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Bone and Mineral Centre, Department of Medicine, University College London, London, United Kingdom
Correspondence: Address reprint requests to Dr. Laurent Bozec, Dept. of Medicine, Rayne Building, 5 University St., London WC1E 6JJ, UK. Tel.: 44-20-76796169; Fax: 44-20-76796219; E-mail: l.bozec{at}ucl.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONSUMMARYACKNOWLEDGEMENTSREFERENCES |
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8 nm that is comparable to the theoretical value. Using force spectroscopy, we established the stretching pattern of the molecule, where both the mechanical response of the molecule and pull-off peak are convoluted in a single feature. By interpreting this response with a wormlike chain model, we extracted the value of the effective contour length of the molecule at (202 ± 5) nm. This value was smaller than that given by direct measurement, suggesting that the entire molecule was not being stretched during the force measurements; this is likely to be related to the absence of covalent binding between probe, sample, and substrate in our experimental procedure. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONSUMMARYACKNOWLEDGEMENTSREFERENCES |
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1-chains and one 2-chain intertwined in an overall right-handed coil, tropocollagen (Ramachandra and Karthan, 1955
). The basic unit of each of the polypeptide chains consists of the repeating sequence, Glycine-X-Y, where X is often proline and Y hydroxyproline. It is the frequent occurrence of proline and hydroxyproline groups in the polypeptide chain that causes the collagen -chains to be tightly packed into a triple helical conformation. Several collagen-related diseases occur in humans, osteoporosis and tendinitis being common examples. Both are linked to the mechanical properties of collagen and its higher order, structurally related forms: collagen fibrils and fibers, and the macroscopic tissues, tendon, and bone.
There have been many studies of collagen fibers using a range of experimental techniques, but more recently atomic force microscopy (AFM) has been used to study this protein under physiological conditions, unattainable by precursor techniques such as x-ray diffraction crystallography or transmission electron microscopy (TEM). The ability of AFM to image as well as to perform force measurements has enabled experimentalists to compare results obtained using molecular scale samples to those obtained at the macroscale using bulk techniques. Early studies involving AFM and collagen fibers were initially carried out by Chernoff et al. (1992), who imaged collagen fibers under dry conditions but were unsuccessful in observing the characteristic D-banding as initially proposed by Schmitt and co-workers in 1942 (Schmitt et al., 1942) based on TEM. This work was later pursued successfully by Baselt et al. (1993) and Revenko et al. (1994), who presented AFM images of collagen fibers displaying the characteristic D-banding pattern. Recently, Gutsmann et al. (2003) published a detailed account of both topological and mechanical behavior of the collagen fibers, isolated from rat tail tendon. One result supported the view that the fibers had the topology of a hollow tube and they suggested that the collagen fiber is composed of a hard shell with a less dense core, giving both flexibility and elasticity to the fiber. In a later publication, Gutsmann et al. (2004) presented some spectroscopic force measurements performed on rat tail collagen fibers. The aim of that study was to link the stretching pattern of the fiber with the D-banding pattern of the fiber, initially proposed by Schmitt et al. (1942). The stretching pattern that was obtained while pulling on the collagen fiber proved to be too complex to identify single characteristic events. Nevertheless, two features were identified with ruptures at 22 nm and 78 nm; these were hypothesized as being related to a possible repeat in the fiber structure, though all of these were not identified by AFM imaging.
There have been few investigations of collagen at the level of the single triple helical tropocollagen molecule structure using AFM. Mertig et al. (1997) presented an analysis of an in vitro generated two-dimensional collagen network formation. In their studies, they managed to isolate single collagen monomers that were prepared on a mica surface and observed that the monomer did not undergo denaturation as it bound the mica surface. More recently, Sun et al. (2002) used optical tweezers to characterize the mechanical behavior of collagen, but more specifically its flexibility. In their study, they used procollagen, the precursor form of the collagen monomer. Procollagen has cysteine groups at both its N- and C-termini, which enables covalent binding to the surface-probe or beads, though not with controlled orientation. They found that the monomer had a persistence length of 14.5 nm and a contour length of 309 nm.
