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Conformational Analysis of Native Fibronectin by Means of Force Spectroscopy York Oberdo¨rfer, Harald Fuchs, and Andreas Janshoff* Physikalisches Institut, Westfa¨ lische Wilhelms-Universita¨ t, Wilhelm-Klemm-Strasse 10, 48149 Mu¨ nster, Germany Received June 12, 2000 Force measurements provide an excellent tool to study the folding and unfolding of single-filamentous proteins. In particular, elastic properties of muscle proteins and proteins of the extracellular matrix are of paramount interest for the understanding of structure-function relationships of fibrous proteins. In this study, we focused on the unfolding of native fibronectin, a modular extracellular matrix protein comprising different types of repeating units exhibiting a large variety of functions. Native fibronectin (220-250 kDa) from blood plasma is a dimer composed of at least three classes of repeating units, FN-I, -II, and -III domains. The domains differing in the number of amino acids all have a β-barrel structure. Statistical analysis of force-extension curves clearly revealed the distinct unfolding of FN-I (45 amino acids), FN-II (60 amino acids), and FN-III (90 amino acids). Besides data about mechanical properties of the filament, the analysis provided also information about the absolute composition and the actual number of amino acids of each domain. Moreover, information about the potential topology may be inferred from computer simulations.
Introduction Since its invention in 1986 by Binnig, Quate, and Gerber1 force microscopy has evolved from a predominantly imaging technique to a versatile tool that allows investigation of molecular forces at interfaces in great detail.2 The cantilever of the microscope acts as a sensor for the local interaction between tip and sample ranging from colloidal forces up to ligand-receptor interactions of biomolecules.3 More recently, force spectroscopy has been utilized to study mechanical properties of synthetic polymers and biomolecules such as DNA, various polysaccharides, and modular protein filaments. Elasticity of polymers has been investigated at the level of interatomic bond flips and conformational changes such as the B-S transition of duplex DNA4,5 and the chair-twisted boat transition of dextran filaments.6-9 Unfolding of protein subunits is of particular interest to unravel the structurefunction relationship of muscle proteins and extracellular matrix proteins involved in cell attachment. A considerable number of data about the mechanical properties of titin, a giant muscle protein consisting of structurally similar immunoglobuline (Ig) and fibronectin (FN-III) subunits adopting a β-barrel structure is available from force spectroscopy by optical tweezers and force microscopes.9-12 Stretching such a modular protein results in a subsequent unfolding of the globular domains: the * To whom correspondence should be addressed. Phone: +49/ 251/8339111. Fax: +49/251/8333602. E-mail:
[email protected]. (1) Binnig, G.; Quate, C. F.; Gerber, C. Phys. Rev. Lett. 1986, 930. (2) Janshoff, A.; Neitzert, M.; Oberdo¨rfer, Y.; Fuchs, H. Angew. Chem., Int. Ed. 2000, 39, 3346. (3) Capella, B.; Dietler, G. Surf. Sci. Rep. 1999, 34, 1. (4) Austin, R. H.; Brody, J. P.; Cox, E. C.; Duke, T.; Volkmuth, W. Phys. Today 1997, 50, 32. (5) Cluzel, P.; Lebrun, A.; Heller, C.; Lavery, R.; Viovy, J.-L.; Chatenay, D.; Caron, F. Science 1996, 271, 792. Smith, S. B.; Cui, Y.; Bustamante, C. Science 1996, 271, 795. (6) Rief, M.; Oesterhelt, F.; Heymann, B.; Gaub, H. E. Science 1997, 275, 1295. (7) Marszalek, P. E.; Oberhauser, A. F.; Pang, Y.-P.; Fernandez, J. M. Nature 1998, 396, 661. (8) Li, H.; Rief, M.; Oesterhelt, F.; Gaub,H. E. Adv. Mater. 1998, 3, 316. (9) Rief, M.; Fernandez, J. M.; Gaub, H. E. Phys. Rev. Lett. 1998, 81, 4764. (10) Rief, M.; Gautel, M.; Schemmel, A.; Gaub, H. E. Biophys. J. 1998, 75, 3008.
