Inhibition of amyloid fibril formation of β-lactoglobulin by natural and synthetic curcuminoids

Article abstract of DOI:10.1039/c8nj03194k

The aggregation of proteins has been associated with several aspects of daily life, including food processing, blood coagulation and many neurodegenerative infections. However, the actual mechanisms responsible for amyloidosis, the irreversible fibril for

Full text of DOI:10.1039/c8nj03194k

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Inhibition of amyloid fibril formation of
b-lactoglobulin by natural and synthetic
curcuminoids†
Cite this: New J. Chem., 2018,
42, 19260
a
Sanhita Maity,a Sampa Pal,a Subrata Sardar,a Nayim Sepay,
Hasan Parvej,a
Shahnaz Begum,a Ramkrishna Dalui,a Niloy Das,b Anirban Pradhan
and
c
a
Umesh Chandra Halder
*
The aggregation of proteins has been associated with several aspects of daily life, including food
processing, blood coagulation and many neurodegenerative infections. However, the actual mechanisms
responsible for amyloidosis, the irreversible fibril formation of various proteins, which is linked to
disorders such as Alzheimer’s disease, Creutzfeldt–Jakob disease and Huntington’s disease, have not yet
been fully elucidated. Curcumin, a potent anti-oxidant, exhibits anti-amyloid activity; however, its activity is
limited due to its instability. Therefore, chemical modifications of curcumin have been performed to obtain
molecules with enhanced stability and superior anti-amyloid activity. Herein, the main objective of this study
is related to the inhibitory effects of three stable analogs of curcumin against bovine b-lactoglobulin (b-lg)
fibrillization. We inferred that a pyrazole derivative of curcumin showed remarkable potency in arresting the
fibrillization of b-lg, as revealed by biophysical techniques. Molecular docking demonstrated that pyrazole-
mediated inhibition of b-lg fibrillogenesis may be initiated by interacting with aggregation-prone regions of
the protein and preventing interactions between monomers, leading to suppression of the overall
aggregation process. This work alludes to a possible broader scope for discovery of other small molecules
that may exert similar effects against amyloid formation and its associated neurodegenerative diseases.
Received 27th June 2018,
Accepted 13th October 2018
DOI: 10.1039/c8nj03194k
rsc.li/njc
dyshomeostasis, oxidative stress, and neurotransmitter system
dysfunction.3–7 Different peptides and proteins generate mor-
Introduction
The anomalous self-assembly and accumulation of misfolded phologically similar or different amyloid fibrils through dimer
proteins is known to have common cellular and molecular and oligomer formation; this has been hypothesized to occur
mechanisms, including protein aggregation, which is the in a stepwise fashion, with a slow phase of nucleation of the
known leading causative agent of a number of conformational precursors of the amyloid fibrils followed by a relatively fast
diseases, such as Alzheimer’s disease (AD), Parkinson’s disease elongation phase.8 Moreover, in the formation of amyloid
(PD), Huntington’s disease (HD) and prion disease.1,2 The fibrils, the incentive to assemble arises from favorable solvation
aggregates consist of fibers with cross b-sheet structures, energies and side-chain interactions accompanying the for-
termed harmful ‘amyloids’, and their morphological features mation of b-sheet structures.9 Currently, numerous clinical
are not associated with behaviors of specific proteins. Although and experimental studies are revealing that soluble oligomeric
the proper aetiology of AD remains controversial, diverse and protofibrillar forms of proteins are potentially neurotoxic.10,11
factors appear to play vital roles in the pathophysiology of
Recently, the most challenging research task has focused on
the disease; these include abnormal b-amyloid (Ab) deposits the inhibition of fibril formation12–14 by the employment of
in extracellular amyloid plaques, which lead to progressive small molecules. These potent modulators are believed to
neuronal death, tau protein hyperphosphorylation, metal ion stabilize monomers by blocking the formation of toxic oligomers
and to divert the monomeric proteins to off-pathway non-toxic
a Organic Chemistry Section, Department of Chemistry, Jadavpur University,
Kolkata 700032, India. E-mail: uhalder2002@yahoo.com
b Department of Chemistry, Durgapur Govt. College, Durgapur,
West Bengal 713214, India
intermediates. Small molecules are being developed to inhibit
aggregation of Ab,15 a-synuclein16 and prions.17
Much evidence has shown that polyphenols, which have
structural constraints, are effective in the inhibition of amyloid
fibrillation.18,19 Curcumin, a classical active yellow lipid-
c Director’s Research Unit (DRU), Indian Association for the Cultivation of Science,
Kolkata-700032, India
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8nj03194k soluble b-diketone dietary polyphenolic component, has been
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extensively studied during the last few decades not only for its
well-known pharmacological attributes, such as antioxidant,
anticancer, antibiotic and anti-amyloidogenic properties, but
also for its protective activities and intrinsic nontoxicity.20
Curcumin has been found to non-specifically bind to amyloid-b
monomers and fibrils and modify protein aggregation
pathways.21 Despite its extensive bioactivities, the potential
efficacy of curcumin is extremely limited for the prevention of
aggregation-related diseases due to its moderate bioavailability
and poor potency related to its insolubility in water as well as
its poor absorption and metabolic instability.22 Many recent
studies have focused on the generation of rationally designed
curcumin analogues and derivatives which have been demon-
strated to have very promising potency and bioactivity compared
to curcumin against amyloid proteins in vitro as well as in vivo.23
Bovine b-lactoglobulin (b-lg), a well-known globular major
whey protein comprising 162 amino acids with a molecular
mass of 18.3 kDa, is predominantly a b-sheet protein.24 In
addition, b-lg possesses two intramolecular disulphide bonds
Fig. 1 Chemical structures of curcumin and its derivatives.
