Mechanical manipulation of Alzheimer’s amyloid β1–42 fibrils

Journal of

Structural Biology Journal of Structural Biology 155 (2006) 316–326 www.elsevier.com/locate/yjsbi

Mechanical manipulation of Alzheimer’s amyloid b1–42 fibrils ´ . Karsai a, Zs. Ma´rtonfalvi a, A. Nagy a, L. Grama a, B. Penke b, M.S.Z. Kellermayer A b

a,*

a Department of Biophysics, University of Pe´cs, Faculty of Medicine, Pe´cs H-7624, Hungary Department of Medicinal Chemistry and Protein Research Group of the Hungarian Academy of Sciences, University of Szeged, Szeged H-6720, Hungary

Received 23 November 2005; accepted 19 December 2005 Available online 2 May 2006

Abstract The 39- to 42-residue-long amyloid b-peptide (Ab-peptide) forms filamentous structures in the neuritic plaques found in the neuropil of Alzheimer’s disease patients. The assembly and deposition of Ab-fibrils is one of the most important factors in the pathogenesis of this neurodegenerative disease. Although the structural analysis of amyloid fibrils is difficult, single-molecule methods may provide unique insights into their characteristics. In the present work, we explored the nanomechanical properties of amyloid fibrils formed from the fulllength, most neurotoxic Ab1–42 peptide, by manipulating individual fibrils with an atomic force microscope. We show that Ab-subunit sheets can be mechanically unzipped from the fibril surface with constant forces in a reversible transition. The fundamental unzipping force (23 pN) was significantly lower than that observed earlier for fibrils formed from the Ab1–40 peptide (33 pN), suggesting that the presence of the two extra residues (Ile and Ala) at the peptide’s C-terminus result in a mechanical destabilization of the fibril. Deviations from the constant force transition may arise as a result of geometrical constraints within the fibril caused by its left-handed helical structure. The nanomechanical fingerprint of the Ab1–42 is further influenced by the structural dynamics of intrafibrillar interactions.  2006 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; Amyloid fibrils; Atomic force microscopy; b-Sheet; Elasticity

1. Introduction Alzheimer’s disease is a neurodegenerative disorder characterized, among others, by the deposition of insoluble filamentous aggregates called neuritic plaques (Mattson, 2004; Selkoe, 1997). The major component of neuritic plaques is the 39- to 42-residue-long amyloid b-peptide (Ab) that forms self-associating fibrillar structures possessing a predominantly cross-b conformation (Serpell, 2000). The structure of Ab has been difficult to explore with conventional methods because of the insolubility and aggregation of the peptide. Recent site-directed spin labeling (To¨ro¨k et al., 2002) and solid-state NMR experiments (Petkova et al., 2002; Tycko, 2004) have formed the basis of a highresolution model of the fibril formed from the Ab1–40 peptide. According to the model, b-strand–turn–b-strand

*

Corresponding author. Fax: +36 72 536261. E-mail address: [email protected] (M.S.Z. Kellermayer).

1047-8477/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2005.12.015

structures (or b-b-arc (Efimov, 1987)) oriented in perpendicular to the fibril axis line up to form two b-sheets that run parallel along the fibril axis. Two of such b-sheet doublets would then form a protofilament (Petkova et al., 2002). A recent work suggested that the b-b-arc is stabilized by intermolecular interactions between neighboring peptides rather than by intramolecular interactions (Lu¨hrs et al., 2005). In this arrangement a single b-sheet doublet forms the amyloid protofilament. Ab protofilaments associate in parallel to form the 4- to 8 nm-wide Ab-fibrils. Amyloid b1–42 peptide (Ab1–42) is the full-length Ab which is the most neurotoxic type of Ab (Ling et al., 2003). Although several short fragments of the Ab peptide have been shown to form filamentous aggregates (Serpell, 2000), Ab1–42 is thought to be primarily responsible for the clinical forms of Alzheimer’s disease (Adlard and Cummings, 2004; Cummings, 2004; Hardy and Selkoe, 2002; Selkoe, 2001). Thus, a detailed exploration of the structural and dynamic properties of Ab1–42 fibrils is warranted. Recently, we investigated the mechanical properties

A´. Karsai et al. / Journal of Structural Biology 155 (2006) 316–326

of different Ab fibrils (Ab1–40 and Ab25–35) using novel, single-molecule manipulation approaches (Karsai et al., 2005; Kellermayer et al., 2005). Single-molecule manipulation experiments provide unique insights into not only the structure and elasticity, but also into mechanically driven transitions of molecular systems (Brockwell et al., 2003; Carrion-Vazquez et al., 2003; Fisher et al., 1999, 2000; Kellermayer et al., 1997, 2005; Liphardt et al., 2001; Rief et al., 1997a,b; Smith et al., 1996; Tskhovrebova et al., 1997). We found that filamentous entities most likely corresponding to subunit sheets can be unzipped from the fibril with constant forces in an equilibrium process, indicating that during mechanical relaxation the subunit sheets rapidly rebind to the fibril surface. In the present work, we extended our experimental approach to investigate the nanomechanical properties of Ab1–42 fibrils. We find that the overall mechanical response of Ab1–42 fibrils is similar to that observed previously for Ab1–40 and Ab25–35, suggesting that the determinants of the fibril’s nanomechanical behavior are similar. However, the forces necessary to unzip subunit sheets of Ab1–42 are significantly lower than in the case of Ab1–40, indicating that the two extra amino acids of Ab1–42 mechanically destabilize the fibril. The rapid, mechanical zipping together of Ab-subunit sheets could be an important general mechanism behind the co-operative formation of amyloid fibrils. 2. Materials and methods 2.1. Samples Amyloid b1–42 peptides were prepared by solid-phase synthesis (Palota´s et al., 2002). 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. Ab1–42 fibrils were grown in solution at room temperature for several days. 2.2. Surface adsorption of amyloid fibrils For AFM and molecular force spectroscopy measurements, fibrils were attached covalently to a silanized glass coverslip (Kellermayer et al., 2005). Briefly, coverslips were cleaned by sonication in acetone, followed by rinsing with distilled water and drying in a stream of high-purity N2 gas. Pre-cleaned coverslips were incubated in toluene vapor containing 2% (3-glycidyloxypropyl)trimethoxysilane (Fluka 50040) for 12 h at room temperature. A 50 ll sample of Ab1–42 fibrils (0.5 mg/ml concentration) preadjusted to pH 9 was pipetted onto the glass surface and incubated at room temperature for 30 min. Unbound fibrils were washed away by rinsing with PBSA buffer. For morphological studies, we used freshly cleaved mica. A 50 ll sample of Ab1–42 fibrils (0.5 mg/ml concentration) was pipetted onto the mica surface and incubated at room temperature for 10 min. Unbound fibrils were washed away by rinsing with PBSA buffer.

