Báo cáo khoa học: Secondary structure conversions of Alzheimer’s Ab(1–40) peptide induced by membrane-mimicking detergents pdf

Secondary structure conversions of Alzheimer’s Ab(1–40) peptide induced by membrane-mimicking detergents Anna Wahlstro ¨ m 1, *, Loı ¨ c Hugonin 1, *, Alex Pera ´ lvarez-Marı ´n 1, *, Ju ¨ ri Jarvet 2 and Astrid Gra ¨ slund 1 1 Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden 2 The National Institute of Chemical Physics and Biophysics, Tallinn, Estonia Introduction The amyloid b peptide (Ab) is the major component of the amyloid plaques, which are found in the brains of Alzheimer’s disease patients. The Ab-peptide is a 39–42 residue peptide cleaved by processing of the amyloid-b precursor protein [1,2]. The Ab(1–40) peptide has a hydrophilic N-terminal domain and a more hydrophobic C-terminal domain, and contains a central hydrophobic cluster (residues 17–21) suggested to play an important role in peptide aggregation. Solu- ble oligomeric peptide aggregates are reported to medi- ate toxic effects on neurons and synapses [1,3] and have attracted growing interest because of their proba- ble link to the pathology of the disease. The formation of aggregates occurs in parallel with a conformational change of the peptide structure to b-sheet. In vitro, the Ab monomer is in a dominating random coil secondary structure in solution at room temperature and physiological pH [4–7]. However, in Keywords amyloid b peptide; CD; NMR; oligomer; SDS Correspondence A. Gra ¨ slund, Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden Fax: +46 8 155597 Tel: +46 8 162450 E-mail: astrid@dbb.su.se *These authors contributed equally to this work (Received 29 April 2008, revised 8 August 2008, accepted 13 August 2008) doi:10.1111/j.1742-4658.2008.06643.x The amyloid b peptide (Ab) with 39–42 residues is the major component of amyloid plaques found in brains of Alzheimer’s disease patients, and solu- ble oligomeric peptide aggregates mediate toxic effects on neurons. The Ab aggregation involves a conformational change of the peptide structure to b-sheet. In the present study, we report on the effect of detergents on the structure transitions of Ab, to mimic the effects that biomembranes may have. In vitro , monomeric Ab(1–40) in a dilute aqueous solution is weakly structured. By gradually adding small amounts of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate to a dilute aqueous solution, Ab(1–40) is converted to b-sheet, as observed by CD at 3 °C and 20 °C. The transition is mainly a two-state process, as revealed by approximately isodichroic points in the titrations. Ab(1–40) loses almost all NMR signals at dodecyl sulfate concentrations giving rise to the optimal b-sheet content (approxi- mate detergent ⁄ peptide ratio = 20). Under these conditions, thioflavin T fluorescence measurements indicate a maximum of aggregated amyloid-like structures. The loss of NMR signals suggests that these are also involved in intermediate chemical exchange. Transverse relaxation optimized spec- troscopy NMR spectra indicate that the C-terminal residues are more dynamic than the others. By further addition of SDS or lithium dodecyl sulfate reaching concentrations close to the critical micellar concentration, CD, NMR and FTIR spectra show that the peptide rearranges to form a micelle-bound structure with a-helical segments, similar to the secondary structures formed when a high concentration of detergent is added directly to the peptide solution. Abbreviations Ab-peptide, amyloid b peptide; D ⁄ P, detergent to peptide ratio; HSQC, heteronuclear single quantum coherence; LiDS, lithium dodecyl sulfate; ppII, polyproline II; SDS-d 25, deuterated SDS; ThT, thioflavin T; TROSY, transverse relaxation optimized spectroscopy. FEBS Journal 275 (2008) 5117–5128 ª 2008 The Authors Journal compilation ª 2008 FEBS 5117 membrane-mimicking environments, such as SDS micelles, the Ab-peptide displays an a-helical structure, with two a-helical segments comprising residues 15–24 and 29–35, separated by a flexible hinge [8], and less structured N- and C-termini. In the presence of phos- pholipid vesicles, a-helical structures as well as b-sheet structures have been reported [9]. Rangachari et al. [10] have described interfacial aggregation of Ab(1–40) at a polar ⁄ nonpolar interface, with a concomitant increase in b-structure content, brought about by SDS micelles. In line with this finding, it was recently shown that the Ab(1–40) and Ab(1–42) peptides form b-sheet- rich aggregates at SDS concentrations significantly below the critical micellar concentration [11]. These aggregates give rise to thioflavin T (ThT) fluorescence and are neurotoxic. In the present study, we report on further properties of soluble oligomeric b-sheet-rich Ab(1–40) aggregates, formed at submicellar SDS or lithium dodecyl sulfate (LiDS) concentrations at detergent ⁄ peptide ratios of approximately 20. The results obtained by CD, NMR, FTIR and ThT fluorescence are compared and inter- preted in terms of mixed micelle-like aggregates with