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Stimulatory and Inhibitory Effects of Alkyl Bromide Surfactants on β-Amyloid Fibrillogenesis Raimon Sabate´ and Joan Estelrich* Departament de Fisicoquı´mica, Facultat de Farma` cia, Universitat de Barcelona, Barcelona (Catalonia, Spain) Received February 22, 2005. In Final Form: May 4, 2005 β-Amyloid peptide (Aβ), in fibrillar form, is the primary constituent of senile plaques, a defining feature of Alzheimer’s disease. In solution assays, fibril formation exhibits a lag time, interpreted as a nucleation/ condensation-dependent process. The kinetics of fibrillogenesis is controlled by two key parameters: nucleation and elongation rate constants. We characterized the time course of Aβ fibril formation by measuring the scattering caused by peptide aggregates. We report here the interaction of Aβ with three alkylammonium bromides (dodecyl, tetradecyl, and hexadecyl) at supra- and submicellar concentrations and their influence on the kinetic constants. We observed a dual behavior: surfactants promoted or retarded fibril formation in a concentration-dependent manner. Below a determined surfactant concentration (close to the corresponding critical micellar concentration in medium without peptide), surfactants favor aggregation, presumably by means of electrostatic interactions that destabilize the native conformation. Beyond such concentration, the stabilizing effects of the monomer predominate. As a general rule, surfactants delay but do not completely inhibit aggregation.
Introduction Alzheimer’s disease (AD) is one of many diseases in which protein form amyloid aggregates. The brains of patients with AD contain a large number of amyloid deposits in the form of senile plaques. The amyloid core of these plaques contain interwoven fibrils that are composed by variants of the β-amyloid (Aβ) peptide. This peptide varies from 39 to 43 amino acids in length, the most abundant forms being 40 and 42 amino acids. Although a causal relationship between Aβ and the development of AD has not been conclusively demonstrated, considerable experimental data suggests that Aβ aggregates are important in the etiology of AD.1-4 Whereas early evidence suggested that Aβ fibrils initiate a cascade of events that result in neuronal cell death,5 many investigators now propose that soluble aggregates of Aβ (also called oligomers or protofibrils), rather than monomers or insoluble amyloid fibrils, may be responsible for synaptic dysfunction in AD.6-10 Nevertheless, Aβ fibrillogenesis is still thought to play a critical role in the development of AD. On the other hand, the conversion of Aβ, which in native form is random, into fibrils, rich in β-form, involves major structural changes leading to the partial or complete disruption of the native fold.11 * Author to whom correspondence should be addressed. Phone: + 34 93 4024559; fax: +34 93 403 59 87; e-mail: joanestelrich@ ub.edu. (1) Harper, J. D.; Lansbury, P. T., Jr. Annu. Rev. Biochem. 1997, 66, 385-407. (2) Murphy, R. M. Annu. Rev. Biomed. Eng. 2002, 4, 155-74. (3) Soto, C. Nature Rev. Neurosci. 2003, 4, 49-60. (4) Gorman, P. M.; Chakrabartty, A. Biopolymers 2001, 60, 381-94. (5) Yankner, B. A. Nat. Med. 1996, 850-852. (6) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353-356. (7) Hartley, D. M.; Walsh, D. M,; Ye, C. P.; Diehl, T.; Vasques, S.; Vassilev, P. M.; Teplow, D. B.; Selkoe, D. J. J. Neurosci. 1999, 19, 88768884. (8) Klein, W. L.; Krafft, G. A.; Finch, C. E. Trends Neurosci. 2001, 24, 219-224. (9) Westerman, M. A.; Cooper-Blacketer, D.; Mariash, A.; Kotilinek, L.; Kawarabayashi, T.; Younkin, L. H.; Carlson, G. A.; Younkin, S. G.; Ashe, K. H. J. Neurosci. 2002, 22, 1858-1867. (10) Kawarabayashi, T.; Shoji, M.; Younkin, L. H.; Lin, W. L.; Dickson, D. W.; Murakami, T.; Matsubara, E.; Abe, K.; Ashe, K. H.; Younkin, S. G. J. Neurosci. 2004, 24, 3801-3809.