317

2.3. Atomic force microscopy Non-contact-mode (AC mode) AFM images of amyloid fibrils bound to silanized glass or mica surfaces were acquired with an MFP3D (Asylum Research, Santa Barbara, CA) AFM instrument. Silicon nitride cantilevers (Olympus) were used for scanning either in air (AC160TS, resonance frequency 300 kHz) or in liquid (BioLever, lever B, resonance frequency 35 kHz). 512 · 512-pixel or 1024 · 1024-pixel images were collected at a typical scanning frequency of 0.7 Hz. 2.4. Single-molecule force spectroscopy Ab1–42 amyloid fibrils were mechanically manipulated by first pressing the cantilever (Olympus BioLever, lever B) tip against the surface, then pulling the cantilever away with a constant, pre-adjusted rate. Typical stretch rate was 300 nm/s, except where otherwise indicated. Experiments were carried out under aqueous buffer conditions (PBSA buffer, pH 7.4). Stiffness was determined for each cantilever by using the thermal method (Hutter and Bechhoefer, 1993). Typical cantilever stiffness was 30 pN/nm. 2.5. In situ force spectroscopy In situ force spectroscopy was carried out by first scanning (under aqueous buffer conditions) the glass-bound sample surface, then pressing the cantilever tip to targeted surface locations identified on the image, and finally re-scanning the surface to test for the effect of the mechanical perturbations (Kellermayer et al., 2005; Oesterhelt et al., 2000). Soft (typical cantilever stiffness 30 pN/nm), high-resonance frequency (35 kHz) cantilevers (Olympus BioLever, lever B) were used. Scanning was carried out in non-contact-mode at high set-point values (1.0–1.2 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. 2.6. Data analysis Force step heights were obtained by measuring the distance between the average force values of consecutive plateaus. For fitting non-linear force curves (with the Marquardt–Levenberg method) we used the wormlike chain model of entropic elasticity (Bustamante et al., 1994) fA z 1 1 ¼ þ  ; k B T L 4ð1  z=LÞ2 4

ð1Þ

where f is force, A is persistence length (measure of bending rigidity), z is end-to-end length, L is contour length, kB is Boltzmann’s constant, and T is absolute temperature. Image processing was carried out using the built-in routines of the MFP3D software. Three-dimensional surface rendering was done with the Argyle toolbox of the MFP3D software.

A´. Karsai et al. / Journal of Structural Biology 155 (2006) 316–326

318

a

b

d

c

e

f

g h

i

j

k

A´. Karsai et al. / Journal of Structural Biology 155 (2006) 316–326

For calculations and statistical analyses we used IgorPro (v. 5.0) and KaleidaGraph (v. 4.0) software packages.

319

surface) of the fibrils. Furthermore, we were unable to resolve a specific topographical change in the mechanically manipulated Ab1–42 fibrils.

3. Results 3.3. Force spectroscopy of Ab1–42 fibrils 3.1. Morphological appearance of Ab1–42 fibrils Images of surface-adsorbed Ab1–42 fibrils, acquired using non-contact AFM, are shown in Figs. 1 and 2. Although the fibrils sometimes had a smooth surface, most often they displayed helical appearance. Fibrils with long helical pitch are shown in Figs. 1a–c. The periodicity in these fibrils was 108.87 ± 14.26 nm (Fig. 1d). The helical appearance of the fibrils could be resolved in fibrils attached to silanized surface and scanned under liquid (Figs. 1e and f). We observed Ab1–42 fibrils with much shorter periodicity as well. In some fibrils a periodicity of 50 nm was observed (Figs. 1g and h), and in some cases a periodicity as low as 25 nm could be discerned (Figs. 1i and j). We observed a left-handed helical arrangement regardless of the periodicity. The left-handed helices can be particularly well identified in Figs. 1b, e, and i. The mean topographical height of the fibrils, which reflects their diameter, was 4.77 ± 1.82 nm (3329 datapoints for 22 fibrils). The height histogram (Fig. 1k) displayed multimodal distribution with peaks at approximately 4, 6, 9, and 12 nm. 3.2. Nanomechanical manipulation of Ab1–42 fibrils Surface-adsorbed amyloid fibrils were mechanically manipulated by pressing the AFM cantilever into the fibril and then pulling the cantilever away from the fibril surface with constant rate. The effect of mechanical perturbation on global fibril structure was investigated with in situ force spectroscopy (Fig. 2). In these experiments the sample surface was scanned prior to (Figs. 2a and c) and following (Figs. 2b and d) the mechanical perturbations. Although the silanized surface has a greater roughness than the mica surface, individual Ab1–42 fibrils could be clearly visualized. The mechanical perturbations were carried out at specific locations along the fibrils’ length (marked with red dots in Figs. 2a and c). As evidenced by the images, the mechanical manipulation did not result in a global perturbation (e.g., material loss, reorientation, or total dissociation from the

