Reversible Mechanical Unzipping of Amyloid β-Fibrils
2005; Elsevier BV; Volume: 280; Issue: 9 Linguagem: Inglês
10.1074/jbc.m411556200
ISSN1083-351X
AutoresMiklós Kellermayer, László Grama, Árpád Karsai, Attila Nagy, Amram Kahn, Zsolt Datki, Botond Penke,
Tópico(s)Hydrogels: synthesis, properties, applications
ResumoAmyloid fibrils are self-associating filamentous structures, the deposition of which is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease and various other disorders. Here we used single molecule manipulation methods to explore the mechanics and structural dynamics of amyloid fibrils. In mechanically manipulated amyloid fibrils, formed from either amyloid β (Aβ) peptides 1-40 or 25-35, β-sheets behave as elastic structures that can be "unzipped" from the fibril with constant forces. The unzipping forces were different for Aβ1-40 and Aβ25-35. Unzipping was fully reversible across a wide range of stretch rates provided that coupling, via the β-sheet, between bound and dissociated states was maintained. The rapid, cooperative zipping together of β-sheets could be an important mechanism behind the self-assembly of amyloid fibrils. The repetitive force patterns contribute to a mechanical fingerprint that could be utilized in the characterization of different amyloid fibrils. Amyloid fibrils are self-associating filamentous structures, the deposition of which is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease and various other disorders. Here we used single molecule manipulation methods to explore the mechanics and structural dynamics of amyloid fibrils. In mechanically manipulated amyloid fibrils, formed from either amyloid β (Aβ) peptides 1-40 or 25-35, β-sheets behave as elastic structures that can be "unzipped" from the fibril with constant forces. The unzipping forces were different for Aβ1-40 and Aβ25-35. Unzipping was fully reversible across a wide range of stretch rates provided that coupling, via the β-sheet, between bound and dissociated states was maintained. The rapid, cooperative zipping together of β-sheets could be an important mechanism behind the self-assembly of amyloid fibrils. The repetitive force patterns contribute to a mechanical fingerprint that could be utilized in the characterization of different amyloid fibrils. Amyloid fibrils are self-associating filamentous structures formed from the 39-43-residue-long amyloid β-peptide (Aβ) 1The abbreviations used are: Aβ, amyloid β-peptide; AFM, atomic force microscopy. or its subfragments (1Serpell L.C. Biochim. Biophys. Acta. 2000; 1502: 16-30Crossref PubMed Scopus (833) Google Scholar). The deposition of amyloid oligomers (2Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D.J. Nature. 2002; 416: 535-539Crossref PubMed Scopus (3721) Google Scholar) and fibrils is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease (3Selkoe D.J. Am. J. 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Single molecule manipulation experiments have in the recent past provided unique and unprecedented insights not only into the structure and elasticity but also into mechanically driven transitions of molecular systems (13Smith S.B. Cui Y. Bustamante C. Science. 1996; 271: 795-799Crossref PubMed Scopus (2381) Google Scholar, 14Kellermayer M.S.Z. Smith S.B. Granzier H.L. Bustamante C. Science. 1997; 276: 1112-1116Crossref PubMed Scopus (1034) Google Scholar, 15Rief M. Gautel M. Oesterhelt F. Fernandez J.M. Gaub H.E. Science. 1997; 276: 1109-1112Crossref PubMed Scopus (2620) Google Scholar, 16Tskhovrebova L. Trinick J. Sleep J.A. Simmons R.M. Nature. 1997; 387: 308-312Crossref PubMed Scopus (668) Google Scholar, 17Fisher T.E. Oberhauser A.F. Carrion-Vazquez M. Marszalek P.E. Fernandez J.M. Trends Biochem. Sci. 1999; 24: 379-384Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 18Fisher T.E. Marszalek P.E. Fernandez J.M. Nat. Struct. 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This rapid, mechanical zipping together of β-sheets could be an important mechanism behind cooperative amyloid fibril formation. Samples—Amyloid peptides were prepared by solid phase synthesis (22Palota ́s A. Ka ́lma ́n J. Palota ́s M. Juha ́sz A. Janka Z. Penke B. Brain Res. Bull. 2002; 58: 203-205Crossref PubMed Scopus (23) Google Scholar). Aβ25-35 and Aβ1-40 peptides contained amino acid residues 25-35 and 1-40 of the β-peptide, respectively. Fibrils were generated by dissolving the peptides in PBSA buffer (10 mm K-phosphate, pH 7.4, 140 mm NaCl, 0.02% NaN3) at 0.5 mg/ml concentration. Aβ1-40 and Aβ25-35 fibrils were grown in solution at 25-37 °C for several days. Surface Adsorption of Amyloid Fibrils—Depending on the type of experiment, different surfaces and methods were employed for attaching amyloid β-fibrils. 