In vitro studies have suggested that Aβ fibrillogenesis occurs in two distinct stages, nucleation and elongation of fibers, and that the kinetics of the process is controlled by two key parameters, nucleation rate (kn) and elongation rate (ke) constants.12 This model is similar to a nucleationdependent polymerization process much like that which characterizes crystal growth. The kinetics of such a fibrillization process can be explained in terms of an autocatalytic reaction mediating the transition from the monomer to the aggregate species.13 On the other hand, numerous studies have documented the importance of β-peptide-lipid interactions in AD.14 As micelles provide a molecular basis for the role of biological membranes in amyloid formation,15 we have studied the effect of surfactants on the propensity of the peptide to form fibrils. As the effects of the most representative anionic surfactant, namely, sodium dodecyl sulfate (SDS), on fibril formation have been extensively described,14,16-19 our study focuses on the effect of cationic surfactants on the aggregation of β-amyloid. Specifically, we have analyzed three of the alkyl trimethylammonium bromides: dodecyl (DTAB), tetradecyl (TTAB), and hexadecyl (HTAB). We have chosen alkyl bromide surfactants instead of phospholipids derivatives because we are interested in the dual behavior of surfactants: the effect of monomer molecules can be extremely different than that observed with micelles. (11) Klein, W. L.; Stine, W. B.; Teplow, D. B. Neurobiol. Aging 2004, 25, 569-80. (12) Jarrett, J. T.; Lansbury, P. T., Jr. Cell 1993, 73, 1055-58. (13) Sabate´, R.; Gallardo, M.; Estelrich, J. Biopolymers 2003, 71, 190-5. (14) Marcinowski, K. J.; Shao, H.; Clancy, E. L.; Zagorski, M. G. J. Am. Chem. Soc. 1998, 120, 11082-91. (15) Henry, G. D.; Sykes, B. D. Methods Enzymol. 1994, 239, 51535. (16) Montserret, R.; McLeish, M. J.; Bo¨ckmann, A.; Geourjon, C.; Penin, F. Biochemistry 2000, 39, 8362-73. (17) Xiong, L.-W.; Raymond, L. D.; Hayes, S. F.; Raymond, G. J.; Caughey, B. J. Neurochem. 2001, 79, 669-78. (18) Pertinhez, T. A.; Bouchard, M.; Smith, R. A. G.; Dobson, C. M.; Smith, L. J. FEBS Lett. 2002, 529, 193-7. (19) Kuroda, Y.; Maeda, Y.; Sawa, S.; Shibata, K.; Miyamoto, K.; Nakagawa, T. J. Pept. Sci. 2003, 9, 212-20.