The aims of our study were to use AFM to investigate type I collagen at the single molecule level to understand the fundamental mechanical behavior of this protein, and to correlate these findings with high-resolution topography imaging.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONSUMMARYACKNOWLEDGEMENTSREFERENCES |
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Imaging collagen by AFM
For single molecule imaging, a droplet 40 µl of the 1:1000 solution was deposited on a mica substrate (mica disks; Agar Scientific, Stansted, UK) that had been freshly cleaved. The solution was left to incubate for 10 min, avoiding droplet evaporation by keeping a water droplet at the side of the sample but not in contact so as to avoid sample dilution. The sample was then rinsed, firstly using PBS and then using ultra high quality (UHQ) water to avoid any salt crystal formation. Finally the sample was dried using a gentle stream of dry N2. The imaging experiments were carried out using a Multimode-Nanoscope IV (Veeco, Santa Barbara, CA), equipped with an E scanner and NSC tips-D lever (MikroMasch, Tallinn, Estonia), with the following characteristics in air: 28 kHz resonant frequency and 0.35 N/m nominal spring constant. The samples were imaged in dry conditions in tapping mode, with the minimum amplitude set point selected to avoid either damaging or altering the sample on the surface. To do so, the probe was brought into contact with a false-engagement (the probe touches the surface and immediately retracts but remains very close), and then the amplitude set point was slowly reduced until the probe remade contact with the surface. This ensures that there is the minimal force applied by the probe onto the sample.
Single molecule pulling experiments
For single molecule force measurements, a single droplet of 80 µl of the 1/100 solution was deposited on a gold-coated glass slide, previously stored in liquid nitrogen. The solution was left to incubate for 5 min, avoiding evaporation. The sample was then gently rinsed using PBS. Finally a droplet of 150 µl PBS was deposited on the sample, before starting the measurements. Force measurements were performed on two different instruments: a Multimode-Nanoscope IV with a Picoforce attachment (Veeco) and a Molecular Force Puller, MFP1 (Asylum Research, Santa Barbara, CA). For consistency, the same type of probe was used on both the instruments during all of the experiments: Microlever-D tips lever (MikroMasch) with the following characteristics in buffer: 3.5 kHz resonant frequency and 0.03 N/m nominal spring constant. The spring constant of the levers was calibrated by performing a thermal tune of the lever in buffer conditions (fitting of the Brownian motion of the lever) (Hutter and Bechhoefer, 1993; Walters et al., 1996). Typical values of the spring constant varied from 0.030 N/m to 0.055 N/m, but the resonant frequency was consistently Fres= 3.5 kHz. The deflection sensitivity of the detector was calibrated by performing a force curve on a bare mica substrate in dry conditions. During the force measurement cycles, a typical load of <5 nN was applied at a constant loading rate of 1.8 µm/s. Series of 100 curves were recorded at a single location on the sample surface, before the probe was moved to a new location with up to 1000 force distance curves being accrued in each experiment. Finally, after the experiment, the samples were imaged using a new probe to evaluate surface coverage by collagen.
The resulting force-distance curves were subsequently analyzed using all the force curves that showed a stretching event and were fitted with the wormlike chain elasticity model (Bustamante et al., 1994) to determine the contour length of the molecule. Once the contour length had been established a frequency plot of the entire data set is produced, to establish the mean of the distribution (Lo: effective contour length). All numerical data are quoted as the mean ± SD of the data set.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONSUMMARYACKNOWLEDGEMENTSREFERENCES |
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). However, producing recombinant collagen in eukaryotic cells with a defined sequence modification is difficult and collagen, being insoluble after enzymatic cleavage of procollagen, cannot easily be chemically modified. Thus, we took the simple approach of binding the collagen to the analysis surface noncovalently by physisorption. Since the strength of this interaction via van der Waals forces is weak, desorption of the adsorbate can easily occur in a buffer environment due to ionic screening lessening binding and to the substantial, though controlled, forces applied to the molecules during the AFM scanning process. This means that imaging collagen monomers in buffer solution in the absence of covalent linkage to the substrate was not possible; nevertheless, results were obtained when collagen was imaged under dry conditions. The first step toward successful imaging of collagen monomers involved the deposition of collagen onto the mica surface with a low surface coverage to single out individual monomers. This implied adjusting both the concentration of the solution used as well as the corresponding incubation times. Fig. 1 presents two images obtained from solutions that were, respectively: a), 1 µg/ml for an incubation time of 10 min; b), 10 µg/ml for an incubation time of 5min; and c), 1 µg/ml for an incubation time of 10 min, the solution left to rest at room temperature for 8 h before incubation.