sequence is determined by the stability of the subunits; the less stable units unfold first. Each unfolding event is accompanied by a lengthening of the filament resulting in a sawtooth profile of the force-extension curve. These kinds of profiles were observed for native and recombinant proteins such as titin,12 tenascin,13 and spectrin.11 The change from the folded to the unfolded state of each domain is highly cooperative without the occurrence of intermediate species and could successfully be modeled by means of Monte Carlo (MC) simulations.9 However, unfolding of the entire filament is not cooperative, which means that each unfolding event of a subunit occurs independently. Many proteins are designed to cope with mechanical load: actin and myosin are responsible for muscle contraction, whereas the stability of connective tissue and cartilage are based on a network of extracellular matrix proteins composed of laminin, entactin, collagen, vimentin, fibronectin, and proteoglycans. Here, we report for the first time on a complete conformational analysis of a modular protein, native fibronectin, consisting of several different classes of globular subunits by means of force spectroscopy employing an atomic force microscope. To our knowledge we are the first to describe force spectroscopy experiments revealing the unfolding of three different subunits in a native protein. Statistical analysis provides a powerful means to determine the number of amino acids of each subunit and the composition of the protein. Our goal is to demonstrate how force spectroscopy of proteins may provide a new means to explore protein composition along with mechanical properties. Fibronectin contains 220 kDa subunits linked into dimers and polymers by disulfide bonds.14 It binds to the cell surface and a number of other extracellular molecules to mediate cell adhesion, binding of collagen, fibrin, heparin, actin, and several other molecules. Fibronectin contains small repeating units of 45-90 amino acids (aa). These regions of primary sequence homology are termed (11) Rief, M.; Pascual, J.; Saraste, M.; Gaub, H. E. J. Mol. Biol. 1999, 286, 553. (12) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. Science 1997, 276, 1109. (13) Oberhauser, A. F.; Marszalek, P. E.; Erickson, H. P.; Fernandez, J. M. Nature 1998, 339, 181. (14) Yamada, K. M. Annu. Rev. Biochem. 1983, 52, 761.
10.1021/la0008176 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/23/2000
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Chart 1. Dimer of Plasma Fibronectin Indicating Structural Domainsa
a Fibronectin is mainly composed of FN-I (45 aa), -II (60 aa), and -III (90 aa) domains. The monomers are connected to each other via disulfide bonds and have a length of ∼62 nm. Cell binding to integrins is mainly provided by FN-III domains, which carry the RGD sequence, while attachment to collagen and fibrin is realized by FN-I subunits.
types FN-I (45 aa), FN-II (60 aa), and FN-III (90 aa). FN-I is present at least 12 times in the fibronectin molecule mainly located near its amino terminus, while FN-III occurs 18 times and FN-II merely 2 times in a single chain.14 Plasma fibronectin as used in this study is composed of A and B chains, which differ slightly in molecular weight and are thought to form disulfide-linked heterodimers. Chart 1 illustrates the composition of plasma fibronectin and sequence of domain occurrence in the filament.15 Experimental Section Bovine fibronectin from plasma (Sigma, Deisenhofen, Germany) was adsorbed to a Petri dish from a 0.2 mg/mL solution in ultrapure water. Before use, the Petri dish was cleaned and exposed to a high-energy argon plasma for 15 min to improve protein adhesion. After the protein was dried, the dish was refilled with 20 mM PBS buffer (pH 7). Measurements were performed with a Bioscope/Nanoscope IIIa microscope (Digital Instruments, Santa Barbara, CA) using oxide-sharpened silicon nitride cantilevers (Nanoscope, Digital Instruments) with nominal spring constants between 0.01 and 0.1 N/m in buffer. The spring constants were determined by means of the thermal noise method.16 The velocity of the cantilever was in the range of 1 µm/s, and contact time on the surface was 0.5 s if not specified otherwise. Each curve was recorded with a resolution of at least 4096 data points. Data reduction was performed with ForceView.17 For the MC simulation a simple two-state model was assumed. The low-energy state is defined as the folded and the high-energy state is the unfolded state. The WLC model (eq 1) was used to describe the force as a function of fibronectin extension. The contour length L was assumed to be a function of the number of unfolded modules Nu with a length of lu and number of folded modules Nf with a length of lf (L ) Nflf + Nulu). The actual polymer length is determined by a kinetic description following transition state kinetics as described in detail by Rief et al.9 The simulation was applied to the stretching experiments of fibronectin with the parameters specified in the legend of Figure 3.
