfolding of the α-helical structure in a single
molecule of calmodulin dimer, the stretching experiment was
repeated under unfolding–refolding cycle conditions. The
immobilization procedure of the protein was similar to the
unfolding experiments except that LC-SPDP was used as
the cross-linker on the substrate and tip. The experiment was
conducted at a scan frequency of 1 Hz in the presence of
5 mM CaCl2. The mean curve differed from the unfolding
curves in Figure 8A in that the first and second peaks observed
in Figure 8A were absent. This indicates that, while native
tertiary structure was not formed within time intervals of
~500 ms (estimated contact time of the tip with the substrate),
some secondary structure, presumably α-helix, was assembled
during approach of the tip.
Discussion
In our previous experience in protein stretching using AFM, the
following problems appeared difficult to resolve unequivocally.
The first was how to ensure that only a single molecule was
being stretched between the tip and substrate, and the second
Spring mechanics of α-helical polypeptide
Fig. 8. (A) Representative F–E curves for calmodulin dimers in the
presence of 5 mM CaCl2. Thin lines represent F–E curves of (Glu)n-Cys at
pH 3.0 with adjustment of extension length. (B) Mean unfolding curve of
calmodulin dimers observed in repeated unfolding–refolding cycles in the
presence of Ca2⫹. Mean PGA F–E curve observed at pH 3.0 (helical state)
and pH 8.0 (coiled state) are also given as references. The refolding curves
were almost identical with the unfolding curves (data not shown).
was how to eliminate unwanted adhesive interactions between
the sample and substrate (or tip) surface. Neither problem has
been solved completely in this work, but the maintenance of
low efficiency of successful interactions between the tip and
the protein and the consistency in the quantitative features of
the force curves under varying conditions including unfolding–
refolding cycles suggest that the force curves obtained in our
experiments were those of single molecule stretching. The
observed fact that, in both one-time and cyclical experiments,
the final rupture took place as a single event may be taken as
a particularly reliable line of evidence for single molecule
stretching.
To reduce unwanted interactions between the sample and
substrate, we used the bifunctional cross-linker NHSPEG3400-MAL, with a long and flexible spacer made of PEG.
The use of PEG cross-linker reduced the problem of strong
adsorption of (Glu)n-Cys to the substrate and allowed us to
obtain reasonably clean F–E curves, but estimation of the
cross-linker extension remained as a problem when it should
be subtracted from the peptide extension curves. Therefore, as
pointed out in the previous section, numerical interpretations
of the F–E curves remain semi-quantitative at the present
stage. A shorter and more homogeneous PEG cross-linker will
be needed to alleviate the last problem in future experiments.
Despite the presence of problems as stated above, the F–E
curves of (Glu)n-Cys obtained in this study provided the
following new observations: (1) a clear dependence on the pH
of the solution, presumably reflecting the extent of α-helix
formation at different pHs (Figure 3B), and (2) under most
conditions, the required force for stretching (Glu)n-Cys
increased smoothly without any noticeable force peaks up to
almost full extension of the chain. This indicates that there is
no sudden break of a rigid conformation to a relaxed one and
that the transition from a contracted form, be it a coil or a
helix, to an extended form is gradual and continuous.
It is clear that more force and work are needed to stretch
(Glu)n-Cys in a solution of pH 3.0 than of pH 7.0 or 8.0. This
is in accordance with the idea that (Glu)n-Cys is in a helical
conformation that is presumably more resistant to a stretching
force than is a flexible random coil. As it has been pointed
out theoretically that the turn-by-turn unfolding scheme with
a more or less constant force of unfolding is energetically
more favorable than the uniform stretching (Rohs et al.,
1999), it was surprising that the stretching force increased
continuously without a plateau region or force peaks, to the
full extension of the molecule. Therefore, in contrast to
the theoretical prediction, these empirical data suggest that a
helical chain can be stretched uniformly along its length, rather
than broken residue-by-residue or turn-by-turn from its two
ends, thus making a gradual transition from a helical to an
almost fully extended conformation.
When the work to stretch the chain to various lengths from
the original contracted form was plotted against solution pH,
we noticed a sigmoidal change in the curve between pH 6
and 4 for the lower three curves in Figure 6. This sigmoidicity
is gradually lost for extensions longer than 17.5 nm (see
also Figure 5). Since this sigmoidal change most probably
corresponds to the helix–coil transition observed in CD spectroscopy, we conclude that the helical chain retains its helical
nature up to 150–200% in relative extension.
In our cyclic unfolding–refolding experiment with (Glu)nCys, the approach and retraction parts of the F–E curves were
almost identical. The shape and quantitative consistency of
F–E curves during more than 200 cycles of extension and
retraction strongly indicate that only a single molecule was
being stretched between the tip and substrate. It confirmed the
reliability of (Glu)n-Cys as a nano-scaled coil spring.
The force measurement performed on engineered calmodulin
dimers revealed similarities between the unfolding and refolding curves of calmodulin and polyglutamic acid, but differences
in their refolding pathway. As expected, the pathway of
mechanical unfolding of the protein was more complex than
that of a simple helix owing, presumably, to the presence of
three-dimensional interactions between helices and distant side
chains on the primary structure.
Finally, the remarkable stability of the spring mechanics of
α-helical polypeptide found in this study will make it a useful
material such as a flexible scaffold, a spring-loaded actuator
or an absorber of large force in our future design of proteinbased nano-machineries.
Acknowledgements
This work was supported in part by grants-in-aid to A.I. from the Japan
Society for the Promotion of Science (Research for the Future Program
99R16701) and from the Japanese Ministry of Education, Science, Culture
and Sports [Scientific Research on Priority Areas (B) 11226202]. The authors
are grateful to Shimadzu for conducting the ESCA analysis of their samples.
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Received June 22, 2000; revised September 25, 2000; accepted October
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