10.1021/la050472x CCC: $30.25 © 2005 American Chemical Society Published on Web 06/18/2005
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In this paper, we report the effect of the indicated surfactants on the nucleation and elongation rate constants of fibrillogenesis, as determined by monitoring the appearance and the growth of fibrils as a function of time. Materials and Methods The peptide used was fragment 1-40 of Aβ (trifluoroacetic acid salt) and was obtained from Bachem (Bubendorf, Switzerland). DTAB, TTAB, and HTAB were purchased from Fluka (Buchs, Switzerland), and molecular sieve (0.4 nm) and 1,1,1,3,3,3hexafluoro-2-propanol (HFIP) were from Sigma (St. Louis, MO). Aβ Preparation. A stock solution was prepared by dissolving 1 mg of Aβ in 500 µL of HFIP, which had been dried at 4 °C over molecular sieve type 4A and then had been centrifuged at 15 000g for 15 min to remove molecular sieve dust. After drying, the solution was incubated at room temperature for 10 min. The HFIP was removed by evaporation under a gentle stream of nitrogen, leaving a slightly yellow film that was then resuspended in 500 µL of anhydrous dimethyl sulfoxide (DMSO) and was bathsonicated for 30 min. Sonication was crucial to remove any traces of undissolved seeds that may resist solubilization. This preparation yielded Aβ in monomeric form.20 Stock solution was divided into aliquots and stored at -20 °C for later use. Aliquots of Aβ stock were added to 10 mmol L-1 Tris-HCl pH 7.4 buffer containing the surfactant to a final peptide concentration of 25.8 µmol L-1. Concentrations of Aβ were determined by absorbance with a calculated molar absorptivity of 1450 M-1 cm-1 at 276 nm. Polymerization Assay. Fresh, nonaggregated Aβ was mixed with surfactant solution and was incubated at 37 °C. Surfactant concentrations used were the following: DTAB, 5-90 mmol L-1; TTAB, 0.5-6 mmol L-1; and HTAB, 0.1-1 mmol L-1. After mixing the peptide with the surfactant solution, 1 mL of this solution was poured into a cuvette, which was then capped tightly and placed inside a temperature-regulated incubator containing a magnetic stirrer. The solutions were stirred continuously (1500 rpm). The solution was briefly mixed by vortexing before each absorbance measurement to suspend the formed fibrils. Absorbances at 276 (tyrosine peak plus scattering) and 400 nm (scattering of the sample) were measured at 20-min intervals in a Shimadzu UV-2401 PC UV-visible spectrophotometer using a matched pair of quartz microcuvettes (1-cm optical length) placed in a temperature-regulated cell holder. This assay was performed in triplicate. Data Processing. Ratios of absorbance values were transformed into fraction of fibrillar form in the system, f. Kinetics of fibrillization has the following equation:13
f)
F{exp[(1 + F)kt] - 1} {1 + F exp[(1 + F)kt]}
(1)
under the boundary condition of t ) 0 and f ) 0, where k ) ke[Aβ]0 and F represent the dimensionless value to describe the ratio of kn to k. [Aβ]0 is the initial peptide concentration. By nonlinear regression of f against t, values of F and k can be easily obtained, and from them the rate constants ke and kn can be obtained. Extrapolation of the linear portion of the sigmoid curve to abscissa (f ) 0) and to the highest ordinate value of the fitted plot afforded two values of time (t0 and t1), which correspond to the lag time and to the time at which the aggregation was almost complete. The time at which half of the Aβ molecules were aggregated (i.e., when f ) 0.5) is the time of half-aggregation (t1/2) (Figure 1).
Results The aggregation behavior of Aβ is extremely sensitive to the presence of trace amounts of fibrils, seeds, and nonsedimenting oligomers often present in commercial samples. In the present study, we ensured a homogeneous starting solution of monomeric Aβ peptides, thereby permitting an examination of the influence of the studied surfactants on peptide aggregation. (20) Sabate´, R.; Estelrich, J. J. Phys. Chem. B 2005, 109, 11027-32.
Figure 1. Logistic curve obtained by plotting the fraction of fibrillar form of Aβ, f, as a function of time. Fraction of fibrillar form was determined by relating any relationship of absorbances at 400 and 275 nm to the maximal relationship observed. From the figure, it can be deduced that t0 indicates the time at which fibrillogenesis begins (f < 0.1), whereas t1 indicates the time at which the process is almost complete (f > 0.9). The time at which half of the Aβ molecules are aggregated (i.e., when f ) 0.5) is the time of half-aggregation (t1/2).