amyloid properties at intermediate detergent concen- trations, where the peptides show dynamic properties, particularly in the C-termini. Results Structural transitions of the Ab-peptide induced by increasing concentrations of membrane-mimicking detergents (SDS or LiDS) were studied by CD, NMR and FTIR spectroscopy at temperatures in the range 3–25 °C. LiDS was used at low temperature measure- ments because it has a higher solubility at lower tem- peratures than SDS; however, the critical micellar concentration is approximately the same for the two detergents [12,13]. CD spectroscopy Detergent titration experiments were performed on a sample with 75 lm Ab(1–40) peptide in 10 mm sodium phosphate buffer at 3 °C and 20 °C and pH 7.2. The structural starting point for Ab(1–40) varies to some extent as a function of temperature. At 3 °C, the secondary structure includes contributions from a poly- proline II (ppII) helix, whereas, at 20 °C, the second- ary structure is almost exclusively random coil, as described previously [5]. Figure 1A shows the titration of the Ab(1–40) pep- tide with microvolumes of LiDS at 3 °C over a deter- gent concentration interval in the range 0.05–20 mm, corresponding to detergent⁄ peptide (D⁄ P) ratios of 0.7–267, respectively. The CD data report on a first structural conversion from a mixture of ppII helix and random coil (weak positive shoulder at approximately 220 nm and negative minimum at 198 nm) occurring at low LiDS concentrations (up to 0.7 mm,D⁄ P=9) to a signal appearing at 1.6 m m LiDS (D ⁄ P = 21) with a maximum at 195 nm and a minimum at 218 nm, indicative of a dominating b-sheet structure. It should be noted that, up to this titration point, the spectra show a relatively well defined isodichroic point, implying a two-state transition between the initial structure and the b-sheet structure. After increasing the LiDS concentration further (3.0 mm LiDS, D ⁄ P = 40), a new state is observed, mostly consisting of a-helix structure. The conversion to a-helix struc- ture reached its final state at 20 mm LiDS with a char- acteristic maximum and two minima at 193 and 208 ⁄ 222 nm, respectively. Figure 1B shows the SDS titration experiment at 20 °C. In the absence of SDS, Ab(1–40) gives a CD spectrum with a minimum at 198 nm, indicating a pre- dominantly random coil secondary structure. As the detergent concentration was increased, the CD signal disappeared in the wavelength region around 198 nm (SDS concentration of approximately 4 mm, D ⁄ P = 53). Further increase of the SDS concentration (up to 5 mm,D⁄ P = 67) yielded a b-sheet spectrum with a positive maximum at 194 nm and a negative minimum at 218 nm. Also at this temperature, there was a relatively well-defined isodichroic point in the titration; however, this was not as clear as in the 3 °C titration. At high SDS concentrations (above 10 mm SDS, D ⁄ P = 133), the secondary structure was mainly a-helix, with a characteristic maximum at 192 nm and two minima at 208 and 221 nm. The mean residual molar ellipticity at 195 nm as a function of detergent concentration at 3 °C and 20 °C is shown in Fig. 1C. The disappearance of an initial weakly structured state and conversion to b-sheet and then to a-helix are evident. The CD intensities at this wavelength allowed us to compare the detergent secondary structure induction at 3 °C and 20 °C (Fig. 1C). Only one transition was visible with a mid- point at 1 mm LiDS at 3 °C. At 20 °C and with SDS, three transitions could be distinguished. The first had a midpoint at 0.7 mm, followed by two more transitions, with midpoints at 2.1 and 4.6 mm SDS. Figure 1D shows the corresponding curves for the mean residual ellipticity at 208 nm as function of detergent concentration. At 3 °C with LiDS, the data show two sigmoidal transitions. The first sigmoidal transition (positive) occurred in the range 0–1.6 mm Detergent-induced Ab(1–40) secondary structures A. Wahlstro ¨ m et al. 5118 FEBS Journal 275 (2008) 5117–5128 ª 2008 The Authors Journal compilation ª 2008 FEBS LiDS with a midpoint at 0.7 mm, corresponding to the transition from initial structure to the structure domi- nated by b-sheet. The second sigmoidal transition (neg- ative) had a midpoint at 2.6 mm. We interpret this as corresponding to the transition from b-sheet to a-helix. In the SDS titration at 20 °C, the intensity at 208 nm again indicated three sigmoidal transitions. Two posi- tive transitions had midpoints at 0.9 and 2 mm, respec- tively, and the third (negative) had a midpoint at 6mm. It should be noted that three transitions are visible at 20 °C at both wavelengths studied, and that the SDS concentration midpoints are in approximate agreement: the first transition at approximately 0.8 mm SDS (D ⁄ P = 11), the second one at approximately 2mm SDS (D ⁄ P = 27) and the third one at approximately 5 mm SDS (D ⁄ P = 67). The third transition probably involves the formation of the partly a-helical state, whereas the two first may involve two similar but distinguishable states with b-sheet structures. Fig. 1. Circular