Force curves obtained during nanomechanical manipulation of Ab1–42 fibrils are shown in Fig. 3. These force curves, which describe a set of molecular characteristics and transitions are referred to as force spectra (Rief et al., 1997a,b). Two basic types of force responses could be discerned. The first type of characteristic force response is the force plateau (Fig. 3a), which often occurs as a set of hierarchical force steps (Fig. 3b). The force plateau is characterized by constant force during extension, although we observed oblique force plateaus as well (see below). The plateau typically ends with an abrupt decrease in force. The second type of force response is non-linear elasticity (Fig. 3c). Sometimes fully reversible non-linear stretch and relaxation curves were observed (Fig. 3c), which could be fitted with the wormlike chain equation of entropic elasticity (Eq. (1)). More frequently, however, the non-linear force curves appeared in combination with force plateaus (Fig. 3d). In some cases we observed a non-linear transition between force plateaus (Fig. 3e). In these force curves a short force plateau was superimposed onto a long plateau, and the short plateau was preceded with a non-linear rise in force. The force plateaus (Fig. 3f) and even more complex force responses (Fig. 3g) were highly repeatable, and similar or nearly identical force curves could be recorded in successive mechanical cycles. We found that the force plateau was reversible. If the cantilever movement was reversed prior to reaching the end of the force plateau, then the relaxation force data traced the extension force data (Fig. 3h). In these experiments often a force step (abrupt force drop) was observed during the relaxation phase. The plateaus observed in the force response of Ab1–42 fibrils were sometimes oblique rather than flat (Fig. 4). Oblique force plateaus are characterized by increasing force during stretch, followed by an abrupt force drop (Fig. 4a inset and b). Repetitive force sawtooth is characteristically observed in case of mechanically manipulated multidomain proteins such as titin (Rief et al., 1997a,b).

b Fig. 1. Structural analysis of Ab1–42 fibrils with AFM. (a) Scanning AFM image of Ab1–42 amyloid fibrils adsorbed to freshly cleaved mica. Height contrast image collected in non-contact mode in liquid. (b) An enlarged portion of the image displayed in amplitude contrast in order to better reveal the helical structure of the fibrils. Arrowheads indicate the location and orientation of the helical twist. As evidenced by the image, these fibrils display a lefthanded helical structure. (c) Three-dimensional rendering of the surface topography of Ab1–42 fibrils. Arrowheads indicate periodic height maxima. (d) Distribution of the distance between neighboring height maxima along the contour of Ab1–42 fibrils. The mean spacing is 108.87 ± 14.26 nm (n = 45). (e) Non-contact-mode AFM image, obtained in aqueous buffer, of an Ab1–42 fibril bound to silanized glass surface. Red line marks the area from which topography data (f) were obtained. (f) Topographical height distribution along the Ab1–42 fibril shown in (e). Arrows point at height maxima separated by 100 nm. (g) Non-contact-mode AFM image, obtained in air, of Ab1–42 fibrils bound to freshly cleaved mica surface. Red line marks the area from which topography data (h) were obtained. (h) Topographical height distribution along the Ab1–42 fibril marked in (g). Arrows point at height maxima separated by 50 nm. (i) Non-contact-mode AFM image, obtained in air, of Ab1–42 fibrils bound to freshly cleaved mica surface. White bracket marks the area from which topography data (j) were obtained. (j) Topographical height distribution along the Ab1–42 fibril marked in (i) Arrows point at height maxima separated by 25 nm. (k) Distribution of topographical height along Ab1–42 fibrils. Mean height measured for 22 filaments at 3329 points is 4.77 ± 1.82 nm. Arrows point at local maxima of the histogram.

A´. Karsai et al. / Journal of Structural Biology 155 (2006) 316–326

320

a

b

500 nm

c

d

100 nm Fig. 2. Non-contact-mode AFM images, acquired in liquid, of mechanically manipulated Ab1–42 fibrils bound to silanized glass surface. The images were acquired prior to (a and c) and following (b and d) targeted mechanical manipulations. Red dots mark the location of mechanical manipulation.

We compared the oblique force steps observed here for Ab1–42 fibrils with the force sawtooth observed for a recombinant tandem-Ig fragment of titin (I55–62) (Grama et al., 2005). There are two main differences in the overall appearance of the two force curves. First, the rising phase of the force sawtooth observed in I55–62 (Fig. 4a) can be fitted with the wormlike chain equation. By contrast, a linear fit is more appropriate for the rising phase of the oblique force plateaus observed for Ab1–42 (Fig. 4b). Second, the force to which the force sawtooth transition decays is progressively increasing with the number of transitions, due to the progressive increase in the fractional extension of the entropic chain. By contrast the force to which the oblique force plateau decays progressively decreases with the number of transitions (Fig. 4b). 3.4. Plateau height statistics From the height of consecutive force plateaus a forcestep-height histogram was constructed for the Ab1–42 amyloid fibril (Fig. 5a). The histogram displayed multimodal distribution with peaks at 23, 45, 70, 90, 110, and 130 pN. For comparison, Fig. 5a also shows the distribution of plateau forces obtained earlier for