1) For AFM imaging under ambient conditions, mica surface was used. A 20-μl sample (0.5 mg/ml concentration) was pipetted onto a freshly cleaved mica surface and incubated at room temperature for 10 min. Subsequently, the surface was rinsed with MilliQ water and dried with a stream of clean N2 gas. 2) For molecular force spectroscopy measurements, fibrils were either adsorbed nonspecifically to a precleaned glass surface or attached covalently to a glass coverslip. The two methods gave similar results. Glass coverslips were cleaned by sonication in acetone, followed by rinsing with MilliQ water and drying in a stream of N2 gas. A 20-μl sample (0.5 mg/ml concentration) was pipetted onto the glass surface and incubated at room temperature for 10 min. Unbound fibrils were washed away by rinsing with PBSA buffer. Rinsing was carried out by repetitively (5×) adding and removing 100 μl of buffer solution. The surface was then left covered with PBSA solution. For covalent modification, precleaned coverslips were incubated in toluene vapor containing 2% 3-glycidiloxypropyl-trimethoxisylane (Fluka) for 12 h at room temperature. Attaching of fibrils to the covalently modified surface was carried out as described above, with an additional step: following the attachment of fibrils, the surface was incubated with PBSA buffer of pH 9 to facilitate covalent binding to the amino-modified surface. Single Molecule Force Spectroscopy—Amyloid fibrils were stretched with an AFM (Asylum Research MFP1D or MFP3D) by first pressing the cantilever (Veeco Microlever or Olympus BioLever) tip against the surface and then pulling the cantilever away with a constant, preadjusted rate. The experiments were carried out under aqueous buffer conditions (PBSA buffer, pH 7.4). Stiffness was determined for each cantilever by using the thermal method (23Hutter J.L. Bechhoefer J. Rev. Sci. Instrum. 1993; 64: 1868-1873Crossref Scopus (3451) Google Scholar). Atomic Force Microscopy—Non-contact mode AFM images of amyloid fibrils bound to freshly cleaved mica were prepared with the MFP3D AFM instrument using silicon cantilevers (Olympus AC160, typical resonance frequency ∼300 kHz). 512 × 512 images were collected at a typical scanning frequency of 1 Hz. In Situ Force Spectroscopy—In situ force spectroscopy was carried out by first scanning (under aqueous buffer conditions) the glass-bound sample surface, pressing the cantilever tip to targeted surface locations identified on the image, and finally rescanning the surface to test for the effect of the mechanical perturbations (24Oesterhelt F. Oesterhelt D. Pfeiffer M. Engel A. Gaub H.E. Muller D.J. Science. 2000; 288: 143-146Crossref PubMed Scopus (607) Google Scholar). Soft (typical cantilever stiffness ∼30 pN/nm), high resonance frequency (typical resonance frequency ∼40 kHz) cantilevers (Olympus BioLever, B Lever) were used. Scanning was carried out in non-contact mode at high set-point values (0.8-1 V) to avoid the binding of sample to the cantilever tip. To correct for drift, the same area was analyzed in several cycles of scanning and mechanical probing. Data Analysis—In the case of non-linear mechanical responses, the relaxation curves were fitted with the worm-like chain equation as shown in Equation 1 (25Bustamante C.J. Marko J.F. Siggia E.D. Smith S.B. Science. 