Laboratory of Biodynamics, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama226-8501, Japan
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Abstract |
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Keywords: -helix mechanics/atomic force microscopy/calmodulin/force-extension relationship/poly-L-glutamic acid
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Introduction |
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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 helixcoil 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 150200% of the pre-stretched 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.
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Materials and methods |
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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)n-Cys 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. MALDI-TOF 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 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 (%) = 100x([
]222/40 000), where [
]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 (1x1 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 cross-linker 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 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 N-terminal 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. Forceextension 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
-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 399400 and 285289 eV revealed the presence of organic nitrogens and carbons, respectively.
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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
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Results |
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A first series of stretching experiments were performed using cross-linkers without polyethylene glycol arms. Figures 3A and B show the forceextension (FE) curves and distribution of extended chain length in such experiments. Parts of the FE curves with negative slopes do not contain actual data but rather correspond to jumps in cantilever movement. Large peaks at the beginning of the FE 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 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.
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To obtain the FE curve for polypeptide stretching without adsorption, we used a PEG cross-linker that was expected to free the polypeptide from adhesive interaction with the substrate. Consequently, the FE curves obtained were devoid of peaks in their initial regions, supporting our strategy to decrease unwanted adhesive interactions. Figure 4A shows mean FE 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 FE 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 FE 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 FE curves in the following sections involves at least ±15% errors due to this inaccuracy.
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An approximate estimate of the Young's modulus of helical PGA may be obtained from the average slope of the FE curve at pH 3 in Figure 4B, which is ~0.03 nN/nm in the region of 510 nm extension. Assuming that the length of helical (Glu)n-Cys is ~10 nm (n = 80, helicity = 80%) and the radius of the
-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)n-Cys. The result in Figure 6
reveals that while the extension is <1520 nm, there is a sigmoidal change in the work of 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 150200% extension but at higher extension the difference between coil and helix becomes obscure as far as the work of extension is concerned.
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FE 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
-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 TrisHCl buffer (pH 7.5) with 5 mM CaCl2, resulted in the FE curves shown in Figure 8A
(the contributions of the PEG cross-linker have been subtracted). In the FE curves in Figure 8A
, a distinct small force peak of 0.4 nN was observed at 4060 nm and multiple force peaks of 0.61.0 nN were observed at 85105 nm of extension before final rupturing. Since the shapes of the above FE curves between the peaks were similar to that of the FE 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.
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Discussion |
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To reduce unwanted interactions between the sample and substrate, we used the bifunctional cross-linker NHS-PEG3400-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 FE 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 FE 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 FE 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 -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 helixcoil transition observed in CD spectroscopy, we conclude that the helical chain retains its helical nature up to 150200% in relative extension.
In our cyclic unfoldingrefolding experiment with (Glu)n-Cys, the approach and retraction parts of the FE curves were almost identical. The shape and quantitative consistency of FE 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 -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 protein-based nano-machineries.
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Notes |
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Acknowledgments |
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References |
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Received June 22, 2000; revised September 25, 2000; accepted October 10, 2000.