Ab1–40 (Kellermayer et al., 2005). To investigate the correlation of the nanomechanical behavior with a structural parameter of Ab1–42 fibrils, we measured the plateau forces as a function of fibril length (Fig. 5b) and fibril height (Fig. 5c) that reflects the fibril diameter. Linear fits did not reveal any correlation between plateau height and either fibril length or topographical fibril height. 3.5. Plateau length statistics To explore the structural and dynamic characteristics of Ab1–42 fibrils in further detail, we analyzed the length of the force plateaus as a function of the plateau height and stretch velocity (Fig. 6). Force plateau length as a function of plateau height is shown in Fig. 6a. We found that the plateau length increased exponentially with increasing plateau height. Plateau length as a function of stretch velocity is shown in Fig. 6b. Plateau length increased linearly with the logarithm of stretch velocity. 4. Discussion Amyloid b1–42 fibrils were mechanically manipulated in the present work by using atomic force microscopy. We

A´. Karsai et al. / Journal of Structural Biology 155 (2006) 316–326

b

c

150 100

ΔF

-50

100

Force (pN)

300

Force (pN)

Force (pN)

a 200

200 100

321

0

50

0

0 -100

0

20

40

60

0

80

10

d 250

e

30

40

50

0

60

100 50

120 80 40

0

0

-50

-40 10

20

30

40

50

60

10

Extension (nm)

g

40

50

60

70

100 50 0 50

0

70

30

150

160

Force (pN)

Force (pN)

150

20

f

200

0

10

Extension (nm)

240

200

Force (pN)

20

Extension (nm)

Extension (nm)

20

30

40

50

60

70

0

20

Extension (nm)

40

60

80

Extension (nm)

h

300

Force (pN)

Force (pN)

200 200 100 0

R 100

S 0

-100

-100 0

10

20

30

40

50

60

Extension (nm)

0

20

40

60

80

Extension (nm)

Fig. 3. Force versus extension curves of Ab1–42 amyloid fibrils. Stretch and release data are displayed in blue and red, respectively, except where otherwise indicated. (a) Representative force plateau. DF marks the height of the force plateau. (b) Force staircase with contiguous, progressively decreasing force plateaus. Dotted lines mark the plateaus. (c) Fully reversible, non-linear elasticity. (d) Force plateaus superimposed onto non-linear force response. (e) Short force plateau superimposed on a long force plateau. A non-linear region precedes the short plateau. Dotted lines mark the plateaus. (f) Repeatability of the force plateau. Data acquired in successive mechanical cycles are indicated with different colors. (g) Repeatability of the force plateau with superimposed short force plateau. Data acquired in successive mechanical cycles are indicated with different colors. (h) Reversible force plateau. The reversible region (R) is indicated with ‘R,’ and the force step during relaxation phase with ‘S.’

obtained high-resolution structural information on Ab1–42 fibrils and characterized their nanomechanical behavior. 4.1. Structure of Ab1–42 fibrils Ab1–42 fibrils appeared on the AFM images as more or less straight or gently curved filaments (Figs. 1 and 2). The fibrils usually displayed a left-handed helical arrangement which manifested in an axial periodicity of height. Periodicity varied in different preparations between 25 and 110 nm. We found a variation in the topographical height of the fibrils as well. Although the average fibril height, which reflects the fibril diameter, was 4.77 ± 1.82 nm (SD), we observed a multimodal height distribution with peaks at 4, 6, 9, and 12 nm. Fibrillar structures with short periodicity (22 nm) and 4.2 nm diameter have

been attributed to protofibrils (Harper et al., 1997), which are intermediates along the path of amyloid fibril formation (Walsh et al., 1997). By this criterion the fibrillar structures shown in Fig. 1i may be identified as Ab1–42 protofibrils. However, protofibrils are typically short (<200 nm) beaded chains that display a bent and folded morphology (Nichols et al., 2002; Walsh et al., 1997, 1999). Considering that the filamentous structures observed in our preparations are typically long (hundreds of nanometers to several micrometers) and straight, and, with exceptions, display long periodicities (up to 110 nm) and diameters of 4–12 nm, it is more likely that the mechanically manipulated structures were mature Ab1– 42 fibrils. The helical appearance, the 2 nm peak-to-peak height variation along the contour of the fibrils (Figs. 1f, h, and j), and the multimodal height histogram (Fig. 1k) sug-

A´. Karsai et al. / Journal of Structural Biology 155 (2006) 316–326

322

200 pN

a

50 nm

200 pN

b

50 nm Fig. 4. Comparison of oblique force plateaus with repetitive force sawtooth observed in the force spectrum of tandem globular domains. (a) Force spectrum of the titin I55–62 tandem immunoglobulin construct (Grama et al., 2005). The individual force peaks were fitted with the equation of the wormlike chain entropic polymer model (Eq. (1)). Inset: Force spectrum of Ab1–42 fibril displaying oblique force plateaus. (b) Magnified view of the force spectrum of Ab1–42 fibril. The oblique force plateaus are indicated with dotted lines.

gest that varying number of protofilaments (Serpell, 2000) make up the investigated the Ab1–42 fibrils. The differences in the protofilament number are likely to arise as a result of the fibril maturation process. Surface-attached individual Ab1–42 fibrils were mechanically manipulated with AFM, in in situ force spectroscopy experiments. In these measurements, the surface of the sample was scanned prior to and following mechanical manipulations in order to identify gross structural changes evoked by the manipulation (Karsai et al., 2005; Kellermayer et al., 2005; Oesterhelt et al., 2000). We found no resolvable structural changes, such as the removal, material loss or dislocation of the fibrils (Fig. 2). The finding indicates either that the dimensions of the structures mobilized during mechanical manipulation fall below the spatial resolution of the method, or that the fibril rapidly recovers following the perturbation. Considering that the manipulated fibrils stayed firmly attached to the silanized surface, the transitions observed (see below) arise from intrafibrillar structural rearrangements. 4.2. Force-driven structural changes in Ab1–42 fibrils Mechanically manipulated individual Ab1–42 fibrils displayed two fundamental types of force responses, similarly to Ab1–40 and Ab25–35 fibrils studied earlier (Karsai et al., 2005; Kellermayer et al., 2005). The typical and most frequently observed force response is the force plateau that often assembles into hierarchical force steps (Figs. 3a and b).