1994; 265: 1599-1600Crossref PubMed Scopus (1832) Google Scholar).fAkBT=zL+14(1−z/L)2−14(Eq. 1) Force plateaus were analyzed by measuring the distance between the average plateau force and the force baseline (Fig. 2A, i, inset). Force steps were analyzed by measuring the distance between the average force values of consecutive plateaus (Fig. 2C, inset). Modeling and Simulation—Amyloid fibril unzipping was simulated with a simple elastically coupled two-state model (26Rief M. Fernandez J.M. Gaub H.E. Phys. Rev. Lett. 1998; 81: 4764-4767Crossref Scopus (424) Google Scholar, 27Kellermayer M.S. Bustamante C. Granzier H.L. Biochim. Biophys. Acta. 2003; 1604: 105-114Crossref PubMed Scopus (65) Google Scholar) in which the activation kinetics are influenced by the mechanical load and the shape of the interaction potential holding the amyloid fibril together. In the model, a β-sheet was gradually pulled off the fibril surface. The apparent contour length of the simulated β-sheet was reduced by the presence of a set of bonds spaced 4.7 Å apart. As the chain was extended, force was generated according to the wormlike chain equation (25Bustamante C.J. Marko J.F. Siggia E.D. Smith S.B. Science. 1994; 265: 1599-1600Crossref PubMed Scopus (1832) Google Scholar). This entropic force is counterbalanced with the unzipping (or desorption) force, which may include electrostatic and non-electrostatic (e.g. hydrophobic interactions) components (28Hugel T. Grosholz M. Clausen-Schaumann H. Pfau A. Gaub H.E. Seitz M. Macromolecules. 2001; 34: 1039-1047Crossref Scopus (235) Google Scholar). In each polling interval (dt) the probability of bond rupture (unzipping) at the given force (f) was calculated according to Equation 2,Pu=ω0dte−(Eau−fΔxu)/kBT(Eq. 2) where w0 is attempt frequency set by Brownian dynamics (29Bell G.I. Science. 1978; 200: 618-627Crossref PubMed Scopus (3449) Google Scholar), kBT is thermal energy, Eau is activation energy of unzipping, and Δxu is distance between the bound and transition states along the unzipping reaction coordinate. Similarly, in the same polling interval the probability of binding (zipping) was calculated according to Equation 3,Pz=ω0dte−(Eaz+fΔxz)/kBT(Eq. 3) where Eaz is activation energy of zipping and Δxz is distance between the dissociated and transition states along the zipping reaction coordinate. The unzipping or zipping processes were permitted or prohibited depending on a comparison of P with a number generated randomly between 0 and 1. Each bond rupture event incremented the apparent contour length with 4.7 Å and vice versa. Force Spectroscopy of Aβ-Fibrils—Amyloid fibrils formed from Aβ1-40 or Aβ25-35 peptides were mechanically manipulated using single molecule AFM (27Kellermayer M.S. Bustamante C. Granzier H.L. Biochim. Biophys. Acta. 2003; 1604: 105-114Crossref PubMed Scopus (65) Google Scholar, 30Rief M. Oesterhelt F. Heymann B. Gaub H.E. Science. 1997; 275: 1295-1297Crossref PubMed Scopus (1014) Google Scholar). The tip of an AFM cantilever was pressed against a glass surface coated with the Aβ-fibrils. Subsequently, the cantilever was pulled away from the surface to stretch the fibrils and then returned toward the surface to relax the fibrils. The force and extension data collected during the stretch-relaxation cycles provided a description of the mechanical response of the fibrils. The mechanical behavior of the two different amyloid fibrils was qualitatively similar. Two fundamental types of mechanical responses were deduced: (a) fully reversible, non-linear elasticity (Fig. 1A) with the contour length often exceeding 100 nm, and (b) force plateau characterized with a constant force level during stretch (Fig. 1B). During extension the force plateau was sometimes preceded with a non-linear elastic response (Fig. 1B, inset) and usually ended with an abrupt force drop. Most frequently the two fundamental processes were combined into complex, hierarchical mechanical responses: force plateaus were superimposed onto one another (Fig. 1C) or onto a non-linear force curve (Fig. 1D). Superimposed force plateaus usually ended with a series of decreasing force steps resulting in a descending staircase pattern. Histograms were constructed from the plateau heights (difference between average plateau force and baseline (Fig. 2A, i, inset). The plateau force histograms revealed multimodal distribution for Aβ25-35 (Fig. 2A, ii) and pointed to multimodality for Aβ1-40 (Fig. 2A, i). Although the peaks were difficult to distinguish in the histogram of Aβ1-40 (Fig. 2A, i), a discrete force plateau distribution was expected based on the presence of plateaus that appeared at integral multiples of ∼30 pN (Fig. 2B). A more detailed analysis of the force step heights in data obtained from in situ force spectroscopy measurements (see below) did reveal the multimodality of plateau force distribution