Curcumin Alters the Salt Bridge-containing Turn Region in Amyloid β(1–42) Aggregates

Background: Curcumin reduces the risk of Alzheimer disease via an unknown mechanism.

Results: Curcumin-incubated Aβ₄₂ aggregates retain the hairpin architecture but have disruptions in the turn region (surprising similarity with Zn²⁺ incubation).

Conclusion: Salt bridge-containing turn region is a major determinant of morphology and toxicity.

Significance: Identification of crucial structural changes provides a checkpoint for developing effective AD therapeutics.

Abstract

Amyloid β (Aβ) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aβ fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid β(1–42) (Aβ₄₂) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23–Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aβ₄₂ fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn²⁺ ions, another agent known to disrupt the fibrils and alter Aβ₄₂ toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aβ₄₂, which is stabilized both by curcumin and by Zn^2+.

Introduction

Curcumin, a phenolic compound from the Asian spice turmeric (Curcuma longa), is of great interest for its plausible role in countering neurodegenerative diseases like Parkinson disease (1–3) and Alzheimer disease (AD)³ (4, 5). In vitro studies have shown that curcumin can retard the process of amyloid β (Aβ) aggregation (5, 6), which is supposed to be the initiator of AD. It disrupts the formation of the long straight Aβ fibrils (5, 7) and reduces Aβ toxicity (8, 9). Moreover, it can also disrupt the preformed Aβ fibrils (6, 7). The reduction of toxicity is possibly related to these effects. Alternative modes of action have also been suggested; it has been shown to inhibit the process that produces Aβ from the amyloid precursor protein (10). In addition, curcumin in general can reduce the concentration of the reactive oxygen species (11), which plays a role in neurodegeneration. Whatever is its main mode of action, information about the conformational changes of Aβ induced by curcumin would be valuable in understanding its role in AD.

There are several modified curcumin analogues that have been developed that show even more potent anti-AD activity in animal models (12), and pharmaceutical development will benefit from this understanding. Curcumin is known to bind to β-sheet-rich Aβ species like protofibrils and fibrils and not to unstructured monomers (7). Theoretical studies have suggested that curcumin can interact with Aβ oligomers to disrupt the β-sheet content and possibly alter the conformation of several regions of the Aβ molecule (13). Experimental studies using curcumin analogues with different linker lengths have shown that there is an optimum linker length that interacts with Aβ, suggesting that the interaction of Aβ with curcumin is site-specific (14, 15). However, it is still not known whether curcumin induces specific structural changes in Aβ aggregates.

Like curcumin, Zn²⁺ ions are also known to alter the toxicity of Aβ and disrupt the Aβ fibrils (16–18). It is known that Zn²⁺ ions preferentially precipitate the amyloid oligomers and can stoichiometrically bind to Aβ (19–21). Zn²⁺ is thought to bind to the N-terminal region, and this is most likely mediated by His-6, His-13, and His-14 (22–25). Using ssNMR studies, we have previously shown that Zn²⁺ binding disrupts the salt bridge between Asp-23 and Lys-28 (26). Although Zn²⁺ and curcumin have a completely different chemical nature, there are interesting parallels between the effects induced by them. It is therefore worthwhile to compare their structural effects. If some of the structural changes are common, then those changes may be key to the disruption of the fibrils and possibly also to the modulation of toxicity.

In this study we examine the structural changes induced by curcumin on Aβ(1–42) (Aβ₄₂) aggregates with ssNMR, probing two different samples that have two different sets of isotopically labeled amino acids (a total of 15 amino acids are labeled). We have previously observed the effects of Zn²⁺ on the same sets of amino acids (26), which gives us an opportunity for a direct atom-by-atom comparison of the effects of these two agents. We also verify the effects of curcumin on the fibrillar morphology of the aggregates using transmission electron microscopy. Significant unexpected similarities emerge between the effects of Zn²⁺ and curcumin, pointing toward a class of structural changes that may be the key to understanding how Aβ fibrillar architecture gets disrupted by small molecules, possibly altering its toxicity.

EXPERIMENTAL PROCEDURES

Aβ₄₂ Synthesis, Purification, and Sample Preparation

Synthesis and purification procedures of two different Aβ₄₂ peptide specimens with different ¹³C and ¹⁵N isotopic labeling schemes (P¹ and P²) are described elsewhere (26). Purified Aβ₄₂ peptides were initially dissolved in pH 11.0 water (adjusted by NaOH) to prepare 2 mm stock solutions. A 4 mm stock solution of curcumin (Sigma) was also prepared by dissolving in pH 11.0 water. To grow Aβ₄₂ aggregates in the presence of curcumin, 1.5 ml of Aβ₄₂ stock (2 mm) and 150 μl of freshly prepared curcumin stock (4 mm) were mixed in pH 11.0 water and immediately diluted with 5.85 ml of HEPES buffer (containing 20 mm HEPES, 146 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl₂·2H₂O, and 0.8 mm MgSO₄·7H₂O) at pH 7.4 such that the total volume was 7.5 ml. This final solution containing 400 μm Aβ₄₂ and 80 μm curcumin was incubated at room temperature (≈24 °C) for 4 days with mild rotation (10 rpm). This resulted in the formation of aggregates. The rest of the materials used in the experiments was same as described in Ref. 26.