During the force plateau a constant force level is maintained during extension. A force plateau ends with an abrupt, stepwise decrease in force. The force plateau has been attributed to the gradual unzipping of Ab-subunit sheets from the underlying amyloid fibril surface (Karsai et al., 2005; Kellermayer et al., 2005). The Ab-subunit sheet is a tandem array of b-strand–turn–b-strand Ab1–42 subunits that form a b-sheet doublet that runs along the fibril axis, and is composed of the peptide’s b-strands in cross-b arrangement (Lu¨hrs et al., 2005; Petkova et al., 2002). Force remains constant during the process because at each time point during extension only a single set of bonds is loaded and broken mechanically. The height of the plateau is related to the energy of the bond(s) holding the Ab-subunit sheets associated to the fibril surface, provided that the system is in thermodynamic equilibrium. If several Ab-subunit sheets are grabbed and unzipped at the same time, then the height of the force plateau increases proportionally. Thus, the plateau height also reflects the number of Ab-subunit sheets unzipped from the fibril surface, which can be calculated based on the single Ab-subunit sheet unzipping force (fundamental unzipping force, see below). The second type of fundamental force response is nonlinear elasticity (Figs. 3c and d). In a truly elastic force response (Fig. 3c) the stretch-and-relaxation force curve is devoid of hysteresis, and the data acquired during the relaxation phase of the mechanical cycle traverses the same path observed during stretch. The appearance of elastic force curves is attributed to stretching Ab-subunit sheets which are otherwise firmly attached at both ends (to the cantilever tip and to the underlying surface). The relatively high forces observed suggest that the hydrogen bonds that hold the Ab-subunit sheets together in the axial direction are stable, or that their dissociation/association occurs on a time scale that is much faster than that of the stretching experiment. Often force plateaus were superimposed on the non-linear elastic curve (Fig. 3d). The observation can be attributed to the simultaneous unzipping and stretching of two independent Ab-subunit sheets. In some instances we observed that a short force plateau, preceded by a non-linear force curve, was superimposed on a long force plateau. The observation can be attributed to mechanical coupling between a long strand of Ab-subunit sheets and a short one. We found that the force plateaus were repeatable and reversible. The repeatability of force plateaus is observed in successive mechanical cycles (Figs. 3f and g). The repeatability of the force response in spite of complete rupture of the Ab-subunit sheets (note force drop to the baseline after the last force step in Figs. 3f and g) indicates that mechanically manipulated Ab-subunit sheets dissociate from the cantilever tip (or rupture near the cantilever tip) rather than from the underlying amyloid fibril. The unzipped Ab-subunit sheets then rapidly reassociate to the fibril surface prior to the subsequent mechanical cycle. The reversibility of the force plateau (Fig. 3h) indeed suggests that the unzipped Ab-subunit sheets may rapidly rebind to the

A´. Karsai et al. / Journal of Structural Biology 155 (2006) 316–326

323

a

b

c

Fig. 5. Analysis of plateau forces. (a) Plateau force step distribution. The plateau force histogram of Ab1–42 is compared with that of Ab1–40 (Kellermayer et al., 2005). Ab1–42 and Ab1–40 histograms are displayed with dark gray and transparent bars, respectively. The numbers and arrows above the histogram peaks indicate the number of Ab-subunit sheets unzipped in a given force plateau. (b) Plateau force displayed as a function of the length of Ab1–42 fibrils. Fibril lengths were measured during in situ force spectroscopy experiments. (c) Plateau force displayed as a function of the topographical height of Ab1–42 fibrils. Equations of the fits are shown above the figures.

fibril surface, in a process that could be seen as zipping the Ab-subunit sheets together. In the reversible regime of the force curve the stretch and relaxation data follow equivalent traces and the molecular system is in thermodynamic equilibrium at each point of extension. Because of equilibrium the free-energy change (DG) associated with the zipping/unzipping process may be calculated from the integral of the force curve, provided that the force necessary to unzip a single Ab-subunit sheet is known (see below). The repetitive force pattern displayed by Ab1–42 fibrils (Fig. 4a inset) sometimes resembled the sawtooth force pattern seen in case of filamentous proteins (Kellermayer

et al., 2003; Rief et al., 1997a,b), such as titin (Labeit and Kolmerer, 1995), which are assembled from tandemly arranged globular domains. To differentiate between mechanical events related to serially linked structural units (tandem domain array) versus structures linked in parallel (unzipping Ab-subunit sheets), we compared the force traces of Ab1–42 fibrils with those observed for a recombinant tandem array of eight globular titin domains (I55–62, Fig. 4a) (Grama et al., 2005). In case of Ab1–42, the force drops to progressively lower levels with each abrupt transition (Fig. 4b), which is strikingly different from that seen in a chain of tandemly arranged domains. Thus, it is unlikely that the oblique force plateaus seen in the force traces of

A´. Karsai et al. / Journal of Structural Biology 155 (2006) 316–326

324

a

y = 3.1787 * e^(0.03539x) R= 0.86239

120

Plat ea u l ength ( nm)

100 80 60 40 20 0 0

20

40

60

80

100

Plateau height (pN)

b

60

y = -162.73 + 80.575log(x) R= 0.77813

Pl a t ea u l e n g t h ( n m )

55

50

45

40

35 300

400

500

600

Velocity (nm/s) Fig. 6. Dependence of the force plateau length on plateau height (a) and stretch velocity (b). Stretch velocity is displayed on logarithmically scaled axis. Equations of the fits are shown above the figures.