dichroism spectra of 75 lM Ab(1–40) peptide in 10 mM phosphate buffer at pH 7.2 in the presence of different concentra- tions of detergent. (A) At 3 °C in LiDS: open square, buffer; open circle, 0.05 m M; open triangle, 0.1 mM; filled square, 0.3 mM; open diamond, 0.5 m M; filled circle, 0.7 mM; filled hexagon, 1.0 mM; open hexagon, 1.3 mM; open star, 1.6 mM; cross, 2.0 mM; filled star, 3.0 m M; open pentagon, 20 mM. (B) At 20 °C in SDS: open square, buffer; open circle, 0.1 mM; filled star, 0.8 mM; open triangle, 2.0 mM; open pentagon, 3.8 m M; filled square, 4.2 mM; open diamond, 4.3 mM; filled circle, 5.0 mM; filled triangle, 6.2 mM; open hexagon, 7.0 mM; open star, 12.2 m M. (C) Plot of the mean residual molar ellipticity at 195 nm for the experiment in LiDS at 3 °C (filled square) and for the experiment in SDS at 20 °C (open circle). (D) Plot of the mean residual molar ellipticity at 208 nm for the experiment in LiDS at 3 °C (filled square) and for the experiment in SDS at 20 °C (open circle). A. Wahlstro ¨ m et al. Detergent-induced Ab(1–40) secondary structures FEBS Journal 275 (2008) 5117–5128 ª 2008 The Authors Journal compilation ª 2008 FEBS 5119 NMR spectroscopy Heteronuclear single quantum coherence (HSQC) and transverse relaxation optimized spectroscopy (TROSY) NMR spectroscopy were used to follow the structural transitions of the Ab(1–40) peptide (75 lm) induced by increasing concentrations of the membrane-mimicking detergent LiDS. The 1 H- 15 N HSQC spectrum of uniformly 15 N-labeled Ab(1–40) in 10 mm phosphate buffer (pH 7.2, 3 °C) at the beginning of a titration is shown in Fig. 2 (left). The corresponding spectrum of the peptide in 128 mm LiDS at the end of a titration is also shown in Fig. 2 (right, green spectrum). There are significant chemical shift differences in comparison to the initial state. Figure 2 (right) also includes the HSQC spectrum of the peptide after direct addition of 150 mm LiDS at 3 °C (red spectrum). The two spectra shown in Fig. 2 (right) were found to overlap very well with one another. However, the intensities (when corrected for different peptide concentrations) were significantly smaller in the spectrum after titration. Assignments of the amide groups of Ab(1–40) in buffer (Table S1) were made by comparison with the previous assignment [14]. Assignment of Ab(1–40) in 150 mm LiDS at 3 °C (Table S1) was performed by starting the NMR experiment at 25 °C where assign- ments are known [8] and decreasing the temperature by 5 °C at a time following the gradual changes of the HSQC spectra. The similarity of chemical shift patterns at 3 °C and 25 °C suggests that the previously determined a-helical regions involving residues 15–24 and 29–35 are the same at the two temperatures after direct addition of a high concentration of detergent [8]. Between the two well defined states shown in Fig. 2 (i.e. at an intermediate detergent concentration), a new state of the peptide characterized by complete NMR signal loss was observed. This state occurred at a criti- cal concentration of LiDS of 1–2 mm, corresponding to D ⁄ P = 13–27. There was no obvious change in chemical shifts, nor linewidth, of the amide HSQC crosspeaks by the grad- ual titration with detergent below the concentration inducing signal loss. To study how the signal was influ- enced by an increasing concentration of detergent, the volume of each crosspeak was integrated. In a titration series with small titration steps (0.05, 0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 10 and 20 mm), most of the signals were unchanged or slowly decayed up to a LiDS concentra- tion of 0.5 mm. However, beyond 0.5 mm LiDS, the signal from every residue abruptly decreased (Fig. 3). Fig. 2. HSQC NMR spectra and assignment of amide crosspeaks for the Ab(1–40) peptide, and the effect of added lithium dodecyl sulfate. Left: 1 H- 15 N HSQC spectrum of 75 lM uniformly 15 N-labeled Ab(1–40) in 10 mM phosphate buffer. The two peaks (V39 and V40) found in the TROSY experiment with 75 l M 15 N-Ab(1–40) in the presence of 2 mM LiDS are indicated with arrows. Right: overlay of HSQC spectra; 75 l M 15 N-labeled Ab(1–40) in 128 mM of LiDS (i.e. the end point in the titration series 0, 0.5, 1, 4, 8, 16, 32, 64 and 128 mM LiDS) (green spectrum) and 300 l M 15 N-labeled Ab(1–40) in 150 mM of LiDS, added in one addition (red spectrum). The peak intensities are corrected in relation to the different peptide concentrations. All measurements were performed in 10 m M phosphate buffer at pH 7.2 and 3 °C. Detergent-induced Ab(1–40) secondary structures A. Wahlstro ¨ m et al. 5120 FEBS Journal 275 (2008) 5117–5128 ª 2008 The Authors Journal compilation ª 2008 FEBS At 1 mm LiDS, corresponding to D ⁄ P = 13, almost all the HSQC crosspeaks had disappeared and, at 2mm, all were lost. At LiDS concentrations of 10 and 20 mm, the crosspeaks reappeared, directly with chemi- cal shifts closely corresponding to those observed after direct addition of 150 mm LiDS (Fig. 2, right, red spectrum). The crosspeaks from the amide groups in the