Solid-state NMR

For ssNMR measurements, solutions containing peptide aggregates were subjected to centrifugation (16,000 × g) for 1 h. The pellets thus collected were washed with de-ionized water twice by resuspending them in water and ultracentrifuging for 1 h each time. The final pellets so obtained were rapidly frozen using liquid nitrogen and then lyophilized. The powdered samples were rehydrated (33 weight %) by adding ≈0.5 μl of de-ionized water per mg of dry peptide aggregates. The hydrated sample was then packed in a 2.5-mm magic angle spinning rotor such that it contains ∼10 mg of sample.

All the ssNMR measurements were performed using a 700 MHz Bruker AVIII NMR spectrometer at a magic angle spinning frequency of 15 kHz using a 2.5-mm triple resonance magic angle spinning probe. Cross-polarization to ¹³C and ¹⁵N from ¹H was implemented using a linear ramped radio frequency field (27) centered around 65 kHz on the ¹H channel and with a 55-kHz field on the ¹³C/¹⁵N channel with a contact time between 2.5 and 4.0 ms. ¹H dipolar decoupling was accomplished using the swept frequency two-pulse phase modulation (φ = 10°) decoupling scheme (28) with a field strength of 85 kHz. Two-dimensional ¹³C-¹³C through-space NMR spectra were recorded using second-order dipolar recoupling schemes of PARIS-xy (m = 1, n = 2) (29) and PDSD (30) and mixing periods of 100 and 1000 ms, respectively. ¹H irradiation of 15 kHz was used while applying PARIS-xy (m = 1, n = 2). A total of 200 points was acquired in the indirect dimension with a dwell time of 12.5 μs amounting to an acquisition time of 2.5 ms. The number of scans per free induction decay and inter-scan delay was fixed to 256 and 2 s, and 512 and 1.5 s in case of PARIS-xy (m = 1, n = 2) and PDSD, respectively. Frequency-selective ¹³C-¹⁵N rotational-echo double-resonance (REDOR) experiments were recorded using the pulse sequence designed by Jaroniec et al. (31). Rotor-synchronized trains of phase-alternated π pulses following xy-8 scheme (32) were applied on the ¹⁵N channel. 466.7 μs long frequency-selective Gaussian pulses were applied at the ¹³Cγ and ¹⁵N_ζ frequencies of Asp-23 and Lys-28, respectively, in the middle of the REDOR-dephasing period (τ_mix). Reference spectra (S₀) were recorded after removing the frequency-selective refocusing pulse on ¹⁵N channel.

The results shown in this study for amyloid aggregates of only Aβ₄₂ and those grown in the presence of 400 μm Zn²⁺ ions (Aβ₄₂-Zn) are based on higher quality ssNMR spectra compared with those reported in our previous article (26). This is a result of better purification and degree of hydration (50 weight %), but they do not change any of the conclusions of the previous study.

NMR Data Analysis

All one-dimensional spectra were processed and analyzed using TopSpin 3.1. All two-dimensional spectra were processed with TopSpin 3.1 and analyzed using CcpNmr Analysis 2.2.2. The data were zero-filled in the t₁ and t₂ dimensions to 512 and 4096 points, respectively. A mixed sine/cosine (ϕ = π/3 at t = 0) apodization function was used in each dimension. All the spectra were externally referenced to tetramethylsilane in methanol (33). TALOS+ (34) was used to obtain predicted ψ and φ backbone torsion angles, based on ¹³C chemical shifts of α, β, and carbonyl carbons.

Calculating Average Chemical Shift Change (Δ̄δ)

¹³C chemical shifts of isotopically labeled amino acids in Aβ₄₂ fibrils (26) were subtracted from that of Aβ₄₂-Zn (26) and Aβ₄₂-Cur aggregates. These chemical shift differences for α, β, and carbonyl carbons (the backbone) and the remaining carbons (the side chain) were then averaged over the number of carbons constituting the backbone and side chain, respectively, to obtain an average chemical shift change (Δδ).

Electron Microscopy

10-μl solutions of Aβ₄₂ (400 μm) and Aβ₄₂-Cur (400 μm Aβ₄₂ + 80 μm curcumin), aggregated for 4 days, were placed on carbon-coated 100-mesh copper grids for 2 min, followed by blotting by a filter paper. The extra salt on the grids was removed by four cycles of mild washing with double-distilled water. Then a drop of 0.1% uranyl acetate was added to each grid and left for 5 min for staining. After removing the extra uranyl acetate solution by a filter paper, the grids were dried under an infrared lamp. The samples were examined with a transmission electron microscope (LIBRA 120, EFTEM, Carl Zeiss, Germany). The width analysis of fibrils was performed with ImageJ (open source software, rsbweb.nih.gov).

Cell Culture

Primary cortical neurons were cultured from pregnant female Wistar rats obtained from the Institute Animal Facility. All animal handling procedures were approved by the animal ethics committee of the Institute. Neuronal cultures were obtained from the cortex of 17-day-old embryos. Cells were grown in Neurobasal media supplemented with 2% B-27 supplement, 0.5% penicillin/streptomycin, and 0.25% l-glutamine. Cell culture media and chemicals were obtained from Invitrogen.