for Aβ1-40 fibrils (Fig. 2C). Accordingly, the peaks corresponding to the smallest plateau forces appeared at 33 pN (±7 S.D., n = 258) and 41 pN (±7 S.D., n = 76) for Aβ1-40 and Aβ25-35, respectively. The rest of the peaks appeared at forces that are integral multiples of the 33- or 41-pN unit forces. We found that the plateau transition was reversible and the force staircases were repeatable. There were several manifestations of reversibility. If the pulling experiment was reversed prior to reaching the end of the force plateau, or if the plateau was preceded by non-linear elastic response (Fig. 3A), then the relaxation force curve followed the stretch force curve. The plateau force was independent of stretch rate, and the lack of hysteresis was maintained across two orders of magnitude of stretch velocity (Fig. 3B). Reversible force staircases, with no hysteresis, were also observed (Fig. 3C). Furthermore, these reversible force staircases often persisted through successive stretch-relaxation cycles (Fig. 3C). In these experiments, fluctuations across force steps were also observed (Fig. 3C, inset), indicating that the rate of the underlying structural fluctuations exceeds the stretch (or relaxation) rate. Time-dependent AFM Imaging and Force Spectroscopy—The force spectroscopy results suggested that during stretch elastic strands are pulled off the Aβ-fibril surface. To explore the nature of these strands, time-dependent AFM imaging and force spectroscopy experiments were performed. Fig. 4 shows the results for Aβ1-40 fibrils. Shortly after dissolution in PBSA buffer, globular aggregates were present, but filamentous structures were not observed (Fig. 4, Day 0). In the corresponding force spectra an occasional force sawtooth was observed, but force plateaus and complex force patterns were absent. The length of the captured molecular species was Day 7, lower panel). These preparations contained an abundance of mature Aβ-fibrils. Thus, the time-dependent AFM imaging and force spectroscopic results allowed the tentative conclusion that the prominent mechanical responses (Fig. 1) are associated with mature Aβ-fibrils. In Situ Force Spectroscopy—To further explore the origin of the elastic strand pulled off the fibril surface, we carried out in situ force spectroscopy experiments. A glass surface with covalently attached mature Aβ1-40 fibrils was first gently scanned in non-contact mode under aqueous buffer conditions. Subsequently, force spectroscopy measurements were carried out at specific target locations of the surface. Fig. 5 shows the results for Aβ1-40 fibrils. Repeated attempts of pulling at the control location (Fig. 5, A and B, spot 1), which was devoid of clearly identifiable amyloid fibrils, produced no characteristic force response. By contrast, if fibrils were targeted (spots 2 and 3), characteristic force plateaus and staircases appeared. Plateau forces exceeding 500 pN were often observed (Fig. 5B, ii and iii). Force plateaus longer than 250 nm were seen (Fig. 5B, iv), and force staircase patterns persisted through successive stretch-relaxation cycles (Fig. 5B, iv) in accordance with earlier observations (Fig. 3C). The fundamental force plateau of ∼30 pN was observed to reach a length of >200 nm (Fig. 5B, v). AFM imaging performed following the mechanical perturbations revealed that the fibrils were not removed in toto by the pulling attempts (Fig. 5A, iii and iv). Furthermore, the gross helical structure of the fibrils was not perturbed either, at least not to an extent resolvable under the experimental conditions (Fig. 5C). Thus, the in situ force spectroscopy experiments indicated that the elastic strands pulled away from the Aβ-fibril represent subfibrillar components of the mature fibril. Identity of Strands Unzipped from the Aβ-Fibril—In the present work we mechanically manipulated surface-attached Aβ-fibrils, formed from either Aβ1-40 or Aβ25-35 peptides, by using single molecule force spectroscopy and in situ force spectroscopy techniques. Various combinations of two main phenomena, non-linear elasticity (Fig. 1A) and force plateau (Fig. 1B), dominated the mechanical response of the fibrils. Our results suggested that the mechanical response is determined by the elasticity and interactions of an element within the structural