When fresh 25.8 µmol L-1 Aβ (1-40) is incubated at 37 °C with or without surfactant, the scattering or the absorbance of the peptide follows a characteristic sigmoidal curve with a clear kinetic lag phase before the rapid development of fibrils occurs. This latter behavior has analogies with the most familiar, extensively studied process of crystal growth and polymer gelation, and it is thought to be associated with the need to develop “nuclei” or “seeds”, small aggregates from which larger molecular assemblies can grow. The sigmoidal curve is consistent with the nucleation-dependent polymerization model12 and can be treated as an autocatalytic reaction.13 The highest absorbance value (at 400 or 276 nm) corresponded to the maximal amount of fibrils formed, that is, to the time of the endpoint amplitude (f ) 1). Lower values corresponded to fibrillar fractions. Values of F were obtained by nonlinear regression of f against t, and from them the rate constants ke and kn were obtained (Table 1). We use the polymerization of Aβ without surfactant as reference. Under these conditions, we obtained a nucleation constant of (0.12 ( 0.01)‚10-3 min-1 and an elongation constant of 1130 ( 60 mol L-1 min-1. When DTAB was present at a concentration from 5 to 30 mmol L-1, the corresponding curves were located to the left of the reference curve, whereas at higher DTAB concentrations they were to the right (Figure 2A). This indicates that low DTAB concentrations favored polymerization and the formation of fibrils, whereas above 40 mmol L-1, the surfactant produced a decrease of the velocities of nucleation and elongation. From Table 1, it can be deduced that DTAB from 5 to 30 mmol L-1 increased kn compared with the value obtained in absence of surfactant, but at higher concentrations, this value seldom varied. The effect of the lowest DTAB concentrations on ke was similar to that observed for kn, but the elongation constant underwent an important reduction when the surfactant concentration increased. Time of half-aggregation (t1/2) is a useful parameter with which to compare the effect of hypothetical inhibitors on peptide aggregation. In the experimental conditions used, t1/2 was 190 min in absence of surfactant. Addition of low concentrations of DTAB reduced this value to 10 or to 124 min (5 mmol L-1 and 30 mmol L-1 respectively), whereas higher DTAB concentrations prolonged this time significantly (on average by 240 min). Plotting t1/2 as a function of DTAB concentration produced a triphasic curve (Figure 2B). Extrapolation of the linear portion of the curve to the prolongation of both
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Table 1. Influence of DTAB on the Polymerization Constants of 25.8 µM Aβa DTAB/mmol L-1 constants
0
5
10
20
30
40
50
70
90
kn/10-3 min-1 0.12 ( 0.01 2.15 ( 0.31 4.93 ( 0.35 5.15 ( 0.38 3.41 ( 0.24 0.15 ( 0.01 0.09 ( 0.01 0.11 ( 0.01 0.08 ( 0.01 ke/L mol-1 min-1 1130 ( 60 1330 ( 90 1760 ( 120 1630 ( 110 1440 ( 100 970 ( 40 890 ( 80 860 ( 80 900 ( 70 t1/2/min 188 ( 1 10 ( 1 11 ( 1 34 ( 1 124 ( 2 213 ( 3 240 ( 4 236 ( 4 244 ( 4 a
Values are the mean ( standard deviation of three independent samples (CMC of DTAB: 16.5 mmol L-1).
Figure 2. Effect of DTAB on the kinetics of Aβ aggregate formation. (A) Time course of the aggregation process. Reaction mixtures containing 25.8 µmol L-1 Aβ (1-40), 10 mmol L-1 Tris-HCl pH 7.4, and 0 (b), 5 (+), 10 ()), 20 (×), 30 (4), 40 (O), 50 (0), 70 (*), or 90 (s) mmol L-1 DTAB were incubated at 37 °C (CMC of DTAB: 16.5 mmol L-1). Points represent means of three independent experiments. At all points, standard errors were within symbols. (B) Variation of the time of halfaggregation as a function of surfactant concentration. The figure of 17.5 mmol L-1 indicates that below this value the aggregation is immediate, while above 42.1 mmol L-1, maximal delay of aggregation is achieved. These values were calculated by the interception of straights obtained by simple linear regression. The 95% confidence interval for the above values is 12.4-22.6 mmol L-1 and 31.8-52.4 mmol L-1, respectively.
