Tài liệu Báo cáo khoa học: The Alzheimer b-peptide shows temperature-dependent transitions between left-handed 31-helix, b-strand and random coil secondary structures doc

The Alzheimer b-peptide shows temperature-dependent transitions between left-handed 31-helix, b-strand and random coil secondary structures Jens Danielsson, Juri Jarvet, Peter Damberg and Astrid Graslund ă ă Department of Biochemistry and Biophysics, Stockholm University, Sweden Keywords amyloid b-peptide; b-strand; left-handed 31-helix; random coil; transition enthalpy Correspondence A Graslund, Department of Biochemistry ¨ and Biophysics, Stockholm University, S-106 91 Stockholm, Sweden E-mail: astrid@dbb.su.se (Received 13 April 2005, revised 26 May 2005, accepted June 2005) doi:10.1111/j.1742-4658.2005.04812.x The temperature-induced structural transitions of the full length Alzheimer amyloid b-peptide [Ab(1–40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy The full length peptide undergoes an overall transition from a state with a prominent population of lefthanded 31 (polyproline II; PII)-helix at °C to a random coil state at 60 °C, with an average DH of 6.8 ± 1.4 kJỈmol)1 per residue, obtained by fitting a Zimm–Bragg model to the CD data The transition is noncooperative for the shortest N-terminal fragment Ab(1–9) and weakly cooperative for Ab(1–40) and the longer fragments By analysing the temperaturedependent 3JHNHa couplings and hydrodynamic radii obtained by NMR for Ab(1–9) and Ab(12–28), we found that the structure transition includes more than two states The N-terminal hydrophilic Ab(1–9) populates PIIlike conformations at °C, then when the temperature increases, conformations with dihedral angles moving towards b-strand at 20 °C, and approaches random coil at 60 °C The residues in the central hydrophobic (18–28) segment show varying behaviour, but there is a significant contribution of b-strand-like conformations at all temperatures below 20 °C The C-terminal (29–40) segment was not studied by NMR, but from CD difference spectra we concluded that it is mainly in a random coil conformation at all studied temperatures These results on structural preferences and transitions of the segments in the monomeric form of Ab may be related to the processes leading to the aggregation and formation of fibrils in the Alzheimer plaques The amyloid b-peptide (Ab) is the major component of the amyloid plaques found in the extracellular compartment in the brains of patients suffering from Alzheimer’s disease The Ab-peptide is a 39–42-residue peptide with the sequence: DAEFRHDSGYEVHHQ KLVFFAEDVGSNKGAIIGLMVGGVVIA(1–42) It is cleaved from the Alzheimer’s precursor protein by the proteases b- and c-secretase [1,2] The Ab(1–40) peptide has a hydrophilic N-terminal region and a more hydrophobic C-terminal region The peptide contains a central hydrophobic cluster, residues 17–21, which is suggested to play an important role in peptide aggregation [3] There is experimental evidence that soluble oligomeric aggregates have toxic effects on neurons and synapses [1,4] The aggregation involves a conformational change of the peptide structure to b-sheet Solid state NMR spectroscopy has shown that fibrils of Ab contain parallel b-sheet structure, whereas shorter fragment fibrils consist of antiparallel b-sheet structure [5,6] In vitro, the Ab monomer is in a dominating random coil secondary structure in solution at room temperature and physiological pH [7–9] Abbreviations Ab-peptide, amyloid b-peptide; PII, polyproline II 3938 FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS J Danielsson et al An NMR study at °C of the Ab(1–40) and Ab(1– 42) peptide with oxidized Met35 showed deviations from random coil behaviour, but only limited information about the solution structure could be derived [10] The details of the high resolution structure or structure propensity of Ab are still not well known In order to understand the early aggregation process and oligomerization of the peptide, further knowledge about the structural energy landscape is needed, and this motivated the present study Earlier studies show that for the fragment Ab(12– 28) the secondary structure of the monomer changes gradually towards a left-handed 31 (polyproline II; PII)-helix when lowering the temperature [11] Also the fragment Ab(1–28) has been shown to adopt a PIIhelical structure in acidic solution [12] For many short peptides, however, not for all, the PII-helix seems to dominate at low temperatures [13–15] The PII-helix is an extended structure with a rotational symmetry of three amino acids per 360-degree turn The torsion angles are (/,w) ¼ ()78°, 146°) The PII-helix differs from PI in the x-torsion angle, where PII is all trans peptide