hierarchy of the mature Aβ-fibril. There are three elements, the Aβ-fibril itself, the protofilament, or individual β-sheets, that may in principle explain the observed mechanics (31Roher A.E. Baudry J. Chaney M.O. Kuo Y.M. Stine W.B. Emmerling M.R. Biochim. Biophys. Acta. 2000; 1502: 31-43Crossref PubMed Scopus (92) Google Scholar). First, an entire Aβ-fibril could, in principle, be manipulated and lifted off the substrate surface, resulting in the observed force responses. However, we excluded this possibility because the fibrils were attached firmly (covalently in many experiments) to the substrate, and in situ force spectroscopy experiments demonstrated that fibrils were not displaced in toto by the mechanical perturbations (Fig. 5, B and C). Second, protofilaments could in principle be lifted off the fibril surface, producing the observed mechanical behavior. We excluded this possibility as well, for two main reasons. 1) A single Aβ-fibril contains five or six protofilaments (1Serpell L.C. Biochim. Biophys. Acta. 2000; 1502: 16-30Crossref PubMed Scopus (833) Google Scholar). By contrast, the high plateau forces (Fig. 5B, ii and iii) indicate that the number of strands pulled off the fibril may be up to at least nine (see also explanation below). 2) The gross helical appearance of the Aβ-fibril, which is thought to be determined by the protofilament structure and arrangement (32Khurana R. Ionescu-Zanetti C. Pope M. Li J. Nielson L. Ramirez-Alvarado M. Regan L. Fink A.L. Carter S.A. Biophys. J. 2003; 85: 1135-1144Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), is not altered significantly by the mechanical perturbations (Fig. 5C). Thus, we concluded that the force responses observed in mechanically manipulated Aβ-fibrils are determined by the elasticity and interactions of individual β-sheets. Non-linear Elastic Behavior—The fully reversible, non-linear force response (Fig. 1A) most likely describes the elasticity of the β-sheet that has been liberated from its lateral confinement within the fibril but held firmly at its ends. We could fit the non-linear force curves with the wormlike chain model of entropic elasticity. The persistence length obtained from wormlike chain fits to force data obtained for 5-day-old Aβ1-40 samples was 0.38 nm (±0.06 nm S.E., n = 165), which is comparable with the persistence length of a fully unfolded protein chain (titin) (15Rief M. Gautel M. Oesterhelt F. Fernandez J.M. Gaub H.E. Science. 1997; 276: 1109-1112Crossref PubMed Scopus (2620) Google Scholar). We assumed that the short calculated persistence length is an apparent value, which is an underestimation of the persistence length of the single β-sheet. The apparent persistence length becomes reduced if multiple strands are pulled in parallel (14Kellermayer M.S.Z. Smith S.B. Granzier H.L. Bustamante C. Science. 1997; 276: 1112-1116Crossref PubMed Scopus (1034) Google Scholar, 27Kellermayer M.S. Bustamante C. Granzier H.L. Biochim. Biophys. Acta. 2003; 1604: 105-114Crossref PubMed Scopus (65) Google Scholar) or if force-dependent interactions of the strand occur (33Kellermayer M.S. Smith S.B. Bustamante C. Granzier H.L. Biophys. J. 2001; 80: 852-863Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The β-sheet (or bundles thereof) can withstand forces up to 600 pN (Fig. 1A). The high tensile strength may be explained by a parallel coupling between the numerous hydrogen bonds (6Petkova A.T. Ishii Y. Balbach J.J. Antzutkin O.N. Leapman R.D. Delaglio F. Tycko R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16742-16747Crossref PubMed Scopus (1656) Google Scholar) holding the peptides together along the β-sheet. Considering that the hydrogen bonds lie in a plane perpendicular to the direction of pulling force, the force is distributed among these bonds. Origin of Force Plateau—The force plateau (Fig. 1, B-D) is interpreted to arise from the unzipping of a β-sheet from the fibril. Force plateaus have recently been observed for single polyelectrolyte chains desorbed from charged surfaces (28Hugel T. Grosholz M. Clausen-Schaumann H. Pfau A. Gaub H.E. Seitz M. Macromolecules. 2001; 34: 1039-1047Crossref Scopus (235) Google Scholar, 34Chatellier X. Senden T.J. Joanny J.-F. Di Meglio J.-M. Europhys. Lett. 1998; 41: 303-308Crossref Scopus (112) Google Scholar, 35Haupt B.J. Ennis J. Sevick E.M. Langmuir. 