Figure 3. Effect of TTAB on the kinetics of Aβ aggregate formation. Reaction mixtures containing 25.8 µmol L-1 Aβ (140), 10 mmol L-1 Tris-HCl pH 7.4, and 0 (b), 2.0 (+), 3.0 ()), 4.0 (×), 5.0 (4), 5.5 (O), 5.75 (0), and 6.0 (*) mmol L-1 TTAB were incubated at 37 °C (CMC of DTAB: 2.7 mmol L-1). Points represent means of three independent experiments. At all points, standard errors were within symbols. (B) Variation of the time of half-aggregation as a function of surfactant concentration. The figure of 4.9 mmol L-1 indicates that below this value the aggregation is immediate. This value was calculated by the interception of two straights obtained by simple linear regression, and its 95% confidence interval is 3.5-6.3 mmol L-1.
inferior and superior straights generated two values of DTAB concentration, 17.5 and 42.1 mmol L-1. These values indicate that below 17.5 mmol L-1 the aggregation is immediate (in this way, we obtained a lag time of close to 0 min when DTAB was present at 5 mmol L-1, in comparison with the 118 min obtained without surfactant), while above 42.1 mmol L-1, maximal delay of aggregation is achieved. In this case, the lag time ranged from 140 to 155 min, depending on the surfactant concentration. Eventually, despite the inhibitory effect of DTAB, complete aggregation was achieved after 330 min. Figure 3A shows the results obtained with TTAB from 0 to 6.0 mmol L-1. The effect was quite similar to that observed with DTAB: low surfactant concentrations acted as promoters of fibril formation and shortened the lag time, while high concentrations inhibited the aggregation kinetically. Reversal of surfactant behavior was observed at 4.9 mmol L-1. It is worth noting the differences in the sharpness of the transition from nonfibrillar to fibrillar form. It was more pronounced at low surfactant concen-
trations than at higher ones. This implies that the surfactant increased or reduced the elongation rate, depending on its concentration. Table 2 shows the variation of elongation and nucleation constants. When t1/2 was plotted as a function of TTAB concentration, Figure 3B was obtained. This figure is different from that obtained with DTAB, since at higher surfactant concentrations than those plotted, the inhibition was total. Figure 4A shows the effect of HTAB from 0 to 0.9 mmol L-1. This surfactant retarded or promoted the conformational transition of Aβ in a concentration-dependent manner. From 0.2 to 0.7 mmol L-1, nucleation constants were higher than those obtained with water (Table 3). HTAB only began to produce an inhibitory effect on aggregation above a concentration of 0.4 mmol L-1. The interception of both lines afforded a value of 0.4 mmols L-1. The elongation constants were consistently higher than those obtained without surfactant. This is in agreement with the observed slope of the curves.
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Table 2. Influence of TTAB on the Polymerization Constants of 25.8 µM Aβa TTAB/mmol L-1 constants
0
2
3
4
5
5.5
5.75
6
kn/10-3 min-1 ke/L mol-1 min-1 t1/2/min
0.12 ( 0.01 1130 ( 60 188 ( 1
7.74 ( 0.62 2020 ( 190 38 ( 1
7.54 ( 0.55 2060 ( 280 49 ( 2
1.06 ( 0.14 2300 ( 210 64 ( 1
0.78 ( 0.06 980 ( 100 137 ( 3
0.02 ( 0.01 860 ( 80 322 ( 4
0.01 ( 0.01 660 ( 50 419 ( 6
0.01 ( 0.01 490 ( 40 >540
a
Values are the mean ( standard deviation of three independent samples (CMC of TTAB: 2.7 mmol L-1).
Figure 4. Effect of HTAB on the kinetics of Aβ aggregate formation. Reaction mixtures containing 25.8 µmol L-1 Aβ (140), 10 mmol L-1 Tris-HCl pH 7.4, and 0 (b), 0.2 (+), 0.3 ()), 0.4 (×), 0.5 (4), 0.7 (O), or 0.9 (0) mmol L-1 HTAB were incubated at 37 °C (CMC of HTAB: 0.43 mmol L-1). Points represent means of three independent experiments. At all points, standard errors were within symbols. (B) Variation of the time of halfaggregation as a function of surfactant concentration. The figure of 0.4 mmol L-1 indicates that below this value the aggregation is immediate. This value was calculated by the interception of two straights obtained by simple linear regression, and its 95% confidence interval is 0.3-0.6 mmol L-1.