Cell Viability Assay

Primary cortical neurons grown in 96-well plates were treated with Aβ₄₂ (40 or 400 μm) on day 5. After 48 h, the cells were assessed for viability. The cells were treated with 0.01 mg/ml propidium iodide (Molecular Probes) for 10 min, washed with phosphate-buffered saline, and imaged with an epifluorescence microscope (Zeiss Axiovert 200, Germany) using a 40× objective. Propidium iodide binds to DNA but is cell-impermeable and hence can penetrate cells with damaged membrane. This gives the dead cell count. The number of live cells (propidium iodide-positive cells were subtracted from total cells counted from transmission images) expressed as a percentage of total cell count gives the percentage cell viability. Cell viability of a control set of cells treated with vehicle is normalized to 100%, and all the results are expressed with respect to that. To investigate the effectiveness of curcumin in reducing the Aβ-induced toxicity, curcumin was added at 1:5 molar ratio (i.e. 8 μm curcumin to 40 μm Aβ₄₂ and 80 μm curcumin to 400 μm Aβ₄₂). Each experiment was performed on six different wells.

RESULTS AND DISCUSSION

We tested the effects of curcumin on Aβ₄₂-induced toxicity on rat primary cortical neuronal cultures. Fig. 1, A–D, shows the superimposed transmission and propidium iodide-stained (marks dead cells, green) images of neurons for different treatments. The results of this study are summarized in Fig. 1E. The cells exposed to 40 μm Aβ₄₂ for 48 h showed a reduced viability of 62 ± 4% compared with vehicle-treated control cells. However, co-incubation with 8 μm curcumin led to an improved viability of 85 ± 2%. This shows that a sub-stoichiometric amount of curcumin (at 1:5 molar ratio to Aβ₄₂) is able to significantly ameliorate the toxic effects Aβ₄₂. This neuro-protective role of curcumin was more evident at a higher concentration of Aβ₄₂. Although the cells treated with only 400 μm Aβ₄₂ for 48 h showed very low viability (4 ± 1%), the addition of 80 μm curcumin improved the cell viability to 74 ± 3%, manifesting a very pronounced effect. Based on these results, we decided to probe the structural alterations of Aβ₄₂ aggregates caused by curcumin, starting from a solution containing 80 μm curcumin and 400 μm Aβ₄₂.

FIGURE 1.

A–D superimposed transmission and propidium iodide-stained (green spots) images of neurons after 48 h of incubation with A, 40 μm Aβ₄₂; B, 40 μm Aβ₄₂ + 8 μm curcumin; C, 400 μm Aβ₄₂; and D, 400 μm Aβ₄₂ + 80 μm curcumin. Scale bar, 10 μm. E, bar graph of the cell viability assay expressed as a percentage relative to vehicle-treated control cells. The graph shows mean ± S.E. (n = 6).

Aβ₄₂ fibrils are characterized by a cross-β architecture in which each Aβ₄₂ monomer adopts a hairpin structure (although not a β-hairpin). The two largely hydrophobic β-strands are connected by a loop, and there is an overhanging unstructured N terminus (35, 36). These β-strands polymerize in a parallel in-register orientation forming intermolecular β-sheets running perpendicular to the direction of the fibril axis. The presence of an intra and intermolecular contact between the side chains of amino acids Phe-19–Leu-34 and Gln-15–Gly-37 (36) and an intermolecular salt bridge between the COO⁻ and NH₃⁺ groups present in the side chains of Asp-23 and Lys-28 (35) are some other prominent features in the available structural models of Aβ₄₂ aggregates. In our previous study (26), we investigated the effect of Zn²⁺ on the molecular structure of Aβ₄₂ fibrils with ssNMR using two Aβ₄₂ peptides, namely P¹ and P², containing ¹³C- and ¹⁵N-labeled amino acids at specific positions along the peptide sequence (26). P¹ has uniformly ¹³C- and ¹⁵N-labeled Gln-15, Phe-19, Ala-30, Leu-34, Val-36, and Gly-38 and uniformly ¹⁵N-labeled His-13, whereas P² has uniformly ¹³C- and ¹⁵N-labeled Ser-8, Val-12, Phe-20, Asp-23, Lys-28, Met-35, and Ile-41 and uniformly ¹⁵N-labeled His-14. We use the same peptides in this work, which allow a direct comparison.

Chemical shifts of ¹³C atoms in isotopically enriched amino acids were obtained from two-dimensional ¹³C-¹³C correlation spectra of Aβ₄₂ aggregates grown in the presence of curcumin (termed as Aβ₄₂-Cur) recorded using PARIS-xy (m = 1) (n = 2) (29) recoupling scheme with a mixing time of 100 ms. Selected regions of these spectra containing aliphatic-carbonyl and aliphatic-aliphatic cross-peaks are shown in Fig. 2, A and B. The corresponding spectra for Aβ₄₂ are shown in Fig. 2, C and D. The chemical shift values are listed in Table 1 along with the values obtained for Aβ₄₂-Zn aggregates. For Aβ₄₂-Cur aggregates, multiple sets of chemical shifts were observed for amino acids Val-12 (two), Asp-23 (three), Lys-28 (two), Ala-30 (three), Val-36 (three), and Gly-38 (two), where each set represents a distinct structural conformation. The rest of the amino acids yield unique sets and hence adopt a unique structural conformation in Aβ₄₂-Cur aggregates. Similar structural heterogeneity prevails in the molecular structure of Aβ₄₂ aggregates (grown in absence of curcumin) with these same amino acids exhibiting multiple structural conformations. Even the number of multiple conformations is same in both cases, except for Val-12 and Lys-28, both of which exhibit an extra conformation in case of Aβ₄₂ aggregates. Fig. 3, A and B, shows the average chemical shift changes (¯Δ̄δ) incurred by the backbone and side-chain carbons of isotopically labeled amino acids in Aβ₄₂ when aggregated in presence of Zn²⁺ ions and curcumin, respectively (see under “Experimental Procedures” for calculation of ¯Δ̄δ values). Only those ¯Δ̄δ values that are ≥0.5 ppm and at least three times larger than the error associated with them are considered as significant, i.e. represent a significant structural change. These are highlighted in gray in Fig. 3, A and B. These changes are generally associated with local structural changes, which can be caused by direct or indirect interaction of the external ligand. These values indicate that both Zn²⁺ ions and curcumin cause structural perturbations of the Val-12 backbone. In addition to these, curcumin also causes chemical shift changes in the Asp-23 and Leu-34 side chains (Fig. 3B).