Ab1–42 are related to serially connected structural units. Rather, they are likely to arise due to a load that increases during unzipping of Ab-subunit sheets. The extra mechanical load may be caused by torsion due to the helical structural arrangement observed in high-resolution images (Fig. 1).

sheet is 1.1 · 1020 J (23 pN * 0.47 nm), which corresponds to 6.63 kJ/mol. It is intriguing that although Ab1–42 and Ab1–40 differ in only two amino acid residues (Ile and Ala) at the C-terminus, their nanomechanical fingerprints are significantly different. Possibly, the two extra residues of Ab1–42 cause a structural destabilization of the fibril. Ab1–42 fibrils form faster, along a distinct pathway, and are more stable than Ab1–40 fibrils (Bitan et al., 2003). The apparently lower mechanical stability of Ab1–42 fibrils than that of Ab1–40 fibrils observed here may have to do with the fact that the direction of mechanical force places constraints on the system (Kellermayer, 2005) which may therefore proceed along a path different from that accessed under thermodynamic equilibrium conditions. The reversibility of the plateau transition indicates that the zipping together of either Ab1–40 or Ab1–41 subunit sheets occurs on a time scale faster than that of the mechanical experiment. Therefore, currently we are unable to detect differences between the zipping rates of these fibril types. Further, high-stretch-rate experiments are required to investigate possible differences in the zipping rates of Ab1–40 and Ab1–42 and how these rates might be related to the kinetics of their fibrillogenesis. The plateau–force distribution has been thought to provide a nanomechanical fingerprint of amyloid fibrils (Karsai et al., 2005; Kellermayer et al., 2005). Indeed, the fundamental unzipping forces are different for the amyloid fibrils so far investigated. By nanomechanical fingerprinting of the various fibrillar forms of Ab, the effect of primary structure on the fibrils’ mechanical and thermodynamic stability may be explored. The plateau force did not correlate with either the length or the diameter of Ab1–42 fibrils (Figs. 5b and c). Fibril length and diameter have been shown to increase with maturation (Harper et al., 1997). The lack of correlation between the plateau height and fibril length and height suggest that either the fibrils studied are in similar stages of maturation or that the intrafibrillar interaction forces do not change significantly during the maturation process. Thus, the factors that determine the intrafibrillar interactions are likely to develop at early stages of amyloid fibrillogenesis. By systematic nanomechanical fingerprinting of structural intermediates of fibril formation, the evolution of intrafibrillar interactions related to amyloid fibrillogenesis may be elucidated.

4.3. Nanomechanical fingerprint of Ab1–42 fibrils 4.4. Structural dynamics of Ab1–42 fibrils The plateau force histogram of Ab1–42 fibrils displayed a multimodal distribution (Fig. 5a) which enabled us to calculate the force necessary to unzip an individual Ab-subunit sheet from the fibril surface. The fundamental unzipping force was 23 pN. In comparison, we measured fundamental unzipping forces of 33 pN for Ab1–40 fibrils (Kellermayer et al., 2005). Considering that the unzipping occurs in equilibrium and that the fundamental force acts ˚ when a single unit (Ab peptide) along a distance of 4.7 A is dissociated from the fibril (Serpell, 2000), the free-energy change associated with the unzipping of Ab1–42 subunit

The abrupt force drop at the end of the plateau corresponds to the complete dissociation of the Ab-subunit sheet. Repetitive force steps appear due to the consecutive dissociation of further subunit sheets. Complete dissociation may occur either because the end of a discontinuous Ab-subunit sheet is reached (structural effect), or because the subunit sheet is suddenly ruptured or broken (dynamic effect). So far there is no literature evidence for a discontinuous arrangement of Ab-subunit sheets within the amyloid fibril. Furthermore, our measurements favor the influence

A´. Karsai et al. / Journal of Structural Biology 155 (2006) 316–326

lifetime of the interactions that hold the captured Ab-subunit sheet attached, the sheet ruptures and a force step appears in the force spectrum (point 3). These interactions include bonds that hold the Ab-peptides together (Hbonds and side-chain interactions), and bonds that keep the b-sheet attached to the AFM tip. Notably, lateral associations between Ab-subunit sheets within the captured strand may stabilize the attached state that results in longer force plateaus (Fig. 6). If all the Ab-subunit sheets become ruptured, the cantilever returns to its equilibrium position and the force to the baseline (point 4). Conceivably, short pieces of Ab-subunit sheets may remain attached to the AFM tip, which may facilitate the re-capture of the same strand in the subsequent mechanical cycle. If the AFM cantilever is allowed to return toward the fibril surface before the Ab-subunit sheets become completely dissociated (at points 2 or 3), then the subunit sheets rebind to the fibril surface with sufficiently large rate that a reversible force plateau is observed (Fig. 3h). Such a zipping process is facilitated by the high local concentration of the binding sites that participate in forming the bonds that hold the Ab-subunit sheet within the fibril. Due to this arrangement an apparent cooperativity arises that facilitates the zipping process. Conceivably, a similar process may take place when amyloid fibrils are being formed in vivo. Altogether, the nanomechanical probing of Alzheimer’s amyloid b-fibrils provides a unique glimpse at their structural dynamics, which may play significant roles in fibrillogenesis and subsequent biological interactions.