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, 1995). Though the mechanism by which collagen molecules aggregates to form a fibrillar network is still not well understood, the presence of the orientation could be the first step toward molecular aggregation and a possible fibrillogenesis. For reference, fibrillogenesis usually takes place under low pH (4.2) and low salt concentrations, resulting in the formation of collagen fibers with the characteristic D-banding (Christiansen et al., 2000). Jiang et al. (2004), have recently published similar results demonstrating that the assembly of collagen monomers into ultrathin highly anisotropic ribbon structures is dependent on the pH of the buffer. In this publication, Jiang and his co-workers observed very similar patterns to that presented in Fig. 1 b, when the pH of the buffer was 7.5. This result was confirmed by considering Fig. 1 c. In this case, a low-concentration solution of collagen molecules was left to rest for a period of 8 h at room temperature. The resulting preparation enables molecular aggregation to occur. In this figure, a general orientation of the molecule is again observed but this time, the molecules have formed ribbons or prefibrillar structures which would support a case for fibrillogenesis to occur under defined conditions (Jiang et al., 2004).
Topographic features of collagen monomers
Topographic features of single type I collagen monomers, as presented in Fig. 1 a, were analyzed and found to have the following characteristics (Table 1). First, the contour length of the monomer, 287 ± 35 nm, corresponds to the value quoted in literature from collagen monomers with comparable numbers of residues. Second, the height of the monomer is smaller, 0.21 ± 0.03 nm, than the theoretical radius of the monomeric collagen triple helix (1.5 nm), whereas the measured width is much larger than predicted, 8.3 ± 0.7 nm (note that no probe deconvolution was performed). This deviation from predicted dimensions is commonly found and has been reported widely for DNA (Fritz et al., 1995; Schabert and Rabe, 1996). In our study, collagen was imaged in a dry environment where the molecule tends to collapse onto a surface, due to either the load applied by the mode of imaging (tapping mode in this case) or the binding forces between the monomer and substrate (stronger van der Waals forces).
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8 nm (n = 20). A possible explanation for this repeat pattern could be that the probe was following the coil-pitch of the collagen molecule. Indeed, the theoretical value of this coil-pitch is known to be 85.5 Å from x-ray analysis (Beck and Brodsky, 1998). However these results should be treated with some caution as similar experiments, carried out on DNA to study the coil-pitch of the double strand, have resulted in observation of apparent axial features (Hansma and Hansma, 1993; Han et al., 1995; Zhang et al., 1996) though proof that they relate to true molecular features is lacking. It is known that the AFM can generate probe-sample artifacts that would convolute real features of the sample with the end of the tip itself (typically 10 nm for tips used in this experiment) resulting in a false impression of the features in either width and/or length (Morris et al., 1999). This tends to occur when the repeat pattern has similar dimensions to that the tip itself. In our experiments, measurements of the coil-pitch induced repeats were taken in directions perpendicular and parallel to the scanning axis, which suggest that these observed repeats could be real surface features on the collagen molecule.
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), and, during this process, the amino and the carboxylic termini are cleaved, leaving the triple helix uncapped at both its ends. Therefore, it is possible that the triple helix could fray, resulting in a much wider end of the monomer, or it could be simply that the free end of the monomer refolds back on itself. Recent x-ray fiber diffraction experiments performed on the axial structure of the C-terminal telopeptide region (Orgel et al., 2000) supports this latter possibility; these showed the occurrence of a hairpin conformation with the C-terminus folded back onto the triple helix.