bonds There are no interresidual hydrogen bonds stabilizing the PII structure The stabilizing factor is proposed to be interactions with the solvent PII can exist only in water and the structure is more stable in D2O than in H2O, suggesting that water–peptide hydrogen bonds are involved in the stabilization [14,16–18] Different residues have different propensities to adopt the PII-helix conformation FTIR, Raman and different CD experiments have led to estimations of the propensity of the amino acids for the PII-helix [14] Using these results to predict the secondary structure of the Ab-peptide shows that the PII content of the full length peptide should be % 40% at low temperature and predominantly in the N-terminal half of the peptide [14] The PII-helix is generally more stable at low temperatures The fraction of PII-helix increases as the temperature decreases, an observation valid both for true polyproline helices and other left-handed 31-helices [11,13] Raising the temperature induces a structural transition This transition has been suggested to be noncooperative for short peptides [19] For longer peptides molecular dynamics simulations of polyalanine suggest a cooperative transition [18]; however, the theoretical results are dependent of the force field used [20] We have earlier observed that the Ab-peptide is more soluble at low temperatures and is stable when kept at low temperature [11,21] suggesting that PIIhelix prevents aggregation of the Ab-peptide The general properties of a PII-helix have been determined by various spectroscopic methods such as FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS Structural transitions of Alzheimer b-peptide CD, NMR, FTIR and Raman optical activity [13] In CD spectroscopy a characteristic positive band appears in the 210–230 nm region [13] This positive band corresponds to an n–p* transition and is at 229 nm in pure polyproline It is shifted towards shorter wavelengths when other residues are involved [22] The CD spectra of PII-helices were earlier often interpreted as random coil spectra However, no positive bands or local maxima should appear in a true random coil CD spectrum [22] The dihedral angles / and w that define the PII structure are also reflected in the J couplings between spins in the residues 3JHNHa couplings can be studied by NMR spectroscopy The 3JHNHa coupling between the amide proton and the a-carbon proton is dependent on the torsion angle / The hydrodynamic properties of peptides reflect structural properties The hydrodynamic radius is related to the diffusion coefficient via Stoke–Einstein’s equation, and is dependent on not only the size but also on the structure of the diffusing Ab-peptide and scales with the molecular mass as the power law RH ¼ n1Mn2 [8] The hydrodynamic radius is also related to the persistence length, which depends on the structural state of the peptide [23,24] In the present investigation, we have explored the structure propensities of Ab(1–40) and selected fragment peptides Using varying temperatures, the energy landscape close to the solution structure is explored and information on the structural transitions of the peptide is obtained The temperature-induced structural transitions also yield information on the background for a potential mechanism for the transition from soluble monomer to aggregated multimer We have used CD as well as NMR at physiological pH, in a temperature range from °C to 60 °C, to study the solution structure of the peptide as well as the PII to coil structural transition The results show that the full length peptide monomer partially adopts a PII-helix structure at low temperatures, particularly in an N-terminal region However, the central hydrophobic cluster, residues 17–21 and particularly the phenylalanine residues 19 and 20, have a tendency towards b-strand formation also at low temperature Results Circular dichroism (CD) spectroscopy Temperature dependence We recorded CD spectra at varying temperatures for the full length Ab(1–40)-peptide, as well as the fragments Ab(1–9), Ab(1–16), Ab (1–28), Ab(12–28), Ab(16–21), Ab(25–35) and the variant fragment 3939 Structural transitions of Alzheimer b-peptide J Danielsson et al [θ] / 10-3 deg cm2 dmol-1 Ab(12–28)G19G20 For each peptide a CD spectrum was obtained at a series of temperatures, ranging from to 60 °C Figure shows the temperature-dependent CD spectra of all the studied peptides Figure 1H shows Ab(25–35) at °C Among the fragments studied here, all N-terminal peptides and the full length peptide are in monomeric form at 10 lm concentrations, as shown earlier with diffusion measurements [8] To ensure that the temperature 0 A Aβ(1-40) -10 Aβ(1-28) -10 Ab(25–35) 200 [θ] / 10-3 deg cm2 dmol-1 B 220 240 velength / nm 200 220 240 Wavelength / nm C D Aβ(1-16) -10 Aβ(12-28) -10 -20 [θ] / 10-3 deg cm2 dmol-1 200 220 240 200 220 240 The hydrophobic fragment Ab(25–35) is different from the other peptides in its CD characteristics It is in an aggregated b-sheet form under these conditions at all temperatures between °C and 60 °C, as the CD spectra show little temperature variation (data not shown) The shape of the spectrum recorded at °C in Fig 1H suggests antiparallel b-sheet secondary structure Molecular mass studies confirmed the aggregated state of the peptide as described below This peptide was not studied further E F Aβ(1-9) Aβ(16-21) -10 -10 -20 200 [θ] / 10-3 deg cm2 dmol-1 dependence of the CD spectra reveals structural transitions a control experiment was performed with 10 lm Sml1(50–104) [25], a 54-residue unstructured peptide in 10 mm phosphate buffer at pH 7.2 This peptide shows no structure transition The CD spectrum shows only small changes, as shown in Fig S1, and these not follow the same pattern as the Alzheimer fragments From the CD experiments we conclude that the N-terminal Alzheimer peptide fragments, as well as the (1–40) peptide, are in equilibrium between a structural state with a large contribution of PII-helix secondary structure (most prominent at °C) and random coil structural state (most prominent at 60 °C) The two state equilibrium is consistent with a relatively welldefined isodichroic region around 208 nm 220 240 40 200 220 240 H G 20 Aβ(25-35) Aβ(12-28)G19G19 -10 PII content estimation Quantification of the PII content of the CD spectra can be carried out in several ways [26,27] Here, the population of PII-helix was calculated from the CD amplitude of the local maximum around 220 nm, [h]max Because the wavelength of this maximum is dependent on the sequence [13] we determined an individual [h]max for each series of recorded spectra From the spectral intensities at [h]max the PII population, xPII was estimated using the relation published by Kelly et al [27]: xPII ¼ ẵhmax ỵ 6100 13700 -20 200 220 240 200 220 240 Fig Far UV CD spectra of the fragments in 10 mM sodium phosphate buffer at pH 7.4 The concentration was 10 lM for all fragments Spectra were recorded at 0, 10, 20, 30, 40, 50 and 60 °C Generally the population of PII-helix decreased as the temperature increased (A) The full length peptide Ab(1–40); (B) Ab(1–28); (C) Ab(1–16); (D) Ab(12–28); (E) Ab(1–9); (F) the central hydrophobic cluster KLVFFA, Ab(16–21); (G) the variant fragment Ab(12– 28)G19G20 In (H) the C-terminal fragment Ab(25–35) fragment at °C is shown, indicating antiparallel b-sheet secondary structure 3940 In the CD spectra of Ab(1–40) in Fig 1A the local maximum at 221 nm, best seen at low temperature, is characteristic of a PII-helix The population is strongly temperature dependent and as the temperature reaches 60 °C only a small fraction of PII-helix remains The PII content was estimated to 43% at °C and reduced to 15% at 60 °C At 37 °C the PII content was 20% The PII population at °C is higher for the shorter fragments compared to Ab(1–40) and ranges from FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS J Danielsson et al Structural transitions of Alzheimer b-peptide 46% for the Ab(12–28) to almost 60% for the Ab(1–9) fragment The calculated populations are in good agreement with the predicted populations based on structural propensities of different residues [14] The structure transition is fully reversible for all peptides (Fig 1A–G) This was shown by lowering the temperature to °C after the temperature increase and then recording a new spectrum at °C This new spectrum was identical to the original low temperature spectrum (data not shown), indicating that no irreversible processes are present We could estimate the location of the PII-helix on the Ab(1–40) peptide by comparing the results on the different fragments The Ab(1–9) peptide shows high propensity to form PII-helix, Fig 1E At °C the population is 58%, and at high temperature it reduces to 42% The central hydrophobic stretch, Ab(16–21) shows a spectrum that has some distinct differences compared to the other fragments, Fig 1F The evaluated PII populations of the fragments except Ab(16– 21) are shown in Fig ωPII / PII population 0.8 0.6 0.4 0.2 200 300 400 Temperature / K Fig Population of PII-helix as a function of temperature The fitted curves are calculated assuming the Zimm–Bragg model using a linearized approximation for s close to the transition temperature The fragments measured are Ab(1–40) (h), Ab(1–28 (.), Ab(1–16) (r), Ab(12–28) (s), Ab(12–28)G19G20 (d) and Ab(1–9) (e) The shortest fragment shows no cooperativity in the transition, in contrast to the full length peptide FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS As already mentioned, the Ab(23–35) fragment aggregated under the conditions used here, and could therefore not represent the monomeric form of the C-terminal segment of Ab However, considering the differences observed between Ab(1–40) and Ab(1–28) one can gain some information about the (29–40) segment of Ab Supplementary Fig S2 shows the difference spectra at °C, 20 °C and 60 °C The difference spectra are dominated by a contribution from random coil structure At °C there is a small contribution (< 10%) from PII-helix Cooperativity for longer and noncooperativity for shorter peptides The amount of PII-helix, hPII, in the peptides is strongly temperature dependent, and the temperature dependence varies within the peptide group From the fitting of hPII according to the linearized model in Eqns (5) and (6) to the data of Fig 2, a transition temperature Tm, an enthaply change DH and a cooperativity r was obtained for each peptide, Table The parameter Tm is the temperature when 50% of the secondary structure of the peptide is populated as PII-helix The full length peptide and the longer fragments exhibit a certain degree of cooperativity in the transition The shorter peptides show a lower degree or no cooperativity The shortest fragment Ab(1–9) shows no cooperativity at all (r ¼ 1.00) in agreement with earlier observations that short segments not exhibit cooperativity in the transition from PII to random coil [19] The shorter N-terminal fragments have a higher Tm, i.e are in a more stable PII conformation than the full length peptide The central hydrophobic stretch Ab(16–21) shows a somewhat different pattern and the temperature dependent population curve cannot be fitted to Eqn (5) These results suggest that this peptide deviates in its behaviour from the others It should be pointed out that these parameters were obtained using a full Zimm–Bragg model The results are not very sensitive to absolute concentrations of the peptides On the other hand, using a simplified Zimm–Bragg model (valid only for very long polymer chains) gives very different values of the cooperativity coefficient r, and is not applicable here CD spectroscopy is a low resolution structural method The CD results could be interpreted in terms of a two state equilibrium between a more structured state with large contributions from a PII-helix and a random coil state However, the small deviations from isodichroic point behaviour, and the anomalous 3941 Structural transitions of Alzheimer b-peptide J Danielsson et al Table Parameters calculated from the temperature dependence of the PII population Cooperativity factor, given by r, where r ¼ is no cooperativity and r 90%, indicating severe aggregation of the peptide Repeating this procedure but at low pH where the peptide is less prone to aggregate shows significantly less loss of peptide, < 30% Structural transition theory The temperature induced transition between PII-helix and random coil can be treated as an ordinary helix–coil transition described by Zimm and Bragg Here we use a slightly modified Zimm–Bragg model [36] Using their matrix method, the partition function, Q, of a peptide with N residues is given by: Q ¼ bMNÀ1 a0 Viscosity correction in diffusion experiments The diffusion coefficient is temperature dependent, primarily because the thermal Brownian motion is directly proportional to the temperature but also because the viscosity of the solvent, here water, is highly temperature dependent, which should be corrected for To account for the viscosity effect two different approaches were taken First, reference molecules with a known hydrodynamic radius were studied Here HDO and a-cyclodextrin were used as references The unknown hydrodynamic radius of the peptide was calculated using: RH (T) ¼ Dref (T) DOBS ð298K) Á RH;ref Dref ð298KÞ DOBS (T) Dref(298K) is the diffusion coefficient of the reference molecule at a reference temperature T ¼ 298K, DOBS(298K) is the measured peptides diffusion at that temperature RH,ref is the reference molecule’s hydrodynamic radius at the reference temperature Dref(T) and DOBS(T) are the diffusion coefficients at temperature T for the reference molecule and the studied peptide, respectively In a second approach, empirical functions for the viscosity of H2O and D2O were obtained from fitting tabulated values to the empirical function [41]: m g ẳ KejT273ị This approach gave the parameters (L, j, t) ¼ (2.41 · 10)3, 0.057, 0.8078) for D2O and (L, j, t) ¼ (1.80 · 10)3, 0.049, 0.8222) for H2O Different mixtures of D2O and H2O 3946 ð2Þ Here, a0 is the vector describing only one residue, which is assumed to be unstructured, b is a row vector consisting only of ones M is the matrix operator that adds one