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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 763 A.Idiris et al. 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 764 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. 765 A.Idiris et al. 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 766 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 767 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 768 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. References Adler,A.J., Greenfield,N.J. and Fasman,G.D. (1973) Methods Enzymol., 27, 675–735. 769 A.Idiris et al. Babu,Y.S., Sack,J.S., Greenhough,T.J., Bugg,C.E., Means,A.R. and Cook,W.J. (1985) Nature, 315, 37–40. Brzoska,J.B., Shahidzadeh,N. and Rondelez,F. (1992) Nature, 360, 719–721 Bustamante,C., Marko,J.F., Siggia,E.D. and Smith,S. 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