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). The protein concentration and incubation times were those that were previously described (Fig. 1 a) and the force measurements were performed in PBS buffer (pH 7.4).
Fig. 4 a, shows a typical force versus distance curve with a complex structure containing features similar to that of those that Gutsmann et al. (2004) reported when studying the stretching pattern of macromolecular complexes of collagen (i.e., fibers consisting of several thousands of molecules) originating from rat tail tendon.
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To understand fully the mechanical properties of collagen, it is essential to know whether a single monomer or a series of monomers are being stretched at the same time. In common with the majority of AFM "pulling" experiments no specific covalent coupling between the AFM tip and collagen was used in our studies; thus there is no control over the binding process and hence no guarantee that single molecules are being examined. Nevertheless, by considering the stretching pattern of repeated experiments, one can differentiate between single and multiple stretching events. As mentioned earlier, two overall types of stretching pattern are observed: a unique stretching peak similar to those observed by Sun et al. (2002) or a complex series of stretching peaks as observed by Gutsmann et al. (2004). Thus, comparing our results with the above, one can consider a single stretching peak as being the mechanical response of a single monomer and a multiple stretching peak pattern to be the convoluted response of multiple monomers attached onto the probe.
Substrate binding and contour length
To understand, the mechanical behavior of the molecule, several parameters have to be assessed. First, it was noticed that the binding affinity of the collagen to the AFM tip was high compared to similar single molecule force measurement experiments. Typically, stretching events were detected in >26% of the force distance curves (picoforce, 26.3%; MFP, 27.4%).
A second point, as illustrated by Fig. 4 c, involves sample desorption. This phenomenon occurs when a plateau appears in the retraction curve after the pull-off of the probe (Conti et al., 2001). In our experiments, this was found to be uncommon (<1%) suggesting that seldom desorption of the molecule from either the tip or the substrate occurs during the stretching process.
To explain the mechanical behavior of the monomer, as expressed by the peaks present in the force-distance curves illustrated in Fig. 4, it was necessary to analyze them quantitatively using a classical entropic treatment such as the wormlike chain (WLC) model (Bustamante et al., 1994). Similar studies involving the characterization of the mechanical properties of DNA (Baumann et al., 1997; Wang et al., 1997) and titin (Rief et al., 1997; Tskhovrebova et al., 1997) suggested that this approach could also be applied to the collagen molecule.
The WLC model is often used to describe the mechanical behavior of a protein as it is being stretched or extended, by establishing a relation between the extension of the protein itself and the entropic force induced by the extension. The WLC can be written as follows:
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). Sun et al. (2002) used the endogenous cysteine residues in procollagen, though this limits observations to the precursor protein (2002). Manipulating the mature form might be problematic as it would require extensive molecular biology to insert specific binding tags into such a complex molecule as collagen—both termini would require differential tagging on one of the components of the triple helix at a location that would be perfectly exposed after proteolytic processing of the procollagen; a more fruitful approach may be to chemically modify the mature triple helix, though the extensive amino acid repeats in the collagen primary sequence would present other challenges.