Results and Discussion Fibronectin was immobilized between tip and sample by nonspecific adhesion and deflection-piezo movement curves were measured in 20 mM PBS buffer. Parts A and B of Figure 1 show typical deflection-piezo movement curves of native fibronectin. In each case, a subsequent unfolding of the filament stretched between tip and substrate is visible by the appearance of a regular sawtooth profile in the retraction curve. In general, two scenarios are conceivable, both explaining the occurrence of a sawtooth profile. In the first scenario, the molecule is stretched until unfolding of the first and weakest subunit occurs accompanied by lengthening of the protein filament determined by the length difference between the folded and unfolded subunit. This procedure is repeated for all subunits located between tip and sample until the filament detaches from either tip or sample surface. Since the filament is picked up by the tip in a random fashion, the
number of subunits stretched between tip and sample differs significantly from experiment to experiment. The average number of observed unfolding events was between 8 and 12 for the fibronectin dimer. The sawtooth profile of force-extension curves originating from subsequent unfolding of modular proteins has been described for titin,9,12 tenascin,13 and spectrin.11 Alternatively to this explanation it is also conceivable that pulling on the molecules leads to a gradual detachment of the modules from the surface, giving rise to several adhesion peaks. Detachment from the surface, however, usually results in the occurrence of an irregular pattern of rupture events with different adhesion forces ranging from a few piconewtons to several nanonewtons representing the nonspecific adhesion of the filament to the substrate. Moreover, even if a regular pattern occurs, it can only be due to the gradual detachment of unfolded modules from the surface.18 This would lead to spacings between the adhesion peaks of merely 3-4 nm. However, the separation observed in our experiments was between 10 and 30 nm. Therefore, we conclude that the appearance of a regular sawtooth profile is due to subsequent unfolding of protein modules rather than caused by a gradual detachment of modules from the surface. Different kinds of unfolding events could be distinguished by different lengthening. Occasionally, differences in lengthening were observed in a single retraction curve as shown in Figure 1A. The distinct unfolding events can be attributed to the unfolding of either FN-I (45 aa), FNII (60 aa), or FN-III (90 aa) subunits varying in length as determined by the number of amino acids. Figure 1B shows a filament that has been stretched and relaxed without detachment from tip or substrate indicated by the fact that the retraction curve does not exhibit a snap-off. Nonspecific adhesion between tip and fibronectin usually exceeded 500 pN ensuring that unfolding takes place before the filament loses contact to either substrate or tip. Typical unfolding forces were in the range of 50-250 pN. Strikingly, the approach curves did not exhibit any refolding events within the time scale of the extensionrelaxation cycle. Altogether, the obtained force-extension curves reveal three main classes of unfolding events characterized by different lengthening of the subunits (Figure 2). Figure 2A shows a force-extension curve where the unfolding of FN-I domains is discernible characterized by a lengthening of 10-12 nm, while Figure 2B displays the subsequent unfolding of six FN-III subunits separated by ca. 25-30 nm. The lengthening was determined from the separation between two unfolding events. This procedure proved to (15) Romberger, D. J. J. Int. J. Biochem. Cell. Biol. 1997, 29, 939. (16) Hutter, J. L.; Bechhoefer, J. Rev. Sci. Instrum. 1993, 64, 1868. (17) ForceView can be downloaded from http:\\www.forceview.de. (18) Haupt, B. J.; Ennis, J.; Sevick, E. M. Langmuir 1999, 15, 3886.