FIGURE 2.

Shown are selective regions of two-dimensional ¹³C-¹³C PARIS-xy (m = 1) (n = 2) spectrum of Aβ₄₂-Cur aggregates, P¹ scheme (A); Aβ₄₂-Cur aggregates, P² scheme (B); Aβ₄₂ aggregates, P¹ scheme (C); and Aβ₄₂ aggregates, P² scheme (D) recorded with a mixing time of 100 ms.

TABLE 1.

¹³C NMR chemical shifts of uniformly ¹³C- and ¹⁵N-labeled amino acids in Aβ₄₂-Cur, Aβ₄₂, and Aβ₄₂-Zn aggregates

All chemical shifts are relative to tetramethylsilane with uncertainties of approximately ±0.1 ppm. Values followed by (†) have an uncertainty ≥ ±0.3 ppm associated with them. The values in the parentheses and braces parenthesis are chemical shift values obtained for Aβ₄₂ and Aβ₄₂-Zn aggregates, respectively. TALOS+ was used to predict backbone torsional angle for each structural conformer in Aβ₄₂-Cur, Aβ₄₂, and Aβ₄₂-Zn aggregates. Only “GOOD” predictions (as explained in Ref. 34) are reported here.

Residue	C′	Cα	Cβ	Cγ	Cδ	Cϵ	Cζ	Torsional angles (φ, ψ)
Ser-8	173.3	57.2	62.7					−
	(174.2)	(57.4)	(62.9)					(−)
	{175.0}	{57.0}	{62.8}					{−116 ± 33, 138 ± 23}
Val-12	173.5	59.6	33.3	19.8,19.8				−115 ± 23, 135 ± 22
	(173.7)	(59.7)	(33.4)	(20.1,20.1)				(−115 ± 23, 135 ± 22)
	{173.9}	{59.6}	{33.5}	{20.1,20.1}				{−115 ± 23, 139 ± 22}
Val-12′	173.2	59.8	32.1	19.9,19.9				−
	(173.9)	(60.5)	(31.9)	(20.1,20.1)				(−)
	{174.6}	{60.1}	{32.1}	{19.9,19.9}				{−104 ± 22, 133 ± 17}
Val-12″	(173.8)	(59.8)	(34.4)	(20.1,20.1)				(−118 ± 18, 132 ± 17)
	{174.9}	{61.3}	{30.8}	{20.3,20.3}				{−103 ± 17, 136 ± 9}
Gln-15	172.6	53.7†	–	32.4	177.8†			−
	(173.2)	(53.8†)	(31.4)	(33.1†)	(177.4†)			(−125 ± 21, 136 ± 22)
	{173.2}	{53.9}	{30.8}	{32.9†}	{177.8†}			{−119 ± 23, 137 ± 23}
Phe-19	172.4	54.6	41.0†	137.8†	130.6	129.8	127.3	−126 ± 16, 138 ± 13
	(171.9)	(54.7)	(41.4)	(137.4)	(130.4)	(129.4)	(127.3)	(−128 ± 16, 137 ± 9)
	{172.1}	{54.7}	{41.3}	{137.6}	{130.2}	{129.4}	{127.0}	{−126 ± 16, 135 ± 9}
Phe-20	171.0†	55.2	41.5†	137.5†	129.8†	129.8	129.8	−120 ± 16, 132 ± 11
	(171.2)	(54.9)	(42.0)	(137.8†)	(130.3)	(130.3)	(130.3)	(−119 ± 18, 137 ± 13)
	{171.3}	{54.9}	{42.4}	{137.7†}	{130.0†}	{130.0†}	{130.0†}	{−119 ± 18, 137 ± 13}
Asp-23	175.2	52.8	40.1	179.0				−100 ± 24, 132 ± 16
	(173.0)	(53.3)	(36.1)	(180.2)				(−)
	{−}	{53.1}	{36.0}	{180.3}				{−}
Asp-23′	174.4	52.2	40.3	178.4				−100 ± 24, 132 ± 16
	(175.3)	(52.3)	(41.6†)	(178.8)				(−99 ± 30, 141 ± 21)
	{174.9}	{52.2}	{41.0†}	{178.5}				{−}
Asp-23″	174.4	51.9	42.9	177.7				−
	(175.1)	(52.1)	(42.0†)	(177.2)				(−)
	{174.6}	{51.9†}	{43.2}	{176.9}				{−}
Lys-28	173.2	54.3	32.6	24.8	28.4	41.2		−124 ± 29, 140 ± 23
	(173.8)	(54.5)	(32.7)	(24.9)	(28.5)	(41.0)		(−124 ± 29, 140 ± 23)
	{173.7}	{54.5}	{32.7}	{25.0}	{28.6}	{41.2}		{−124 ± 29, 140 ± 23}
Lys-28′	175.2	54.2	32.2	23.4	27.8	41.0		−116 ± 26, 135 ± 24
	(175.0)	(54.8)	(32.2)	(23.7)	(27.9)	(41.2)		(−111 ± 30, 137 ± 24)