Discussion Under the experimental conditions used, Aβ produces an aggregated β-structure in less than 5 h. Spectrophotometric analysis of conformational transition and amyloid fibril formation by the peptide showed a protracted lag time in monomer or in oligomer form, followed by a sigmoidal (cooperative) structural transition and fibril growth. Cationic surfactants have a dual effect, depending on concentration: they both stimulate and inhibit the formation of aggregates and amyloid material. The aggregation curves establish a boundary concentration for this behavior: 17.5 mmol L-1 for DTAB, 4.9 mmol L-1 for TTAB, and 0.4 mmol L-1 for HTAB. Previously, we used conductimetry to determine the critical micellar concentrations (CMC) of surfactants in 10 mmol L-1 TrisHCl, obtaining the following values: 16.5 mmol L-1 (DTAB), 2.7 mmol L-1 (TTAB), and 0.43 mmol L-1 (HTAB). However, the true values may be slightly different, since peptides have been suggested to lower the CMC of surfactants, because of the formation of mixed peptidesurfactant micelles.21 Comparing these values with those
obtained from aggregation curves, it is clear that the boundary concentration is close to that of the corresponding CMC, and we can deduce that the stimulating effect was due to the monomeric form of the surfactant, whereas micelles substantially reduced the propensity to aggregate. Submicellar concentrations of alkyl bromides promote rapid fibril formation despite the fact that the initial species are monomeric. A similar conversion of soluble state to β-rich aggregates has been observed previously for several peptides in SDS solution,18 as well as for the protein acylphosphatase under moderate concentrations of TFE, although at high concentrations aggregation was strongly inhibited.22 Under these circumstances, monomers of surfactant bind to the peptide through electrostatic interactions. In this way, the positive charge of the ammonium group of alkyl bromide surfactants interacts with the negatively charged amino acids of the peptide. At pH 7.4, 15% binding sites are negatively charged, since the side chains of Asp1, Glu3, Asp7, Glu11, Glu22, and Asp23 are deprotonated. This charge neutralization can reduce the electrostatic repulsion within the polymer molecule, making a conformational transition to β conformation more likely. In the presence of alkyl bromides, fibril formation can be stimulated not just by unfolding but also by an enhanced stability of hydrogen-bonding interactions. It is also likely that the alkyl chains stimulate aggregation. Before and after neutralization of the acidic groups of glutamic and aspartic acids, the hydrocarbon tails of surfactants induce cagelike ordering of the first shell of water molecules, strengthening their H-bonding and causing significant decrease in the entropy of the water.23 If hydrophobic interactions occur between uncharged residues and the hydrophobic tail, the entropy of the water increases. Thus, the driving force of the stimulated aggregation is the same as that which explains micelle formation, that is, the hydrophobic effect. Once such aggregates are formed, intermolecular β-sheet interactions are likely to develop readily, such that the energy barrier for nucleation is overcome. Hence, β-aggregates of peptides are formed as a consequence of submicellar concentrations. On the other hand, positive charges are also present on the peptide sequence at neutral pH. According to this fact, the electrostatic interactions between positively and negatively charged residues would be an essential feature of the aggregation mechanism. It is likely that neutralizing the negative charges hampers the electrostatic aggregation, making more favorable the aggregation in β-structures. Beyond the boundary concentration, the antiaggregant effects of the surfactant begin to appear. Now, two surfactant species coexist in solution: micelles and monomers (to simplify, we have overlooked the existence of premicellar aggregates, such as those observed in DTAB solutions).24 Each one of these species exerts an antagonist (21) Tessari, M.; Foffani, M. T.; Mammi, S.; Peggion, E. Biopolymers 1993, 33, 1877-87. (22) Chiti, F.; Webster, P.; Taddei, N.; Clark, A.; Stefani, M.; Ramponi, G.; Dobson, C. M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3590-5. (23) Tanford C. The Hydrophobic effect: Formation of micelles and biological membranes; Wiley Publishing: New York, 1980.