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Figure 1. Typical force experiments of native fibronectin. The deflection-piezo movement curves display the extension of plasma fibronectin accompanied by an irreversible unfolding of different subunits. (A) Deflection-piezo movement curve of a single molecule detaching from tip or substrate after 195 nm. A fully extended monomer exhibits a length of ∼1.2 µm. The curve shows the typical sawtooth profile indicative of the unfolding of FN-I and FN-III domains. The inset shows two different force curves, one displaying mainly FN-I unfolding events and the other one predominately showing unfolding of FN-III. (B and C) The filament is kept suspended between tip and substrate revealing the irreversible nature of the unfolding events upon relaxation. This is evident from the shape and length of the extension curves. Unfolding of FN-I and -III domains is discernible upon extension of the filament. (C) The approach curves do not display any refolding events as expected from the asymmetric potential of the folded and unfolded states.9 The molecule could be stretched and relaxed several times. The number of unfolding events decreases with time. The deflection offset in Figure 1C was set to arbitrary values in order to display all extension-relaxation cycles in one figure.
be more reliable than fitting each extension curve with the wormlike chain (WLC) model9 and extracting the contour length differences since in some cases the number of data points on the curve was too low to provide reliable fitting results. However, we corroborated the lengthening
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Figure 2. (A) Typical force-extension curve displaying the unfolding of FN-I domains. The unfolding forces are 80-90 pN with a pulling velocity of 1 µm/s. Lengthening of FN-I subunits is limited to 10-12 nm. (B) Subsequent unfolding of six FN-III domains occurring at forces of 100-150 pN at a pulling velocity of 1 µm/s. (C) Histogram showing the lengthening of different domains due to subsequent unfolding of the modular filament fibronectin. Three Gaussian peaks were fitted to the data with peak maximums at 12.9 ( 0.6 nm representing FN-I, 18.2 ( 0.9 nm for FN-II, and 25.1 ( 0.5 nm for FN-III units. The integral of the corresponding Gaussian curves is related to the percentage of subunit occurrence in native fibronectin, i.e., 153.7 for FN-I, 38 for FN-II, and 280.5 events for FN-III subunits.
obtained from fitting the contour lengths of the WLC model to the data and determination of the separation between two unfolding events for particular cases, in which fitting gave trustworthy results. For both procedures we obtained almost the same results. Statistical analysis of different unfolding events with respect to the lengthening of the subunits was performed to determine the composition of the filament (Figure 2C). The number of unfolding events with a particular spacing is plotted versus the lengthening of the domain. From structural data of native fibronectin, we expected to find a distribution composed of three Gaussians. This is based on the fact that the protein consists of three classes of modules.14 Fitting of three Gaussian curves to the histogram data results in domain lengthening of 12.9 ( 0.6 nm for FN-I, 18.2 ( 0.9 nm for FN-II, and 25.1 ( 0.5
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nm for FN-III units. This is in good agreement with the results of Rief et al., who investigated filaments containing solely FN-III domains obtaining values between 28 and 32 nm depending on the construct.9-12 They demonstrated that the resolution of force measurements is sufficient to distinguish between subunits differing in a single amino acid.10 Integration of the Gaussian peaks (Figure 2C) corresponds to the number of unfolding events of the different subunits, which is in excellent agreement with the average natural occurrence of FN-I, -II, and -III domains in plasma fibronectin (Chart 1).15 FN-I (45 aa) occurs 12 times, equivalent to 38% of all subunits in native FN monomers in agreement with the statistical unfolding of FN-I of 32% of all unfolding events. FN-II (60 aa) is found merely two times in native FN monomers14,15 (6%), and unfolding of type II units was only observed in 8% of all cases. FN-III domains (90 aa) are found 18 times in fibronectin (56%), which is in good agreement with the experimental data revealing