	{174.8}	{54.6}	{32.5}	{23.8}	{23.8}	{41.2}		{−109 ± 29, 139 ± 24}
Lys-28″	(173.6)	(55.4)	(34.1)	(26.2)	(30.0)	(40.8)		(−134 ± 21, 144 ± 20)
Ala-30	174.4	48.7	20.8					−124 ± 17, 134 ± 15
	(174.3)	(48.8)	(20.7)					(−120 ± 20, 132 ± 16)
	{174.3}	{48.8}	{20.8}					{−120 ± 20, 132 ± 16}
Ala-30′	174.3	50.1	21.5					−
	(174.6)	(49.9)	(21.0)					(−)
	{174.7}	{50.1}	{21.5}					{−}
Ala-30″	175.6	50.8	18.7†					−98 ± 11, 121 ± 9
	(175.8)	(50.5)	(19.0)					(−100 ± 11, 122 ± 8)
	{176.1}	{50.6}	{18.8}					{−99 ± 10, 122 ± 8}
Leu-34	173.1	52.5	45.0	26.0	23.0,23.0			−137 ± 11, 145 ± 11
	(172.5)	(52.5)	(44.4)	(26.7)	(24.4,24.4)			(−129 ± 19, 144 ± 12)
	{172.6}	{52.4}	{44.4}	{26.7}	{24.0,25.3}			{−131 ± 18, 145 ± 11}
Met-35	172.5	53.1	34.2	30.9		18.1		−129 ± 21, 137 ± 13
	(172.8)	(53.2)	(34.2†)	(30.9)		(18.3)		(−123 ± 27, 138 ± 14)
	{172.8}	{53.3}	{34.4}	{31.0}		{18.3}		{−123 ± 27, 139 ± 14}
Val-36	173.1	59.8	33.3	21.1,21.1				−126 ± 13, 130 ± 4
	(173.2)	(60.1)	(33.2)	(21.5,21.5)				(−125 ± 13, 129 ± 6)
	{173.2}	{60.1}	{33.2}	{21.6,21.6}				{−124 ± 14, 127 ± 7}
Val-36′	173.4	59.4	31.5	19.9,19.9				−105 ± 13, 132 ± 7
	(173.6)	(59.2)	(31.3)	(20.0,20.0)				(−104 ± 13, 132 ± 8)
	{174.3}	{59.2}	{31.5}	{20.0,20.0}				{−106 ± 13, 130 ± 6}
Val-36″	172.9	58.0†	34.2	20.2,20.2				−129 ± 9, 141 ± 15
	(172.9)	(58.3)	(34.4)	(19.8,19.8)				(−128 ± 9, 145 ± 15)
	{172.6}	{59.2}	{34.4}	{19.7,19.7}				{−130 ± 9, 151 ± 14}
Gly-38	170.3	42.6						−
	(170.1)	(42.3)						(−)
Gly-38′	{170.2}	{42.4}						{−}
	169.6	44.6						−
	(169.5)	(44.7)						(−)
	{169.5}	{44.7}						{−}
Ile-41	172.8	58.7	37.8	26.2,16.4	12.2			−
	(173.2)	(58.9)	(38.1)	(26.3,16.4)	(12.3)			(−)
	{173.0}	{58.9}	{38.1}	{26.4,16.5}	{12.3}			{−}

FIGURE 3.

Average chemical shift difference (¯Δ̄δ) is shown between the backbone and side-chain carbons of various structural conformers (indicated along the x axis) of isotopically labeled amino acids in Aβ₄₂ when aggregated in the presence and in the absence of 400 μm Zn²⁺ ions (A) and 80 μm curcumin (B). Structural conformers showing significant chemical shift changes (as explained in the text) are highlighted using light gray filled rectangles. C–G, selected regions of ¹⁵N one-dimensional and ¹³C-¹³C two-dimensional PARIS-xy (m = 1) (n = 2) (100-ms mixing time) spectra of Aβ₄₂ (black), Aβ₄₂-Zn (blue), and Aβ₄₂-Cur (orange) aggregates, respectively. Dashed arrows in C and D highlight peaks in ¹⁵N one-dimensional spectra of Aβ₄₂-Zn aggregates corresponding to ϵ² nitrogen in imidazole ring of His-13 (C) and δ¹and ϵ² nitrogens in imidazole ring of His-14 (D). Correlations highlighted in two-dimensional spectra are between the following: E, C_β and C′ carbons in Ser-8 (S8 (cross signs)); F, C_α and C_β carbons in Val-36 (V36 (cross signs), V36′ (plus signs), and V36″ (circled cross signs)); G, C_α and C′ carbons in Gly-38 (G38 (cross signs) and G38′ (plus signs)).