been used as potent ligands of fibrillar Ab-42 aggregates34 and
also have the ability to prevent tubulin self-assembly.35 It has
been reported that curcumin diacetate (DAC) also has an
influence on hIAPP aggregation.23 However, by employing
several biophysical techniques, for the first time, we have
evaluated the effects of curcumin derivatives on the inhibitory
mechanism of b-lg fibril formation in vitro.
Results and discussion
SDS-polyacrylamide gel electrophoresis study
(Cys66–Cys160, Cys106–Cys119) and one free thiol group (Cys121
which is buried between the b-barrel and the major a-helix.25
)
Bovine b-lg is an extensively used model carrier protein for SDS-PAGE analysis is a convenient technique to observe protein
hydrophobic ligands due to its pH-dependent opening, excellent aggregation under non-reducing conditions. In the SDS-PAGE
encapsulating properties and unique resistivity to acidic pH.26 study, the native b-lg appeared as a single band (Lane 2) in the
Due to its carrier properties, this protein is a good candidate to gel around 18.3 kDa, which corresponds to the monomeric
serve as a model transporter for delivering important hydrophobic state of b-lg. The b-lg sample in Fig. 2A was incubated at 75 1C
nutrients to improve their bioavailability. The protein b-lg in 10 mM phosphate buffer at pH 7.4 for 6 h; a series of protein
presents an interesting conundrum because it forms fibrillar bands appeared, suggesting the highly packed nature of
structures upon thermal treatment at physiological pH.27,28 the aggregates formed after incubation (Lane-3). This useful
Despite the increasing quantity of studies on this topic, the technique was employed to dissociate all the non-covalently
mechanisms of fibril formation and identification of the building bonded aggregates into monomers while the disulphide-linked
blocks of fibrils is still a subject of debate.29 The primary cause of aggregates remained intact. The thiol group of cys121 of b-lg
initiation of thermal aggregation of b-lg is thought to involve forms S–S linkages when incubated at 75 1C to 80 1C, which is
disulfide bridge formation by exchange between free cysteine and associated with the formation of dimers and other covalently
one of the two disulfide bridges.30 A two-step model has been bound ‘‘intermediates’’ during aggregation48,49 by scrambling
proposed for the aggregation process at neutral pH, in which of disulphide bonds (creation of new intramolecular and inter-
small particles are formed first and then further aggregate into molecular disulphide bridges and rearrangement of existing
large clusters that exhibit self-similarity in certain cases.31
intramolecular disulphide bridges).50
A recent proposal by Ma et al. suggests involvement of the
When b-lg was incubated at 75 1C for 6 h, bands appeared
lag time for the formation of stable nuclei (nucleation) and the in the gel corresponding to the formation of self-assemblies of
apparent rate constant for the growth of fibrils (elongation) in b-lg (Lane 3, bands II–V) and to the monomer, with lower
the kinetics of the fibrillation process at neutral pH.32 However, intensity (Lane 3, band I). The SDS-PAGE analysis of heat-
the aggregation process usually involves conformational changes treated b-lg in the presence of curcumin derivatives at a molar
of the whole protein or of a specific domain; this is often concentration ratio of 1 : 1 under non-reducing conditions
attributed to the association of partially unfolded molecules.33 showed a very different result. In the presence of curcumin
Extensive research has been carried out to establish the mode and DAC (Lane 6 and Lane 7) at molar concentration ratios of
of action of curcumin and the chemical moiety responsible for 1 : 1, trimeric states were also formed. Stabilization of the
its anti-fibrillogenic properties; however, the actual mechanism dimeric state with a less intense band in the trimeric state
responsible for these activities remains elusive due to the instability was observed when b-lg was incubated with the IOC derivative
of curcumin in solution. Moreover, efforts to explore the potency at a molar concentration ratio of 1 : 1 (Lane 4). The dimeric
of stable curcumin analogues as inhibitors of b-lg aggregation state of b-lg was stabilized in the presence of PY derivative
are lacking. Herein, we therefore screened curcumin derivatives, (Lane 5) at a molar concentration ratio of 1 : 1; this is consistent
isosteric isoxazoles (IOC), pyrazoles (PY) and curcumin diacetate with its lower electrophoretic mobility. The stabilization of the
(DAC) (Fig. 1), as ideal small molecular candidates to achieve dimeric state by the PY derivative may be due to blocking of the
better pharmacological attributes against b-lg aggregation formation of toxic oligomers and prevention of the primary nuclea-
in vitro. Pyrazole (PY) and isoxazole (IOC) derivatives have also tion process to destabilize the formation of oligomers of b-lg.