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Table 3. Influence of HTAB on the Polymerization Constants of 25.8 µM Aβa HTAB/mmol L-1 constants
0
0.2
0.3
0.4
0.5
0.7
0.9
kn/10-3 min-1 ke/mol-1 L min-1 t1/2/min
0.12 ( 0.01 1130 ( 60 188 ( 1
1.22 ( 0.33 3280 ( 260 49 ( 1
1.10 ( 0.21 3150 ( 250 53 ( 1
1.19 ( 0.16 3100 ( 280 59 ( 1
0.43 ( 0.06 3200 ( 270 91 ( 1
0.20 ( 0.02 2120 ( 140 148 ( 1
0.07 ( 0.01 1540 ( 70 218 ( 1
a
Values are the mean ( standard deviation of three independent samples (CMC of HTAB: 0.43 mmol L-1).
effect, and the observed effect will be that of the predominant species. For instance, DTAB at a concentration of 30 mmol L-1 (well above the CMC) theoretically contains 16.5 mmol L-1 monomers, and the rest will be surfactant in micellar form. Bearing in mind that at this concentration the DTAB micelles have an aggregation number, N, of 61,25 the concentration of micelles will be 0.22 mmol L-1. Hence, we have approximately nine micelles per molecule of Aβ, but under these conditions, there are also more than 600 molecules of surfactant per Aβ. As the stoichiometry of the interaction is six surfactant molecules per peptide molecule (at pH 7.4, there are six amino acids charged negatively), there are more than 100 reactive units per peptide molecule. These figures suggest that the effect of surfactant monomers will be greater than that afforded by the micelles, as shown in Figure 2A. At this concentration, the aggregation curve is displayed to the right of that obtained without surfactant, but its slope is closer to water than that observed at 10 mmol L-1. At 50 mmol L-1, the delaying effect of the surfactant on aggregation is clear. Now, the micelle concentration is 0.59 mmol L-1, corresponding to 25 micelles per molecule of peptide (at this concentration, the aggregation number is 63). From Figure 2B, we deduced that the theoretical concentration for obtaining the maximal delay of aggregation was 43.4 mmol L-1. This concentration corresponds to 17 micelles of surfactant per peptide molecule (N ) 62). Thus, the maximal effect of DTAB under the assay conditions is achieved with a ratio of 17 micelles per molecule of Aβ. The effect of the aliphatic tail length is important not only for micellar characteristic but also for those concerning the interaction with Aβ. The TTAB molecule has two more methyl groups than DTAB, thereby reducing the CMC to 2.7 mmol L-1 and the boundary concentration to 4.9 mmol L-1. Figure 3A shows that at 5.5 mmol L-1 the retardation of aggregation overcame the propensity of the monomers to aggregate. At this concentration, the concentration of micelles is 0.037 mmol L-1 (N ) 75), which corresponds to 1.4 micelles per molecule of Aβ. At the boundary concentration, the stoichiometric ratio between micelles and peptide molecules is approximately 1. HTAB, with an aliphatic tail containing 16 methyl groups, has a CMC of 0.43 mmol L-1 and a boundary concentration similar to the CMC (0.4 mmol L-1). However, the antiaggregant effect is not very apparent below 0.9 mmol L-1 of surfactant. At this concentration, the micelle concentration is 0.004 mmol L-1 (N ) 106) and the stoichiometry is 0.17 micelles per Aβ molecule. Takanashi et al.26 showed that HTAB retarded or inhibited the structural transition of a peptide undergoing R f β transition and amyloid fibrillogenesis at concentrations over 0.5 mmol L-1. These data suggest that the length of the aliphatic chain plays an important role in the antiaggregant effect. In this way, micelles of 12-carbon alkyl bromides are weaker delayers (24) Sabate´, R.; Gallardo, M.; Estelrich, J. Electrophoresis 2000, 21, 481-5. (25) Sabate´, R.; Estelrich, J. J. Phys. Chem. B 2003, 107, 4137-42. (26) Takanashi, Y.; Ueno, A.; Mihara, H. Chem. Eur. J. 1998, 4, 2475-84.