amino acid residues in the N- and C-terminal ends returned with the strongest signals upon titration with detergent (Fig. 3). This is probably due to an increased mobility in the N- and C-terminal end segments (i.e. residues up to G9 and beyond G37). The chemical shifts observed at detergent concentrations of 10 and 20 mm were retained in the presence of the higher LiDS con- centrations of 64 and 128 mm, which all coincide with the chemical shifts found at 150 mm LiDS (Fig. 2). The disappearance of all NMR peaks at detergent concentrations of 1–2 mm may have more than one explanation. An obvious reason for signal loss is that Fig. 3. The crosspeak signal intensity of assigned residues of 15 N-labeled Ab(1–40) in the 1 H- 15 N HSQC spectra as a function of LiDS con- centration (0, 0.05, 0.1, 0.2, 0.3, 0.5, 0.7, 1, 2, 10, 20 and 128 m M) at pH 7.2 and 3 °C. The volumes of the HSQC crosspeaks of 75 lM of 15 N-Ab(1–40) were integrated. The amino acids are divided into different figures according to the earlier findings indicating that residues 15–24 and 29–35 have a-helical structure, whereas the regions in the ends and in between are unstructured [8]. The x-axis (LiDS concentra- tion) is shown as a logarithmic scale. A. Wahlstro ¨ m et al. Detergent-induced Ab(1–40) secondary structures FEBS Journal 275 (2008) 5117–5128 ª 2008 The Authors Journal compilation ª 2008 FEBS 5121 large, probably heterogeneous, aggregates of peptide and detergent are formed. Exchange on an intermedi- ate time scale between aggregates of different sizes may also contribute. To study this state further, TRO- SY experiments and translational diffusion NMR experiments were performed. TROSY makes it possi- ble to study larger proteins or complexes because it reduces transverse relaxation rates [15]. In the TROSY spectrum in the presence of 2 mm LiDS (i.e. at the same conditions where all HSQC signals disappeared), two C-terminal residues were observable (V39 and V40) (Fig. 2, left). This observation suggests a higher mobility of the charged carboxy terminus of Ab(1–40) in the aggregated state. NMR translational diffusion experiments were per- formed to investigate whether NMR-visible complexes of different sizes could be observed during the deter- gent titration. The results (Fig. 4) revealed a diffusion coefficient of 1.29 · 10 )10 m 2 Æs )1 for Ab(1–40) in D 2 O buffer (pH 7.4, 25 °C), indicating that the observable peptide is monomeric [4]. The diffusion coefficient did not change significantly from this value in the presence of 0.1, 0.5 and 1 mm deuterated SDS (SDS-d 25 ). The NMR signal disappeared abruptly at 2 mm SDS-d 25 , and a diffusion coefficient could not be determined for this condition. At 5 mm, the resonances had reap- peared in the ‘new’ positions and the associated diffusion coefficient had decreased. This implies forma- tion of an assembly of peptides, probably also in complex with detergent molecules (Fig. 4). At the same time, some fibrils could be seen in the sample solution. The diffusion coefficient for Ab(1–40) in the presence of 10 mm SDS-d 25 was 0.85 · 10 )10 m 2 Æs )1 , which can be compared with the diffusion coefficient 0.48 · 10 )10 m 2 Æs )1 for 100 lm Ab(1–40) in directly added 150 mm SDS, which comprises a state when one peptide is bound to one micelle [8]. FTIR spectroscopy FTIR spectroscopy was used to obtain further infor- mation about the striking changes in the secondary structure of Ab(1–40) observed at concentrations in the range of 0–4 mm SDS or LiDS. The amide I¢ region in the IR spectrum is indicative for the second- ary structure of the peptide. It has been shown that Ab(1–40) has a strong secondary structure concentra- tion dependence [16]. To increase the signal-to-noise ratio and to eliminate contributions of the baseline drift, the concentration of peptide was as low as possi- ble (100 lm), and only slightly higher than the CD and NMR concentrations. The negative second derivative of the spectra in the amide I¢ band is shown in Fig. 5. Assignment of different secondary structures was performed according to Byler and Susi [17]. The spectra indicate that, at 20 °C, the peptide had a mixture of random coil and b-sheet secondary struc- ture in the absence of SDS and with SDS at a low D ⁄ P ratio of 1 (0.1 mm SDS). At 1.4 mm, the random coil contribution disappeared, the b-sheet contribution decreased and a-helix structure became evident. At a Fig. 4. The translational diffusion coefficient (D t )of75lM Ab(1–40) versus increasing SDS-d 25 concentration (0, 0.1, 0.5, 1, 5 and 10 m M). The experiment was performed in 10 mM phosphate buffer at 25 °C and pH 7.4. , diffusion coefficient for 100 lM Ab(1–40) in 150 mM SDS at 25 °C [8]. The gray box indicates the conditions for which a diffusion coefficient could not be determined due to signal loss. Fig. 5. Secondary structure induction by SDS of 100 lM Ab(1–40) in 10 m M phosphate buffer at pH 7.2 and 20 °C. The negative second derivative of the peptide in the presence of different SDS concentrations is shown: thick black line, 0 m