residue to the vector of possible conformations with their statistical mass For a system where M is a · matrix, as it is here, Eqn (2) becomes:  NÀ1   1 rs 3ị Q ẳ ẵ1 1 s s is the intrinsic statistical weight for a residue to be in PII conformation r is a cooperativity coefficient The factor rs is then the statistical weight for a residue PII conformation following after an unstructured residue If r is there is no cooperativity in the transition, and if r ( the cooperativity is high Calculating the partition function from Eqn (3) gives: kN k0 sị ỵ kN ðs À k1 Þ k0 À k1  q 1 ỵ s ặ ỵ sị2 ỵ 4rs ẳ Qẳ k0;1 4ị Here the eigenvalues k0,1 of M are introduced At equilibrium the fraction of residues in PII conformation is given by the power of s in the partition function divided by the total number of residues The fraction of PII is then xPII ¼ (N-1))1 d(lnQ) ⁄ d(lns) and can be calculated from Eqn (4) Performing this calculation we obtain: FEBS Journal 272 (2005) 39383949 ê 2005 FEBS J Danielsson et al xPII ẳ Structural transitions of Alzheimer b-peptide      s Nc0 ỵ c001 kN k0 sị ỵ Nc1 þ c11À1 kN ðs À k0 Þ k Às k Às k0 k1 À 1ÞðkN ðk0 ðN sðc0 À c1 Þ À ðN À 1Þðk0 À k1 ị References sị ỵ kN s k1 ịị ð5Þ c0,1 are the derivatives of k0,1 with respect to s: > > 1< ỵ s þ 2r = À qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi c0;1 ¼ > 2> : ỵ sị2 ỵ 4rs; 6ị The parameter s is, as mentioned above, related to the equilibrium constant and thus to the enthalpy change due to the structural transition The temperature dependence of the transition can be studied, and if the populations of the structural entities can be determined, the parameters s and r can be determined from Eqn (5) Close to the transition temperature Tm the parameter s may be approximated as a linear function of T [36] sẳ1 DH T Tm ị RTm At s ¼ the two states are equally populated Hydrodynamic radius from simulations Simulations were performed to obtain hydrodynamic data As a model for the peptides a peptide chain with no side chains was used Three bonds with fixed bond lengths represented every residue The bond lengths used were 1.33, 1.45 and ˚ 1.52 A and the fixed angles between the bonds were: 58°, 64° and 75° The rotations around the bonds were controlled: for a random coil peptide the rotation was free and for defined structures the rotation was constrained The simulation was ˚ performed using a hard sphere model with a 1.3 A van der Waals radius of the heavy atoms Fifteen hundred structures of each length were generated with different distributions of torsion angles The random coil had free rotation in all three torsion angles The PII-helix was simulated using equally distributed angles centred at (/,w,x) ¼ ()78°, 146°, 180°) and with a arbitrary width of 10° for all angles A b-strand was simulated in the same way with the torsion angles centred at (/,w,x) ¼ ()139°, 146°, 180°) and with the same flexibility The hydrodynamic radius was calculated as the radius of gyration, the mean distance from the centre of mass Acknowledgements This study was supported by a grant from the 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arctic mutant carriers J Mol Biol 339, 145–159 Zhang S, Iwata K, Lachenmann MJ, Peng JW, Li S, Stimson ER, Lu Y, Felix AM, Maggio JE, Lee JP (2000) The Alzheimer’s peptide aa adopts a colˆ FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS J Danielsson et al lapsed coil structure in water J Struct Biol 130, 130–141 40 Damberg P, Jarvet J & Graslund A (2001) Accurate ă measurement of translational diffusion coefficients: a practical method to account for nonlinear gradients J Magn Reson 148, 343–348 41 Cho C, Urquidi J, Singh S & Robinson G (1999) Thermal offset viscosities of liquid H2O, D2O and T2O J Phys Chem 103, 1991–1994 FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS Structural transitions of Alzheimer b-peptide Supplementary material The following supplementary material is available online: Fig S1 Far UV CD spectra of 10 lM Sml1 in 10 mM sodium phosphate buffer at pH 7.4 Fig S2 Far UV CD difference spectra between 10 lM Ab(1–40) and Ab(1–28) in sodium phosphate buffer at pH 7.4 3949 ... between the bonds were: 58°, 64° and 75° The rotations around the bonds were controlled: for a random coil peptide the rotation was free and for defined structures the rotation was constrained The. .. conformation already at °C, and go directly from there to random coil This high b-strand propensity is in good agreement with the predicted structures [14] The temperature dependence of the hydrodynamic... fi b-strand fi coil similar to residues 2–8, whereas Ala21 goes directly from PII-rich state to random coil Val18, Phe19, Phe20 and Val24 on the other hand seem to start in a b-strand- rich conformation