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18% of the entire data set. The WLC fitted the lower part of the curvature of the peak below the discontinuity well, whereas the upper part did not (a similar behavior is observed with peaks that do not show this discontinuity). This suggests that the curvature of the peak contains two mechanical behaviors, of which the WLC can only model one effectively. In force-distance measurements as performed on titin (Rief et al., 1997), for example, it is common not to include the last peak in the fitting of the WLC, as it is often considered as the pull-off the probe. However, in most of the force curves recorded herein, there is only one peak, compatible with collagen not being a modular protein. Thus, it is probable, as argued above, that information regarding the stretching of the monomer is included in the same peak as the pull-off peak and the presence of this discontinuity marks a transition between two mechanical regimes. The first one is situated below the discontinuity and corresponds to the portion of the curves that can be fitted by the WLC where there is true elastic stretching of the monomer. Other physical techniques will be required to examine the changes in topology that occur during this transition. Though we have no direct evidence, it is may be that it represents stretching and/or unwinding of the monomer. The second regime present in the stretching peak is more generally known as the pull-off peak. This occurs when the probe is still being retracted and therefore still applying an increasing force to the molecule, despite the molecule itself having undergone a transition. In this regime, the molecule is inelastic and the WLC model does not fit, meaning that if the binding forces in the probe-molecule-substrate system are strong enough, the internal structure of the monomer may undergo irreversible changes. Once the force exerted by the retraction of the probe exceeds that of the binding of the probe-molecule-substrate system, there is a rupture either of the probe-monomer bound or substrate-monomer bound. There have been several accounts in the literature where partial relaxation or even plateau occurring in the stretching curve of single molecule could be related to conformational changes in the system. Marszalek et al. (2002) have, for example, studied the chair-boat transitions occurring while stretching polysaccharides molecules. They found that structural rearrangement may occur when the molecule of amylose is submitted to large extension force. The molecule undergoes that release of tension by changing its native conformation (chair) to a longer conformation (boat). In the case of the collagen molecule, the nature of the phenomenon that leads to a discontinuity in the stretching peak or in the transition in the mechanical behavior is yet to be elucidated, but could be related to the winding of the monomer. As mentioned earlier, the tropocollagen molecule consists of three left-handed coiled polypeptide chains intertwined in an overall right-handed coil. As the molecule is being stretched, it is possible that the right-handed coil unwinds, leading to a small extension of the molecule length. Once this occurs, further stretching may involve a stress being applied to the hydrogen bonds that link the polypeptide chains together. A large stress may induce breakage in those bonds and therefore prevent the molecule for regaining its original conformation. A more rational explanation may come from structural variations that occur throughout the molecule. It is possible for the monomer to experience some micro-unfolding if there is an interruption in the continuity of the polypeptide chains due to the absence of prolines and hydroxyprolines at the respective X- and Y-sites of the Gly-X-Y repeat sequence. These localized structural differences could affect the local flexibility of the molecule sufficiently to influence the mechanical properties measured by AFM pulling. Perret et al. (2001) have assayed unhydroxylated recombinant collagen I homotrimer, native collagen I homotrimer and heterotrimer using dynamic light scattering. They found the absence of hydroxylated groups does influence the elasticity of the whole system, reinforcing that the basis of the inherent flexibility of the collagen molecule, is not only due to its conformation, but also to the its nature and the amino-acids contents. |
SUMMARY |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONSUMMARYACKNOWLEDGEMENTSREFERENCES |
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). It is interesting that the conditions under which these occurs are not similar to those used in published work on collagen fibrillogenesis (Christiansen et al., 2000).
The main focus of this publication revolves around understanding the mechanical behavior of the collagen monomer under applied stress. The force-distance curves obtained included those with multiple stretching peaks that were also observed in a more complex manner in rat tail collagen fibers in the recent publication by Gutsmann et al. (2004). The apparent simplicity of the force-distance curves used in our study emerged from the fact single monomers were pulled whereas others used macromolecular complexes of collagen fibers. The generic aim of this study was to further the understanding of the mechanical behavior of the basic building block, collagen, of skeletal tissues. There have been many studies involving macro- or mesoscale mechanical studies of these collageneous tissues, but none include the contribution of the monomeric mechanical response. Understanding the mechanical response of type I tropocollagen could, for example, may contribute to a more complete knowledge of the characteristic stress-strain curve of tendons. The implication of such data could lead to a better understanding of the tendon damage such as occurs in tendinitis or partial tendon rupture (Karlsson et al., 1992; Uhthoff and Sano, 1997). These experiments offer a precursor approach to a complete model of collagen biomechanics ranging from single molecules to entire tissues.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTS AND DISCUSSIONSUMMARYACKNOWLEDGEMENTSREFERENCES |
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Submitted on November 30, 2004; accepted for publication March 15, 2005.
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) indicates when the molecule starts being stretched. The lack of deflection between the two markers indicates that the monomer is being straightened with such a low force that the cantilever cannot detect it. (c) Stretching pattern presenting a plateau after the pull-off indicating the desorption or peel-off of the molecule from the surface. (Inset) Zoom on plateau after the pull-off.