M; thin black line, 0.1 m M; gray line, 1.4 mM; light gray line, 10 mM. The spectra were normalized for trifluoroacetic acid intensity (as indicated by an aster- isk). The wavenumber intervals corresponding to the specific secondary structures are also indicated. Detergent-induced Ab(1–40) secondary structures A. Wahlstro ¨ m et al. 5122 FEBS Journal 275 (2008) 5117–5128 ª 2008 The Authors Journal compilation ª 2008 FEBS high SDS concentration (10 mm), the peptide showed a predominantly a-helix structure, with a shoulder in the b-sheet region. ThT interactions The titration of 75 lm Ab(1–40) with increasing amounts of SDS at 20 °C was monitored by ThT (15 lm) fluorescence, a classical probe for aggregated amyloid material [18]. Figure 6 shows a titration curve yielding maximum ThT fluorescence (approximately 8 · initial intensity) at 2.2 mm SDS. Further addition of SDS decreased the fluorescence intensity to approxi- mately 3 · initial intensity (at 45 mm SDS). The lack of complete reversal of ThT fluorescence indicates the presence of some remaining amyloid-like material also at higher SDS concentrations, although most of the aggregation appears to have been reversed. As a control, an SDS titration of ThT in the absence of Ab(1–40) was also performed (Fig. 6). This experiment showed that ThT fluorescence is generally low com- pared to the results in the presence of the peptide. However, also in the absence of peptide, ThT fluores- cence under SDS titration follows a sigmoid curve, with a midpoint at approximately 2 mm. This behav- iour of ThT is in general agreement with previous results obtained for ThT interacting with anionic micelles [19]. To further characterize the state of the Ab(1–40) during the SDS titration, five representative SDS titration points were chosen (0, 1.1, 2.2, 4.6 and 25.5 mm SDS) for investigation by native-PAGE (Fig. S1). A preliminary qualitative assessment of the gels could be performed with respect to the presence of low and high molecular weight species in the different samples [20]. Whereas the 0 and 25.5 mm SDS samples had a relatively high population of low molecular weight species (presumably peptide monomers), the samples prepared with intermediate SDS concentra- tions showed mainly high molecular weight (aggre- gated) peptide species. Discussion By combining CD, NMR and FTIR experiments, we have shown that the aggregation process of Ab(1–40) induced by LiDS or SDS detergent gives rise to a variety of secondary structure states, each of which is relatively stable under its given conditions. It is demonstrated that the extreme variability of the secondary structure of the peptide is dependent on its environment. The CD results reveal that, in a dilute aqueous solu- tion, Ab(1–40) has a dominating random coil second- ary structure with a low contribution of ppII helix and b-sheet at low temperature. Titrations with SDS or LiDS show that a secondary structure conversion of Ab(1–40) can be described essentially as a two-state process, involving conversion of the initial weak struc- ture to b-sheet-rich structures. Continued addition of SDS or LiDS, reaching concentrations close to the critical micellar concentration, induces rearrangement of the peptide structure to a structure with a-helix con- tributions. The NMR results at 3 °C show that the Ab(1–40) peptide retains its random coil ⁄ ppII structure free in solution in the presence of low detergent concentra- tions in the range 0.05–0.5 mm. At a detergent concen- tration of 1–2 mm, on the other hand, the NMR signal is essentially lost and the results suggest peptide aggre- gation and possibly intermediate chemical exchange. Preliminary light absorption observations (data not shown) suggest considerable light scattering under these conditions, in agreement with the formation of large particles. A high molecular weight state induced by submi- cellar concentrations of detergent was also recently observed by Tew et al. [11] using CD and NMR. They observed that the 1D 1 H-NMR spectrum disappeared at a certain SDS concentration but showed up again at SDS concentrations above the critical micellar concen- tration. In the present study, we aimed to analyze the aggregated state further after assignment of the amide Fig. 6. SDS titration monitored by ThT fluorescence. SDS titrations in the absence of peptide (open squares) and in the presence of 75 l M Ab(1–40) (open and filled circles) showing the fluorescence changes of 15 l M ThT in 10 mM phosphate buffer at pH 7.3 and 20 °C. The SDS concentrations are: 0, 0.09, 0.4, 0.6, 1.1, 1.7, 2.2, 2.8, 4.6, 6.5, 8.4, 10.3, 14.1, 17.9, 25.5 and 45 m M. Full circles indi- cate the concentrations analyzed by native-PAGE (see Supporting information, Fig. S1). A. Wahlstro ¨ m et al. Detergent-induced Ab(1–40) secondary structures FEBS Journal 275 (2008) 5117–5128 ª 2008 The Authors Journal compilation ª 2008 FEBS 5123 HSQC crosspeaks. We were able to follow the intensity changes for the individual amino acids at increasing LiDS concentrations. We conclude that the signals only partly