1999; 15: 3886-3892Crossref Scopus (81) Google Scholar, 36Conti M. Bustanji Y. Falini G. Ferruti P. Stefoni S. Samori B. Chemphyschem. 2001; 10: 610-613Crossref Scopus (38) Google Scholar, 37Seitz M. Friedsam C. Jostl W. Hugel T. Gaub H.E. Chemphyschem. 2003; 4: 986-990Crossref PubMed Scopus (67) Google Scholar, 38Friedsam C. Becares A.D.C. Jonas U. Seitz M. Gaub H.E. New J. Phys. 2004; 6: 1-16Crossref Scopus (36) Google Scholar) or for non-electrolyte chains pulled out of a collapsed globule due to Rayleigh instability in poor solvent conditions (39Haupt B.J. Senden T.J. Sevick E.M. Langmuir. 2002; 18: 2174-2182Crossref Scopus (109) Google Scholar). It is unlikely that our findings are related to Rayleigh instability, because amyloid fibrils are highly structured and the observed force plateaus are reversible. During unzipping, the β-sheet, pulled perpendicular relative to the fibril axis, is desorbed, peeled, or unzipped from the fibril. Because of the pulling geometry, the bonds laterally connecting the β-sheet to the underlying fibril surface are loaded and broken individually at the same average force, one after the other as the pulling progresses. Each bond rupture releases a short section of the β-sheet and increases the length of the already liberated β-sheet segment. Bond rupture events were not resolved, because the bonds are too closely spaced (axial β-hairpin spacing ∼4.7 Å) (1Serpell L.C. Biochim. Biophys. Acta. 2000; 1502: 16-30Crossref PubMed Scopus (833) Google Scholar). We observed a significant difference between the unit unzipping forces for Aβ1-40 and Aβ25-35 fibrils (p < 0.0001). The multiple peaks in the plateau force histograms (Fig. 2) were interpreted to arise from the simultaneous unzipping of strands containing different numbers of β-sheets. Accordingly, the smallest unit forces of 33 or 41 pN correspond to the unzipping of single Aβ1-40 or Aβ25-35 β-sheets from the fibril, respectively. It is important to point out that a single β-sheet can interact with the underlying substrate at both of its ends, so as to form a loop, which results in unzipping force that is twice the unit force (see also "Model and Simulation" below). With the unit unzipping forces it is possible to calculate the number of β-sheets involved in a given force plateau transition. For example, the prominent ∼130-pN force plateau seen in Fig. 5B, iv is caused by the unzipping of a minimum of two (if both form loops) and a maximum of four β-sheets (if none forms a loop). At present we can only speculate about the origin of the differences between the unit unzipping forces of Aβ1-40 and Aβ25-35 β-sheets. Considering that amino acid side chain interactions determine the strength of interaction between parallel β-sheets, the origin of the unzipping force difference could be because of differences in the arrangement of the exposed side chains. One possibility is that Lys-28, which participates in forming a salt bridge with Asp-23 that stabilizes the hairpin structure of the Aβ1-40 peptide (6Petkova A.T. Ishii Y. Balbach J.J. Antzutkin O.N. Leapman R.D. Delaglio F. Tycko R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16742-16747Crossref PubMed Scopus (1656) Google Scholar), is exposed in Aβ25-35 and may form a relatively strong interaction with Met-35 of the neighboring peptide (40Terzi E. Holzemann G. Seelig J. Biochemistry. 1994; 33: 7434-7441Crossref PubMed Scopus (177) Google Scholar), resulting in greater unzipping forces. Force Steps and Staircases—The unzipping process continues until the entire β-sheet is liberated, which is marked by an abrupt decrease in force. Descending force staircase (Fig. 1C) arises during the simultaneous unzipping of several β-sheets followed by their gradual, one-by-one (or group-by-group) release from the underlying fibril surface. The presence of force staircase patterns suggests that the β-sheets terminate at different positions, probably because of a staggered arrangement within the fibril. The structural implications for a possible staggered β-sheet arrangement within the amyloid fibril are not fully understood. The position of the force step along the length axis allows the estimation of the length of a β-sheet within the fibril. We found unit unzipping force plateaus up to 