of dynamic effects that determine the termination of the force plateau (Fig. 6). We observed that the length of the force plateau, which corresponds to the time spent during the unzipping transition, increases with both the plateau height (number of subunit sheets unzipped in parallel, Fig. 6a) and stretch velocity (Fig. 6b). The findings are inconsistent with a structural model of discontinuous Absubunit sheets, because in this case a constant plateau length is expected. Rather, the plateau length is influenced by dynamic effects related to the lifetime and dynamic equilibrium of the interactions that hold the captured subunit sheets in place. The observation that a force step may occur in the reverse direction (Fig. 3h) lends further support to this idea. Although further experimentation is required to map the role of dynamic effects, our observations indicate that Ab fibrils display a structural dynamism that is likely to influence its structural transitions and interactions within the biological environment. 4.5. Model of Ab1–42 nanomechanics The events of the nanomechanical experiments on Ab1– 42 fibrils may be summarized in the following phenomenological model (Fig. 7). A strand of Ab-subunit sheets is first captured and mobilized with the tip of the AFM cantilever (point 1). As the cantilever is pulled away from the fibril, the subunit sheets become gradually unzipped from the fibril surface (point 2). Considering that, due to the pulling geometry, at each point of extension a single set of bonds, which hold the Ab-subunit sheet attached, are broken, unzipping occurs at a constant force level. Because unzipping occurs in small steps (step size compa˚ (Serpell, 2000)), the rable with b-strand spacing, 4.7 A details of the individual bond-rupture events remain hidden within the force plateau. The helical arrangement of the Ab-subunit sheets on and/or within the fibril may result in geometric distorsions during the unzipping process, which manifests in deviations from a horizontal force plateau (e.g., oblique plateau, Fig. 4). Depending on the

1

2

325

Acknowledgments This work was supported by grants from the Hungarian Science Foundation (OTKA T049591), Hungarian Ministry of Education (OM-110/2002), Hungarian Ministry of Health (ETT-440/2003) and the South Trans-Danubian Co-operative Research Center. M.S.Z.K. is a Howard Hughes Medical Institute International Research Scholar.

3

4 AFM cantilever *

Aß1-42 fibril

Aß-subunit sheets

Functionalized glass surface Fig. 7. Schematics of the subfibrillar events underlying the nanomechanical behavior of Ab1–42 fibrils. Numbers indicate steps of the Ab1–42 mechanical manipulation. (1) Two Ab-subunit sheets (indicated with black and gray lines) on the fibril surface are captured with the AFM tip. (2) The subunit sheets are being unzipped from the fibril surface, giving rise to a force plateau. The helical arrangement of the Ab-subunit sheets on the fibril surface may result in oblique force plateaus. (3) One of the subunit sheets ruptures or dissociates from the AFM tip. The event results in a force step. The dissociated sheet may rapidly reassociate to the fibril surface. (4) The second subunit sheet ruptures or dissociates from the AFM tip. The cantilever returns to its equilibrium position and hence the force to the baseline. The dissociated sheet may reassociate to the underlying fibril surface. Small fragments of the Ab-subunit sheets may remain adsorbed on the AFM tip (indicated with asterisk) and facilitate the capture of subunit sheets in subsequent mechanical cycles.

326

A´. Karsai et al. / Journal of Structural Biology 155 (2006) 316–326

References Adlard, P.A., Cummings, B.J., 2004. Alzheimer’s disease—a sum greater than its parts? Neurobiol. Aging 25, 725–733. Bitan, G., Kirkitadze, M.D., Lomakin, A., Vollers, S.S., Benedek, G.B., Teplow, D.B., 2003. Amyloid b-protein (Ab) assembly: Ab40 and Ab42 oligomerize through distinct pathways. Proc. Natl. Acad. Sci. USA 100, 330–335. Brockwell, D.J., Paci, E., Zinober, R.C., Beddard, G.S., Olmsted, P.D., Smith, D.A., Perham, R.N., Radford, S.E., 2003. Pulling geometry defines the mechanical resistance of a beta-sheet protein. Nat. Struct. Biol. 10 (9), 731–737. Bustamante, C.J., Marko, J.F., Siggia, E.D., Smith, S.B., 1994. Entropic elasticity of k-phage DNA. Science 265, 1599–1600. Carrion-Vazquez, M., Li, H., Lu, H., Marszalek, P.E., Oberhauser, A.F., Fernandez, J.M., 2003. The mechanical stability of ubiquitin is linkage dependent. Nat. Struct. Biol. 10 (9), 738–743. Cummings, J.L., 2004. Alzheimer’s disease. N. Engl. J. Med. 351 (1), 56–67. Efimov, A.V., 1987. Pseudo-homology of protein standard structures formed by two consecutive b-strands. FEBS Lett. 224, 372–376. Fisher, T.E., Carrion-Vazquez, M., Oberhauser, A.F., Li, H., Marszalek, P.E., Fernandez, J.M., 2000. Single molecular force spectroscopy of modular proteins in the nervous system. Neuron 27 (3), 435–446. Fisher, T.E., Oberhauser, A.F., Carrion-Vazquez, M., Marszalek, P.E., Fernandez, J.M., 1999. The study of protein mechanics with the atomic force microscope. Trends Biochem. Sci. 24 (10), 379–384. Grama, L., Nagy, A., Scholl, C., Huber, T., Kellermayer, M.S., 2005. Local variability in the mechanics of titin’s tandem Ig segments. Croat. Chem. Acta 78, 405–411. Hardy, J., Selkoe, D.J., 2002. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297, 353–3556. Harper, J.D., Wong, S.S., Lieber, C.M., Lansbury, P.T., 1997. Observation of metastable Abeta amyloid protofibrils by atomic force microscopy. Chem. Biol. 4 (2), 119–125. Hutter, J.L., Bechhoefer, J., 1993. Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 64 (7), 1868–1873. ´ ., Nagy, A., Kengyel, A., Ma´rtonfalvi, Z., Grama, L., Penke, B., Karsai, A Kellermayer, M.S.Z., 2005. Effect of lysine-28 side chain acetylation on the nanomechanical behavior of Alzheimer amyloid b25–35 fibrils. J. Chem. Inf. Model. 45 (6), 1641–1646. Kellermayer, M.S.Z., 2005. Visualizing and manipulating individual protein molecules. Physiol. Meas. 26, R119–R153. Kellermayer, M.S., Bustamante, C., Granzier, H.L., 2003. Mechanics and structure of titin oligomers explored with atomic force microscopy. Biochim. Biophys. Acta 1604 (2), 105–114. Kellermayer, M.S., Grama, L., Karsai, A., Nagy, A., Kahn, A., Datki, Z.L., Penke, B., 2005. Reversible mechanical unzipping of amyloid beta-fibrils. J. Biol. Chem. 280 (9), 8464–8470. Kellermayer, M.S.Z., Smith, S.B., Granzier, H.L., Bustamante, C., 1997. Folding–unfolding transitions in single titin molecules characterized with laser tweezers. Science 276 (5315), 1112–1116. Labeit, S., Kolmerer, B., 1995. Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 270 (5234), 293–296. Ling, Y., Morgan, K., Kalsheker, N., 2003. Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer’s disease. Int. J. Biochem. Cell Biol. 35 (11), 1505–1535.