More differences are observed when other types of spectral features, like changes in the peak intensities in both one-dimensional and two-dimensional spectra, are considered. These changes are shown in Fig. 3, C–G, which shows selective regions of one-dimensional and two-dimensional spectra of Aβ₄₂ (black), Aβ₄₂-Zn (blue), and Aβ₄₂-Cur (orange) aggregates. Amino acids showing other type of spectral changes are Ser-8, His-13, and His-14 in the case of Zn²⁺ ions and Val-36 and Gly-38 in the case of curcumin. Our previous study has shown that the presence of Zn²⁺ ions imparts increased structural order to the otherwise less ordered side chains of His-13 and His-14 (26). This observation was based on observation of broad peaks around 174 and 208 ppm in ¹⁵N one-dimensional spectrum of Aβ₄₂-Zn aggregates (Fig. 3, C and D, blue). These broad peaks arise from the imidazole ring nitrogens, ϵ² and δ¹, present in the side chain of histidine. The absence of signal in the case of Aβ₄₂ aggregates grown in the absence of Zn²⁺ ions (Fig. 3, C and D, black) is attributed to the structural heterogeneity associated with these side chains, which become structurally more ordered due to Zn²⁺ binding. However, the presence of curcumin does not impart any structural order to these flexible His side chains as is evident from the absence of NMR peaks in the region of interest (Fig. 3, C and D, orange). Fig. 3E highlights the C_β-C′ correlations in Ser-8 as observed in the PARIS-xy (m = 1) (n = 2) spectrum recorded with a mixing time of 100 ms. Clearly, a much stronger correlation is observed in the case of Aβ₄₂-Zn aggregates (Fig. 3E, blue) compared with the Aβ₄₂ (black) and Aβ₄₂-Cur (orange) aggregates. The intensity of such through-space ¹³C-¹³C correlations produced by second-order recoupling schemes like PARIS-xy (m = 1) (n = 2) depends strongly on the molecular properties of the system as well as the experimental parameters used (37). Because all spectra were recorded under similar experimental conditions, this change in intensity of C_β-C′ correlations in Ser-8 must have its origin in changes occurring at the molecular level. It most likely reflects that the Ser-8 backbone acquires more structural order when Aβ₄₂ is aggregated in presence of Zn²⁺ ions but not curcumin.

Fig. 3F highlights the C_α-C_β cross-peaks observed for three conformers of Val-36 as observed in PARIS-xy (m = 1) (n = 2) spectrum using a mixing time of 100 ms. In the case of Aβ₄₂ (Fig. 3F, black) and Aβ₄₂-Zn (blue) aggregates, the Val-36 conformer yields the most intense cross-peak (cross signs) followed by Val-36′ (plus signs) and Val-36″ (circled cross signs), and hence it is the most populated conformer among the three. However, in the case of Aβ₄₂-Cur aggregates (Fig. 3F, orange), cross-peaks of both Val-36 and Val-36′ conformers are nearly equally intense and that of Val-36″ was not observed at all (only observed in PDSD spectrum recorded with long mixing time of 1 s). It is thus clear that curcumin disturbs the population equilibrium of various structural conformers of Val-36. Similar observations exist in the case of Gly-38, where population distribution between the two conformers of Gly-38 (Fig. 3G), Gly-38 (cross signs) and Gly-38′ (plus signs), gets perturbed only in the presence of curcumin but not in the presence of Zn²⁺ ions.

In our previous study, it was shown that Zn²⁺ ions disrupt the Asp-23–Lys-28 salt bridge in Aβ₄₂ aggregates by stabilizing only the non-salt bridge-forming conformations of Asp-23 and Lys-28 (26). Interestingly, presence of curcumin also causes major population redistribution between different conformers of Asp-23 and Lys-28, leading to the near disappearance of the Asp-23 and Lys-28″ conformers as assigned in Aβ₄₂ aggregates (see Table 1). This effect is clearly shown in Fig. 4, A and B, which shows an overlay of selected regions of the one-dimensional ¹³C and ¹⁵N spectra of Aβ₄₂ (black) and Aβ₄₂-Cur (orange) aggregates, respectively. It is further exemplified in Fig. 4, C and D, which shows the connectivity between the aliphatic carbons (β, γ, δ, and ϵ) of the three conformers of Lys-28 in Aβ₄₂, and the only two conformers of Lys-28 in Aβ₄₂-Cur aggregates, respectively. These are two-dimensional ¹³C-¹³C PARIS-xy spectra recorded with a mixing time of 100 ms. However, because of the extremely fast decaying nature of C_γ resonance associated with the curcumin-stabilized conformers in Aβ₄₂-Cur aggregates (see Fig. 5B, orange), a REDOR dephasing curve lasting long enough to comment on the presence or absence of the salt bridge could not be derived in this case. A comparative REDOR dephasing signal for Aβ₄₂ aggregates is shown in Fig. 5A. All the observations made so far are summarized in Fig. 4, E and F, where amino acids having significant chemical shift changes are highlighted using dark cyan filled circles and those exhibiting other type of spectral changes are highlighted in circles with red outline. The effect of curcumin and Zn²⁺ on the molecular structure of the β-sheet regions of Aβ₄₂ is therefore somewhat complementary, with curcumin targeting amino acids mainly in the C terminus and Zn²⁺ ions mainly in the N terminus. However, both of them affect the salt bridge-forming amino acids Asp-23 and Lys-28 present in the loop region connecting the two β-sheets.