recover towards high detergent concentrations. At and above the critical micellar concentration, the recovered spectrum displays amide chemical shifts very similar to those seen in a sample after direct addition of a high concentration of deter- gent. The amide groups of N- and C-terminal residues return to visibility with the strongest signal intensities, implying higher mobility at the peptide terminus. This observation is also strengthened by the TROSY mea- surement showing weak amide crosspeaks from V39 and V40 at conditions where no peaks are visible in the HSQC experiment. Under the same conditions with high detergent concentrations, the CD spectra provide evidence of significant a-helix formation. The a-helical state of Ab(1–40) in SDS micelles has been characterized previously by NMR. It was found to consist of two a-helical segments, involving residues 15–24 and 29–35, respectively, of which the C-terminal helix is inserted into the micelle [8]. It is interesting to compare this structure with two earlier proposals of full length Ab structures. (1) In complex with an affi- body protein, Ab(1–40) forms a hairpin between resi- dues 17–36, where residues 24–29 apparently form the loop connecting the two b strands [21]. (2) A model based on solid state and solution state NMR for a fibril formed by Ab(1–42) showed a parallel ⁄ in-register b-sheet arrangement between residues 18–26 and 31–42 [22]. Obviously, there are two segments of Ab [approx- imately 16–25 and 30–36 ⁄ 42 in Ab(1–40) and Ab(1–42), respectively] that are prone to form stable hydrogen bonds. We hypothesize that these segments therefore easily form secondary structures; with affibodies or in fibrils, they may form a b-sheet and, with dodecyl sulfate, they may form a-helices. The NMR diffusion measurements revealed that, up to a detergent concentration of 1 mm,Ab(1–40) is monomeric and then a state follows that cannot be characterized by diffusion NMR, but probably involves large aggregates. Continued titration with SDS, reaching a concentration of 5–10 mm, induces micelle-like formations, which appear to have a more rapid translational diffusion than the better defined state at an SDS concentration of 150 mm, when one peptide is associated with one micelle of normal SDS micellar size [8]. FTIR spectroscopy indicates the presence of some b-sheet structure in addition to random coil when Ab(1–40) is dissolved in dilute aqueous buffer. The NMR and CD measurements report mainly random coil under similar conditions. This is despite the careful procedures performed when preparing the peptide solutions as described in the Experimental procedures. A possible explanation for this discrepancy is the exis- tence of small amounts of very large aggregates, or seeds, in the sample, which remain in the sample prep- aration and are not detectable by NMR or CD. At 1.4 mm SDS (D⁄ P = 14), the IR band indicating a b-sheet is transformed into two shoulders, which might represent the seeds and the b-sheet containing com- plexes of Ab(1–40) and detergent molecules, respec- tively. These observations emphasize the problems encountered in spectroscopic studies with respect to an aggregating peptide displaying heterogeneous proper- ties. Different techniques visualize different compo- nents of the sample, even when great care has been taken to ensure that the same (or very similar) state of the sample is investigated in all experiments. The ThT and electrophoresis experiments provide further evidence of an aggregated and amyloid-like state of Ab(1–40) at SDS concentrations of approxi- mately 2 mm. Obviously, these properties are not fully reversed when the titration continues towards higher concentrations of SDS, above the critical micellar concentration. The b-sheet containing aggregates of Ab(1–40) and detergent formed at a detergent concentration of 1–2 mm (corresponding to detergent ⁄ peptide ratios of 13–27) may have different hypothetical arrangements. The sample is not homogeneous in this state, as is evident from the ThT and native-PAGE experiments (Fig. 6; see also Fig. S1). The potential occurrence of chemical exchange between aggregates characterized by different structures and sizes, with intermediate kinet- ics, contributes to making NMR characterization diffi- cult. The kinetic exchange effects may in fact be the major reason for the loss of NMR signal intensity towards high SDS concentrations in the titration experi- ments, where only one fraction of the sample is NMR visible (i.e. the fraction where detergent micelles solubi- lize individual peptides and induce partial a-helical sec- ondary structure). The major fraction of the peptide molecules remains NMR invisible, suggesting that the aggregates are only partly dissolved after the titration. The situation may be compared to that of a partly- folded molten globule structure of a protein like a-lact- albumin [23]. In that case, the collapse of a core region of partly-folded protein structure gave rise to extreme NMR line broadening due to chemical exchange, whereas completely