unfolding of modules with a length difference between folded and unfolded states of 25 nm in 60%. This result demonstrates how accurately the composition of the native protein can be unraveled by means of simple stretching experiments. Assuming that a lengthening of 25.1 nm corresponds to a subunit of 90 amino acids as deduced from extension curves of FN-III domains, 18.2 nm lengthening computes to 65 amino acids for FN-II and 46 amino acids for FN-I domains. This is in good accordance to the real number of amino acids, i.e., 60 aa for FN-II and 45 aa for FN-I. Mechanical parameters are obtained from fitting the parameters of a WLC model to the data (Figure 3)9
F(x) )
kBT 1 1 (1 - x/L)-2 + x/L lp 4 4
[
]
(1)
L denotes the contour length of the filament and lp the persistence length. This model has been proven well-suited to describe the elasticity of protein filaments from muscle and extracellular matrix up to forces of several hundred piconewtons.10 The solid lines shown in Figure 3 are the fitting results of the WLC model providing the persistence length as a measure for the bending rigidity lp of the molecule and the contour length L. The persistence length obtained from fitting the parameters of the WLC model to the data is 0.4 ( 0.1 nm for FN-III as shown in Figure 3, in good agreement with previous work.10 Generally, the persistence lengths of all three classes are in the same range of 0.3 ( 0.2 nm indicative of domain homology as far as mechanical properties are concerned. It should be mentioned that the persistence length at low force (50 pN) characteristic for a conformational change upon stretching.10 Deviations in persistence lengths (up to 50% error) are mainly due to inaccuracy of spring constant determination (20-30% error) and the sometimes limited number of data points preventing reliable fitting procedures. Unfolding forces for all subunits were in the range of 70-150 pN (Figure 2A,B) as previously reported by Rief et al. for FN-III domains (113 pN for tenascin-FN-III at a pulling velocity of 0.5 µm/s).10 In particular, the unfolding force observed for FN-I (70-100 pN) was slightly lower than that for FN-II, and FN-III (90-150 pN) modules and therefore unfolding of FN-I occurs before FN-II or -III modules break off. The lower unfolding forces for FN-I
Figure 3. Comparison of a simple Monte Carlo simulation (top) of the subsequent unfolding of three FN-III domains with experimental data (bottom):9 pulling velocity, 1 µm/s; potential width, 15 nm (xf) and 0.55 nm (xu); persistence length (lp), 0.4 nm; rate constant in the absence of an external force, 3 × 10-5 s-1 for unfolding and 2 s-1 for refolding.9 The solid lines in the bottom figure are the results of the WLC fit to the data revealing a persistence length of 0.4 ( 0.1 nm. Unfolding forces are between 80 and 130 pN. The difference between folded and unfolded length of the domain in the MC simulation was set to 26 nm in accordance to the statistical analysis shown in Figure 2C.
domains are indicative of a larger value for xu (potential width of unfolding) of about 1 nm as shown by MC simulations. Unfolding of fibronectin is irreversible within the time scale of one single extension-relaxation cycle in accordance with the behavior of other proteins such as spectrin, titin, and tenascin. No refolding events were observed upon moving the cantilever again toward the surface (Figure 1B,C approach curves). None of the approach curves displays refolding events. Reduction of the contact time to zero seconds results in a significant suppression of refolding events during contact; thus fewer unfolding events were observed in the second approachretraction cycle (Figure 1C). The number of discernible unfolding events increases with contact time indicative of the fact that the protein needs time to refold in its native conformation. In summary, force spectroscopy of modular proteins in conjunction with statistical analysis provides a quantitative measure of protein composition, structure, and potential topology along with mechanical properties. The study shows that force spectroscopy of proteins may be employed to derive their composition, structural integrity, and mechanical properties such as the bending modules. Acknowledgment. The authors are very much indebted to H.-J. Galla for his extraordinary support, C. Steinem for many fruitful discussions, and M. Neitzert for his valuable initial work. LA0008176