220 nm long (Fig. 5B, v), indicating that the length of individual β-sheets within the fibril can be well in excess of 200 nm. Considering that protofibrils, the putative precursors of amyloid fibrils, have a maximum observed length of 200 nm (11Walsh D.M. Lomakin A. Benedek G.B. Condron M.M. Teplow D.B. J. Biol. Chem. 1997; 272: 22364-22372Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar), our findings raised the intriguing possibility that during amyloidogenesis protofibrils fuse by the annealing of their component β-sheets. The observed long β-sheet unzipped from the fibril raises a topological problem. β-sheets are embedded in hierarchically wound helices with a pitch of ∼46 nm (41Malinchik S.B. Inouye H. Szumowski K.E. Kirschner D.A. Biophys. J. 1998; 74: 537-545Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 42Serpell L.C. Sunde M. Benson M.D. Tennent G.A. Pepys M.B. Fraser P.E. J. Mol. Biol. 2000; 300: 1033-1039Crossref PubMed Scopus (299) Google Scholar). Unzipping a β-sheet, the length of which exceeds the helix pitch, is expected to cause torsion or rotation of or within the fibril. Considering that the rotation of the entire fibril was prevented by its firm attachment to the substrate surface, it may be possible that the component protofilaments possess a certain degree of torsional or rotational freedom within the fibril. Reversibility of Force Plateau and Force Step—The lack of force hysteresis often seen in the force responses (Fig. 3) indicates that the mechanically perturbed amyloid fibril system passes through identical structural states during stretch and release and is in thermodynamic equilibrium at each point of extension. The absence of stretch-rate dependence of plateau force (Fig. 3B) indicates that the equilibrium is highly dynamic and the system fluctuates between the (associated and dissociated, or zippered and unzipped) states on a time scale that is much faster than that of the pulling experiment. Because of the equilibrium, the average mechanical energy invested in driving the transition reflects the associated free energy change (ΔG) (43Liphardt J. Dumont S. Smith S.B. Tinoco Jr., I. Bustamante C. Science. 2002; 296: 1832-1835Crossref PubMed Scopus (965) Google Scholar). Considering the mean plateau forces for the single β-sheet and a 4.7 Å monomer spacing (1Serpell L.C. Biochim. Biophys. Acta. 2000; 1502: 16-30Crossref PubMed Scopus (833) Google Scholar), the ΔG of lateral β-sheet binding is ∼1.6 × 10-20 J and ∼2 × 10-20 J/monomer (i.e. ∼9.6 kJ mol-1 and ∼12 kJ mol-1) for Aβ1-40 and Aβ25-35, respectively. The prerequisite of reversibility is coupling, via the β-sheet, between the bonds holding the β-sheet laterally within the fibril. In lieu of coupling the dissociated monomers would diffuse away and reassociation would be unlikely. Coupling is serial in the case of a single β-sheet that is gradually unzipped from the fibril surface. However, parallel coupling can also occur, as in the case of parallel-associated β-sheets. A tight coupling between parallel-arranged β-sheets can result even in the reversal of the force staircase related to complete β-sheet dissociation, and a completely released β-sheet can rebind to the fibril in a reversible reaction (Fig. 3C). Because the complete release and rebinding of a β-sheet results in large force steps, the rapid fluctuation between the states becomes visible in the force traces (Fig. 3C, inset). Repeatability of Mechanical Response—We found that the complex mechanical response of the amyloid fibril was repeatable through successive stretch-relaxation cycles. That is, the force patterns recorded in successive mechanical cycles were identical or very similar (Figs. 3C and 5B, iv). The observation indicates that the structural features of the manipulated strand (e.g. number and arrangement of β-sheets) are preserved and that the mechanical events occur not randomly, but in a sequential manner (44Case R.B. Chang Y.P. Smith S.B. Gore J. Cozzarelli N.R. Bustamante C. Science. 