Figure 5. Scheme of the interaction of alkyl bromide surfactants with the peptide β-amyloid. In the absence of surfactant, the time needed to reach a complete aggregation is t, in the presence of submicellar surfactant concentrations is ts, whereas in the presence of supramicellar concentrations is tm. Experimental data shows that ts < t < tm.
of the peptide aggregation than micelles with chains of 16-carbons. The molecular mechanism by which micellar alkyl bromides stabilize secondary structure is unclear. Even in the case of the most extensively studied surfactant, SDS, the details of the molecular mechanism of stabilization are still lacking.16 For hydrophobic membrane peptides, it is generally reported that peptides penetrate deeply into the hydrophobic core of SDS micelles and adopt an R-helical structure as a result of the hydrophobic interactions.27 For amphipathic peptides, SDS micelles provide a hydrophobic environment, presumably allowing folding and stabilization to be driven by hydrophobic effects.28 Thus, as Aβ is an amphipathic peptide that undergoes electrostatic interactions with the monomer or the micelle of alkyl bromides, we can raise the question of whether the peptide is anchored via electrostatic bonds at the surface of the micelle or instead is incorporated into its hydrophobic core. The dual behavior of these positive surfactants leads us to propose (Figure 5) that the monomer, mainly in random coil, in our conditions as well as in physiological ones, will be able to undergo a change to β-sheet conformation. Then, the folded monomers will aggregate and form the amyloid. In the presence of surfactants at submicellar concentrations, the surfactant monomers aid to a more rapid folding, and, consequently, the peptide has a propensity to aggregate. On the contrary, the presence of micelles induces a delay or a inhibition of the aggregation. It is possible that micelles concentrate peptide close to their surface, and this fact avoids the interaction among the peptide monomers. A similar behavior has been observed in the amyloid formation of apolipoprotein C-II in the presence of short-chain phospholipids.29 Overall, the finding that cationic surfactants can both stimulate and inhibit fibril formation reflects the sensi(27) Pervushin, K.; Orekhov, V. Yu.; Popov, A. I.; Musina, L. Yu.; Arseniev, A. S. Eur. J. Biochem. 1994, 219, 571-83. (28) Waterhous, D. V.; Johnson, W. C. Biochemistry 1994, 33, 21218. (29) Hatters, D. H.; Lawrence, L. J.; Howlett, G. J. FEBS Lett. 2001, 494, 220-224.
Effects of Surfactants on β-Amyloid Fibrillogenesis
tivity of this process to the solution environment, undoubtedly a major factor in the deposition, and hence the toxicity of fibrils in biological systems.30 Conclusions In this work, we have demonstrated that the interaction of alkyl bromide surfactants with the β-amyloid peptide can either increase or decrease the relative rates of amyloid fibril formation. Submicellar concentrations induce a more rapid and extensive change in scattering compared to data (30) Bucciantini, M.; Giannoni, E.; Chiti, F.; Baroni, F.; Formigli, L.; Zurdo, J. S.; Taddei, N.; Ramponi, G.; Dobson, C. M.; Stefani, M. Nature 2002, 416, 507-11.
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obtained for β-amyloid alone. Conversely, these surfactants at supramicellar concentrations cause a systematic reduction in the scattering increase over the sample time scale. This reduction of the number or size of aggregates can be accompanied by a delaying of the time needed to reach the end point amplitude. Acknowledgment. This work was supported in part by funds from the Spanish Ministerio de Sanidad y Consumo (FIS 00/1121). LA050472X