FIGURE 4.

A, carbonyl region of one-dimensional ¹³C spectra of Aβ₄₂ (black) and Aβ₄₂-Cur (orange) aggregates, highlighting peaks yielded by γ carbon of Asp-23. B, selective region of ¹⁵N one-dimensional spectrum of Aβ₄₂ (black) and Aβ₄₂-Cur (orange) aggregates, highlighting peaks yielded by ζ nitrogen of Lys-28. Peaks in A and B are labeled according to side chains of various conformers of Asp-23 and Lys-28 as explained in Ref. 26. Selective region of two-dimensional ¹³C-¹³C correlation spectra (PARIS-xy, 100 ms) of Aβ₄₂ (C) and Aβ₄₂-Cur (D) aggregates. Also shown is the connectivity between aliphatic carbons (β, γ, δ, and ϵ) of three and two conformers of Lys-28 in Aβ₄₂ and Aβ₄₂-Cur aggregates, respectively. E and F, amino acids showing structural changes in the presence of Zn²⁺ ions and curcumin are highlighted over a schematic of a hairpin model of Aβ₄₂. Structural changes causing significant chemical shift changes are highlighted using dark cyan filled circles. Amino acids showing other type of spectral changes are highlighted using circles with red outline.

FIGURE 5.

Carbonyl regions of ¹³C spectra of Aβ₄₂ (A) and Aβ₄₂-Cur (B) aggregates. The topmost spectrum (one-dimensional) in each case is recorded by simply acquiring the signal after cross-polarization from protons. Other spectra are recorded using frequency selective REDOR pulse sequence, without applying the frequency selective pulse on ¹⁵N channel (reference spectra, S₀) and spin-echo periods as indicated.

A recent ssNMR study by Masuda et al. (38) has shown that curcumin interacts with the residues from 17 to 21, as well as with residue 12 present in the N-terminal β-sheet. Masuda et al. (38) postulate that curcumin targets the C-terminal β-sheet in a nonspecific fashion. Our results substantiate this speculation with three amino acids in this region, namely Leu-34, Val-36, and Gly-38, undergoing either structural reorganization or population redistribution between different conformers in the presence of curcumin. Regarding the N terminus, our results are partially in agreement with their findings. We also found that Val-12 was affected by curcumin. However, Phe-19 and Phe-20, the only isotopically labeled amino acids in the 17–21 region, remain unaffected by the presence of curcumin. This discrepancy most likely has its origin in different way these studies have been performed. Masuda et al. (38) incubated curcumin with preformed fibrils, whereas we precipitated Aβ₄₂ from the solution in the presence of curcumin. In a nut shell, curcumin does target the β-sheet-rich regions of Aβ₄₂, but these are not the only regions affected by it, as shown by our results.

Secondary chemical shifts (Δδ) of Aβ₄₂ and Aβ₄₂-Cur aggregates, defined as the difference between the observed and random coil chemical shifts, were calculated for C_α, C_β, and carbonyl carbon atoms and are shown in Fig. 6, A and B, respectively. A positive value for C_β carbon and negative values for C_α and carbonyl carbon atoms is indicative of the amino acid being part of a β-sheet region. Most of the amino acids follow these criteria, thus indicating dominance of the β-sheet structural elements in both Aβ₄₂ and Aβ₄₂-Cur aggregates. With the current isotopic labeling schemes, it seems that these regions run roughly between amino acids Val-12 and Phe-20 (N-terminal β-sheet) and Ala-30 and Ile-41 (C-terminal β-sheet). Even though multiple conformations are observed for some amino acids in this region, all of them seem to adopt β-sheet secondary structure except Gly-38. The presence of curcumin increases the population of the non-β-sheet conformer (Gly-38′) of Gly-38. Among the remaining amino acids, Ser-8 clearly seems to be part of the unstructured N terminus in both Aβ₄₂ and Aβ₄₂-Cur aggregates. The salt bridge-forming conformer of Asp-23, denoted D23 in figures, adopts a totally random structure. Its other two conformers in Aβ₄₂ aggregates also adopt non-β-sheet conformation. However, in case of Aβ₄₂-Cur aggregates, two out of its three conformers (Asp-23′ and Asp-23″) seem to adopt the β-sheet structure. Similarly, in the case of Lys-28, the disappearing conformer, denoted K28″ in figures, adopts a structural conformation that is significantly different from the remaining two conformers (K28 and K28′). The structural conformation of the other two conformers of Lys-28 is not completely like β-sheet, but this conformation is more or less retained in the presence of curcumin. It seems that in Aβ₄₂ aggregates, both Asp-23 and Lys-28 adopt a secondary structure that is partially β-sheet in nature, and hence, they are most likely part of the turn region connecting the N- and C-terminal β-sheets. The presence of curcumin results is an increase in the β-sheet content of both Asp-23 and Lys-28 in Aβ₄₂-Cur aggregates. However, as both of them still contain conformers that are not strictly β-sheet-like, they might still be part of the turn region. To summarize, even though presence of curcumin causes local disturbances in the molecular structure, Aβ₄₂-Cur aggregates still retain the basic secondary structural motifs of Aβ₄₂ aggregates.