Liphardt, J., Onoa, B., Smith, S.B., Tinoco, I.J., Bustamante, C., 2001. Reversible unfolding of single RNA molecules by mechanical force. Science 292 (5517), 733–737. Lu¨hrs, T., Ritter, C., Adrian, M., Riek-Loher, D., Bohrmann, B., Do¨beli, H., Schubert, D., Riek, R., 2005. 3D structure of Alzheimer’s amyloidb(1–42) fibrils. Proc. Natl. Acad. Sci. USA 102, 17342–17347. Mattson, M.P., 2004. Pathways towards and away from Alzheimer’s disease. Nature 430 (7000), 631–639. Nichols, M.R., Moss, M.A., Reed, D.K., Lin, W.L., Mukhopadhyay, R., Hoh, J.H., Rosenberry, T.L., 2002. Growth of beta-amyloid (1–40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy. Biochemistry 41 (19), 6115–6127. Oesterhelt, F., Oesterhelt, D., Pfeiffer, M., Engel, A., Gaub, H.E., Muller, D.J., 2000. Unfolding pathways of individual bacteriorhodopsins. Science 288 (5463), 143–146. Palota´s, A., Ka´lma´n, J., Palota´s, M., Juha´sz, A., Janka, Z., Penke, B., 2002. b-Amyloid-induced increase in the resting intracellular calcium concentration gives support to tell Alzheimer lymphocytes from control ones. Brain Res. Bull. 58 (2), 203–205. Petkova, A.T., Ishii, Y., Balbach, J.J., Antzutkin, O.N., Leapman, R.D., Delaglio, F., Tycko, R., 2002. A structural model for Alzheimer’s b-amyloid fibrils based on experimental constraints from solid state NMR. Proc. Natl. Acad. Sci. USA 99 (26), 16742–16747. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M., Gaub, H.E., 1997a. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276 (5315), 1109–1112. Rief, M., Oesterhelt, F., Heymann, B., Gaub, H.E., 1997b. Single molecule force spectroscopy on polysaccharides by atomic force microscopy. Science 275 (5304), 1295–1297. Selkoe, D.J., 1997. Images in neuroscience. Alzheimer’s disease: from genes to pathogenesis. Am. J. Psychiatry 154 (9), 1198. Selkoe, D.J., 2001. Alzheimer’s disease: genes, proteins and therapy. Physiol. Rev. 81 (2), 741–766. Serpell, L.C., 2000. Alzheimer’s amyloid fibrils: structure and assembly. Biochim. Biophys. Acta 1502 (1), 16–30. Smith, S.B., Cui, Y., Bustamante, C., 1996. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271 (5250), 795–799. To¨ro¨k, M., Milton, S., Kayed, R., Wu, P., McIntire, T., Glabe, C.G., Langen, R., 2002. Structural and dynamic features of Alzheimer’s Abeta peptide in amyloid fibrils studied by site-directed spin labeling. J. Biol. Chem. 277 (43), 40810–40815. Tskhovrebova, L., Trinick, J., Sleep, J.A., Simmons, R.M., 1997. Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 387 (6630), 308–312. Tycko, R., 2004. Progress towards a molecular-level understanding of amyloid fibrils. Curr. Opin. Struct. Biol. 14, 96–103. Walsh, D.M., Hartley, D.M., Kusumoto, Y., Fezoui, Y., Condron, M.M., Lomakin, A., Benedek, G.B., Selkoe, D.J., Teplow, D.B., 1999. Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates. J. Biol. Chem. 274 (36), 25945–25952. Walsh, D.M., Lomakin, A., Benedek, G.B., Condron, M.M., Teplow, D.B., 1997. Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate. J. Biol. Chem. 272 (35), 22364–22372.