unfolded protein structures allowed NMR observations. However, the experiments performed in the present study do not allow us to defi- nitely decide whether there are one or more reasons for the NMR line broadening during the SDS Detergent-induced Ab(1–40) secondary structures A. Wahlstro ¨ m et al. 5124 FEBS Journal 275 (2008) 5117–5128 ª 2008 The Authors Journal compilation ª 2008 FEBS titration. By contrast to the a-lactalbumin molten globule study, where the overall molecular weight is constant, there is both increased molecular weight in the aggregates and heterogeneity in the present study. Both effects may contribute to the loss of NMR signals. In one hypothetic structural scenario, the aggregates are constituted by micelle-like oligomers of Ab(1–40) peptide surrounded by detergent molecules. b-sheet structures would be induced by peptides interacting with other peptides. In this model, the complete loss of structure and higher mobility of the C-terminus of the peptide may be due to its positioning in a hydrophobic interior of the structure (analogous to the hydrophobic interior of a lipid bilayer). In another structural sce- nario, the aggregate is formed by assembled Ab(1–40) peptides, where each peptide is embedded by detergent molecules. Presumably, this model is less likely because it seems improbable that a b-structure should be induced in a peptide surrounded by detergent mole- cules. The detailed molecular properties of the aggregated complex may represent the state of the peptide when it aggregates at a crowded cell membrane surface. In turn, this situation may reflect the state of a peptide that is closely related to the oligomeric toxic species thought to be involved in the pathology of Alzheimer’s disease [3]. It is interesting to note that similar aggre- gation ⁄ solubilization effects of anionic detergents have also been described for other molecules, including chlorin p 6 , a natural porphyrin compound [24], other membrane interacting short peptides, such as dynor- phin neuropeptides [25] and antimicrobial peptides [26], or the intrinsically disordered protein a-synuclein implicated in misfolding and fibril formation in Parkinson’s disease [27]. Experimental procedures Materials Ab(1–40) used in the CD measurements was produced by Neosystem Laboratoire (Strasbourg, France). The peptides studied were unmodified at the N- and C-termini. It is known that, in physiological preparations, both non-modi- fied and C-terminally amidated forms of the peptide are found. For NMR HSQC and TROSY measurements, uniformly 15 N-labeled Ab(1–40) from Alexo-Tech AB (Umea ˚ , Sweden) was used. In the diffusion NMR study, unlabeled Ab (1–40) was obtained from rPeptide (Athens, GA, USA). The peptides were used without further purifi- cation. SDS was purchased from ICN Biomedicals Inc. (Irvine, CA, USA), LiDS was from Sigma (Stockholm, Sweden) and deuterated SDS-d 25 was obtained from Cam- bridge Isotope Laboratories (Andover, MA, USA). Preparation of the peptide CD To remove aggregation seeds in the sample, the peptide was dissolved in HFiP for 1 h, followed by freezing and lyophi- lizing. The lyophilized peptide was dissolved in 10 mm NaOH, 4 mgÆmL )1 , and sonicated in water bath for 1 min. NMR The peptide was stored at –18 °C and thawed before use. In the titration experiments, the concentration of the peptide was 75 lm, which was determined by weight. When prepar- ing the sample, a previously described protocol was used [6]. NaOH (10 mm) was added to the peptide yielding a concen- tration of 2 mgÆmL )1 and the sample was sonicated in ice bath for 1 min. Cold distilled water and D 2 O (10% of D 2 O was added for signal locking) were added to half the final sample volume and, again, the sample was sonicated for 1 min. Sodium phosphate buffer (20 mm) was added to reach the final sample volume. The peptide concentration in the assignment experiment was 300 lm and, for that reason, the peptide was dissolved directly in LiDS in distilled water to avoid aggregation. After sonication in an ice bath, D 2 O was added and, after another sonication, 20 mm sodium phos- phate buffer was added. For all experiments, the pH was adjusted to 7.2 by adding small amounts of NaH 2 PO 4 and Na 2 HPO 4 using the pH meter Orion PerpHecT LogR meter (San Diego, CA, USA). All sample preparations were performed on ice. For diffusion NMR measurements, the peptide was dissolved as described, but at pH 7.4. FTIR The sample was prepared in the same way as for the CD experiments but using deuterated reagents. Preparation of detergent solution The 200 mm SDS solution was prepared in 10 mm sodium phosphate buffer (pH 7.3) or 10 mm Tris–HCl buffer (pH 7.3). LiDS was dissolved in 20 mm of sodium phos- phate buffer and two stock solutions (50 and 500 mm) were made to minimize the dilution effects. SDS-d 25 was dis- solved in D 2 O and two stock solutions were used (10 mm and 100 mm). CD spectroscopy CD spectra were recorded at 3 °C and 20 °C in LiDS and SDS, respectively, and for different titration steps in deter- A. Wahlstro ¨ m et al. Detergent-induced Ab(1–40) secondary structures FEBS Journal 275 (2008) 5117–5128 ª 2008 The Authors Journal compilation ª 2008 FEBS 5125 gent at concentrations in the range 0.05–20 mm. The spec- tral region was recorded from 190–250 nm, with a 0.2 nm step resolution, on a Jasco J-720 CD spectropolarimeter (Jasco Inc., Easton, MD, USA) equipped with a PTC-343 temperature controller using quartz cells of 1.0 mm optical path length. At 20 °C, the scanning speed was 100 nmÆ min )1 and the spectra were collected and averaged over 20 scans. At 3 °C, a scanning speed of 50 nmÆmin )1 was used and ten scans were employed. The background signals were subtracted from the CD spectra of the peptides. The peptide concentration was 75 lm in all experiments. The same peptide sample was used in one titration series. NMR spectroscopy The NMR measurements were used to follow the structural changes in the Ab(1–40) peptide caused by LiDS titration. Experiments were performed on a Varian Inova 600 MHz spectrometer at 3 °C (Varian NMR, Inc., Palo Alto, CA, USA). 1 H- 15 N HSQC experiments were acquired in 1 H dimension in a 6 kHz window centered at 4.98 p.p.m. using a 0.12 s acquisition time and eight scans. In the 15 N dimen- sion, 256 increments were acquired in a 2.5 kHz window centered at 118.5 p.p.m. These parameters were used also in the assignment experiment and, after every temperature change of 5 ° C, the sample equilibrated for at least 20 min before the next run. The TROSY experiment was per- formed to study the state characterized by NMR signal loss in the HSQC spectra. The same parameters as for HSQC were used and 96 scans were averaged. Solvent suppression was performed with excitation sculpting. NMR data pro- cessing and integration of peak volume were performed in Varian vnmr software, whereas the spectra were presented using sparky [28]. The diffusion experiments were per- formed with the pulsed field gradient spin-echo experiment (pulsed field gradient longitudinal eddy–current delay) with the 600 MHz Varian Inova spectrometer, which is equipped with a z-axis gradient coil. Thirty different linearly spaced gradient strengths were used with a delay between the gradient pulses of 150 ms and a gradient length of 2 ms. Calibration of the pulsed field gradients was performed by means of a standard sample, 1% H 2 OinD 2 O and 1mgÆmL )1 GdCl 3 , and the knowledge that the diffusion coefficient of HDO in D 2 Oat25°C is 1.90 · 10 )9 m 2 Æs )1 [29]. FTIR spectroscopy FTIR spectra were collected in a Bruker Tensor 37 spec- trometer (Bruker, Ettlingen, Germany) at 20 °C with a 4cm )1 spectral resolution. Three series of 100 scans each were acquired and averaged, and the second derivative was performed with nine smoothing points using opus software (Bruker). For clarity of the results, the negative second derivative of the spectra normalized for the trifluoroacetic acid band (approximately 1675 cm )1 ) is shown. To improve signal-to-noise ratios, the peptide concentration was 100 lm. ThT and native–PAGE experiments For ThT measurements, the final ThT concentration was kept at 15 lm for all samples (10 mm sodium phosphate buffer, pH 7.3, 20 °C). SDS titrations were performed both in the absence and presence of peptide (75 lm). Samples were excited at 450 nm (1 nm slit width) and single wavelength emission measurements at 483 nm (1 nm slit width) were performed in a Jobin-Yvon Flu- oroMax spectrofluorometer (HORIBA Jobin-Yvon Inc., Edison, NJ, USA). Titrations were carried out in the same way as for the CD experiments. During the SDS titration in the presence of peptide, aliquots were sampled at 0, 1.1, 2.2, 4.6 and 25.5 mm of SDS. These aliquots were analyzed in a 10–20% Tris–HCl native-PAGE (gel ran for 3 h) and subsequent silver staining was per- formed using a silver staining kit (Bio-Rad, Hercules, CA, USA). Acknowledgements We thank Andreas Barth for generous access to the FTIR spectrometer, and L. E. Go ¨ ran Eriksson for helpful discussions. We thank Torbjo ¨ rn Astlind for technical help with the NMR experiments. This study was supported by the Swedish Research Council and by the Catalan Government postdoctoral fellowship ‘Beatriu de Pino ´ s’ (2005 BP-A 10085 to A.P M.). Further support was obtained from the European Commission (contract LSHG-CT-2004-512052), the Carl Trygger Foundation, the Marianne and Marcus Wallenberg Foundation and the Swedish Foundation for Strategic Research (Bio-X). 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Secondary structure conversions of Alzheimer’s Ab(1–40) peptide induced by membrane-mimicking detergents Anna Wahlstro ¨ m 1, *, Loı ¨ c Hugonin 1, *,. structural transitions of the Ab(1–40) peptide (75 lm) induced by increasing concentrations of the membrane-mimicking detergent LiDS. The 1 H- 15 N HSQC spectrum of uniformly 15 N-labeled Ab(1–40) in 10. b-sheet structures would be induced by peptides interacting with other peptides. In this model, the complete loss of structure and higher mobility of the C-terminus of the peptide may be due to its