FIGURE 6.

Secondary chemical shift plot for backbone carbons of all structural conformers of isotopically labeled amino acids are shown in Aβ₄₂ (A) and Aβ₄₂-Cur (B) aggregates. C, two-dimensional ¹³C-¹³C PDSD spectrum of Aβ₄₂-Cur aggregates (P¹ scheme) recorded with a mixing time of 1000 ms. Cross-peaks between γ carbon of Leu-34 and aromatic (δ/ϵ) carbons of Phe-19 are highlighted. Ambiguous cross-peaks between aromatic carbons of Phe-19, and α and β carbons of Leu-34 are also highlighted using plus and cross signs, respectively.

It was seen that despite some major structural changes, Aβ₄₂ aggregates grown in the presence of Zn²⁺ ions also retain both the secondary structure as well as the hairpin structure of Aβ₄₂ fibrils (26). In the hairpin structure of Aβ (both 40 and 42) fibrils, the side chains of amino acids present in the two β-sheets interdigitate to form a hydrophobic steric zipper (35, 36, 39–43). This interdigitating pattern differs from one structural model to another (35, 36, 39, 40, 42, 43). In other words, even though each model proposes the same hairpin structure for Aβ, they differ in terms of inter-residue contacts proposed between the two β-strands. However, intramolecular Phe-19–Leu-34 side-chain contacts have been proposed in most of the Aβ₄₂ and Aβ₄₀ models (36, 40, 42, 43), and they have even been used to verify the hairpin structure of Aβ (26, 40). We also looked for this contact in Aβ₄₂-Cur aggregates. Because it is a long range contact, a two-dimensional ¹³C-¹³C through-space correlation spectrum was recorded using the PDSD recoupling scheme (30) with a long mixing time of 1 s. A selected region of this spectrum is shown in Fig. 6C. The presence of the unambiguous cross-correlation between the aromatic (δ/ϵ) carbons of Phe-19 and γ carbons of Leu-34 is indicative of their side chains being spatially proximal. Hence, just like the secondary structure, the tertiary structure also seems to be conserved in Aβ₄₂-Cur aggregates.

We note that the Phe-19–Leu-34 contact by itself does not prove that the peptide is bent into a hairpin shape. This contact constrains the possibilities to either a hairpin or to an open state with the monomeric strands in antiparallel arrangement, with Phe-19 and Leu-34 in register. This alternative possibility would imply a fibrillar width that is much larger. We examined the width of the Aβ₄₂ and Aβ₄₂-Cur aggregates by transmission electron microscopy. The Aβ₄₂ aggregates show a fibrillar morphology with the dominant form showing long, straight, unbranched fibrils that extend frequently beyond 1 μm in length (Fig. 7A). The Aβ₄₂-Cur aggregates, however, show only short fibrils or quasi-fibrillar structures (Fig. 7B). An analysis of the width of the fibrillar Aβ₄₂ and quasi-fibrillar Aβ₄₂-Cur aggregates (a few representative positions are marked in red in Fig. 7, A and B) yields similar values (7.9 ± 0.9 and 8.1 ± 1.1 nm, respectively). This implies that the Aβ₄₂-Cur aggregates have a similar hairpin architecture as the Aβ₄₂ fibrils. The disruption of the mesoscopic structures observed in the presence of curcumin is consistent with those reported earlier (5, 7) and also as reported for Zn²⁺ (26). It is possible that both of these agents precipitate the aggregates of Aβ₄₂ at an early stage and reduce their toxicity by locking them into a nontoxic conformation, and/or by simply removing them from the solution. Evidence for such a mode of action has indeed been seen for Aβ₄₀-Zn²⁺ (17, 21) and also for Aβ₄₀-Cur.⁴

FIGURE 7.

A, amyloid fibrils formed by 400 μm Aβ₄₂ aggregated for 4 days. Long fibrillar structures are clearly visible. B, aggregates formed by 400 μm Aβ₄₂ in the presence of 80 μm curcumin. Fibrillar structures are clearly disturbed in the presence of curcumin. Red lines show a few representative positions where the fibrillar widths were measured.

CONCLUSION

Significant changes in the Asp-23 and Lys-28 conformation is a major feature of the curcumin-induced disruption of the Aβ₄₂ conformation. We note that the Phe-19–Leu-34 contacts in Aβ₄₂ are thought to be intramolecular, whereas the salt bridge connecting Lys-28 and Asp-23 is thought to be between neighboring strands (35). Thus curcumin (like Zn²⁺) seems to preserve the gross intramolecular conformation, while disrupting the intermolecular arrangements. This is consistent with its ability to disrupt the fibrillar architecture (as observed by transmission electron microscopy in Fig. 7) (5, 7). There are also specific changes in the intramolecular structure, with the C-terminal residues mostly affected by curcumin, whereas the N-terminal residues are mostly affected by Zn²⁺ ions. Both of these therefore appear to stabilize a nonfibrillar or partly fibrillar family of aggregate structures whose morphology and toxic properties are very different from that of the regular fibrillar aggregates. These structural differences highlight potential target regions of the peptide for designing therapeutics for Alzheimer disease.