2004; 305: 222-227Crossref PubMed Scopus (43) Google Scholar). Repeatability is easily reconciled if an elastic coupling persists between the cantilever tip and the fibril surface that may guide the β-sheet rebinding through consecutive mechanical cycles (Fig. 3C). However, if the bundle of β-sheets is completely removed by the tip between stretch-relaxation cycles (such as happened in Fig. 5B, iv), the β-sheets first need to be delivered back to the fibril surface and allowed to rebind in a fast reaction. Possibly, under these conditions the underlying fibril surface serves as a spatial guide for structurally correct reassociation. Considering the added complexities due to helical protofibril arrangement, much further work is required to understand the details of the process. Model and Simulation—We have proposed the following model to explain our observations (Fig. 6A). During mechanical manipulation of surface-adsorbed Aβ-fibrils, β-sheets are lifted off the fibril surface (Fig. 6A, i). As the pulling progresses, the β-sheets become unzipped via the sequential rupture of side chain interactions, which convert the β-sheets from an associated to a dissociated state. Because each rupture event increases the length of the unzipped β-sheet with a uniform and very small distance, the unzipping process proceeds at an apparent constant force that results in a force plateau (Fig. 6A, i). Because the β-sheets within a bundle are parallel-coupled, the plateau force scales with the effective (i.e. two in case of a loop) number of β-sheets. If a β-sheet becomes unzipped entirely, the force drops in a unit step. Numerous sequential steps result in a descending force staircase (Fig. 6A, ii). If the β-sheet bundle is parallel-coupled to an elastic element (e.g. a β-sheet already liberated but held at its ends, Fig. 6A, iii), then the shape of the force staircase is distorted by the superimposed non-linear elastic behavior. If the β-sheet is allowed to retract, it reassociates to the underlying fibril in a rapid and an apparently cooperative process. The driving force of the rapid rebinding is the very high local concentration of the conjugate binding sites, which increases with each rebinding event. The rapid rebinding is probably further facilitated by the correct orientation of the conjugate binding sites defined by the structural order of the β-sheet. As a result, the β-sheets are rapidly zippered together. A simulation based on a simple elastically coupled two-state model (26Rief M. Fernandez J.M. Gaub H.E. Phys. Rev. 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Implications—We have mechanically manipulated individual amyloid β-fibrils and argued that their complex mechanical responses are most plausibly explained by a reversible unzipping of long elastic β-sheets from the fibril. The findings are thought to have important implications for understanding the structure, structural dynamics, and mechanisms of formation of amyloid fibrils. The reversible rebinding of β-sheets at high loads and loading rates to the underlying fibril surface indicates that the associated state is strongly favored and a mechanically perturbed amyloid fibril is rapidly recovered by zipping together the β-sheets. This or a similar process may be important in the structural rearrangements that are thought to occur during the final formation of the amyloid fibril. The repeatable force patterns provided a mechanical fingerprint of the Aβ-fibril. This fingerprint was observed to be different for Aβ1-40 and Aβ25-35 fibrils, probably because of underlying differences in the arrangement and interactions of their β-sheets. Although we tentatively excluded that protofibrils, the precursors of mature amyloid β-fibrils (9Harper J.D. Wong S.S. Lieber C.M. Lansbury P.T. Chem. Biol. 1997; 4: 119-125Abstract Full Text PDF PubMed Scopus (631) Google Scholar, 10Nichols M.R. Moss M.A. Reed D.K. Lin W.L. Mukhopadhyay R. Hoh J.H. Rosenberry T.L. Biochemistry. 2002; 41: 6115-6127Crossref PubMed Scopus (166) Google Scholar, 11Walsh D.M. Lomakin A. Benedek G.B. Condron M.M. Teplow D.B. J. Biol. Chem. 1997; 272: 22364-22372Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 12Walsh D.M. Hartley D.M. Kusumoto Y. Fezoui Y. Condron M.M. Lomakin A. Benedek G.B. Selkoe D.J. Teplow D.B. J. Biol. 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Terenius L. Nordstedt C. J. Biol. Chem. 1996; 271: 8545-8548Abstract Full Text Full Text PDF PubMed Scopus (846) Google Scholar) that interfere with amyloidogenesis. We thank Ildikó Konrád for assistance.
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