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Fig. 2 (A) SDS-PAGE (12%) patterns of native b-lg (Lane 2) and b-lg incubated at 75 1C for 6 h in the absence (Lane 3) and presence of 1 : 1 molar
concentration ratios of IOC (Lane 4), PY (Lane 5), curcumin (Lane 6) and DAC (Lane 7). Lane 1 represents the SDS PAGE pattern of marker proteins with
known molecular weights (Page Rulert, Prestained Protein Ladder, 10, 17, 26, 34, 43, 55, 72, 95, 130 and 170 kDa, respectively; Fermentus Life Science,
#SM0671) run parallel. (B) Relative fluorescence intensities of tryptophenyl residues (emitted at 335 nm) of b-lg incubated at 75 1C for 6 h in the absence
(1) and presence of curcumin (2, 3), DAC (4, 5), IOC (6, 7) and PY (8, 9) at molar concentration ratios of 1 : 0.5 and 1 : 1, respectively in 10 mM phosphate
buffer at pH 7.4 containing 2% ethanol. The samples were excited at 295 nm and the emission was recorded in the range of 310 to 400 nm. The protein
concentrations during intrinsic fluorescence were 13.6 mM. The results are the mean of three different experiments performed in duplicate. Standard
deviations are within the range of ꢀ2.0.
Lane 1 represents the SDS-PAGE pattern of marker proteins presence of PY derivative (Fig. 2B, circle 9) at a 1 : 1 molar
with known molecular weights (Page Rulert, Prestained Protein concentration. The decreases in the intrinsic fluorescence
Ladder, 10, 17, 26, 34, 43, 55, 72, 95, 130, and 170 kDa, intensity suggest that curcumin, DAC, IOC and PY derivatives
respectively; Fermentus Life Science, #SM0671) run parallel.
interacted with b-lg, resulting in conformational changes of
the protein; PY derivative caused the maximal change in the
protein conformation.
Tertiary conformational alterations probed by tryptophan
fluorescence
Disassembly of b-lg aggregates with curcumin derivatives
monitored by ThT assay
Intrinsic fluorescence parameters, such as fluorescence inten-
sity (FI) and emission maxima (lmax), provide a very sensitive
mean for studying the structural dynamics and polarity of The thioflavin T (ThT) assay is believed to be a reliable
protein chromophores, viz. Trp, Tyr and Phe. The two trypto- technique for quantification of amyloid fibrils; therefore, we
phan residues of b-lg, Trp19 and Trp61 provide a convenient monitored the changes in dye fluorescence to investigate the
spectroscopic means for assessing the conformational states of inhibitory effects of these derivatives against b-lg aggregation
proteins. In particular, Trp61 is quenched by the close proximity in vitro. All the samples were blank corrected (data not shown)
of the neighbouring disulphide bond, Cys160–66, and Trp19 because coloured compounds can suppress ThT fluorescence;
is situated within the centre of the hydrophobic calyx of also, we considered the percent increase in ThT intensity
the protein in the native state.51 Trp19 was therefore the most instead of the absolute intensities. The extent of aggregation
useful probe for tryptophan fluorescence of bovine b-lg. In our was monitored by sampling at regular intervals. Fig. 3A shows
study, the b-lg incubated at pH 7.4 exhibited a prominent the kinetics of b-lg aggregation in the absence and presence
emission peak around 335 nm in the wavelength range of of curcumin derivatives monitored by ThT binding assay.
300 to 400 nm when excited at 295 nm. Hence, the relative The percentage increases in ThT fluorescence for all the
changes in the fluorescence intensity around 335 nm can be compounds were calculated using eqn (1). The kinetic curves
used as a probe for changes in the microenvironment in the of the ThT fluorescence intensity at 485 nm are consistent with
vicinity of the tryptophan residues of proteins arising due to a nucleation-dependent polymerization model in which the lag
altered conformations. Fig. 2B reveals the relative fluorescence corresponds to the nucleation phase and the exponential part
intensities at 335 nm of b-lg after co-incubation with curcumin, corresponds to the elongation phase (fibril growth).32 In our
DAC, IOC and PY derivatives at molar concentration ratios experimental conditions, aggregated b-lg displayed a lag phase
of 1 : 0.5 and 1 : 1 for 6 h at 75 1C. The maximal quenching of of B150 min (Fig. 3A). The significantly decreased ThT fluores-
the fluorescence intensity (Fig. 2B) of b-lg was observed in the cence for PY derivative indicates that it suppressed fibril
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Fig. 3 (A) Time course of b-lg aggregation in the absence (curve a) or presence of curcumin derivatives as monitored by ThT assay. b-lg was
co-incubated at 75 1C with curcumin (curve b), DAC (curve c), IOC (curve d) and PY (curve e) at molar concentration ratios of 1 : 1 for 6 hours in 10 mM
phosphate buffer at pH 7.4 containing 2% ethanol. The ability of these compounds to prevent aggregation is shown as the percentage increase in ThT
fluorescence intensity. Aggregated samples were incubated with 50 mM of ThT for 30 min at 25 1C. The samples were excited at 440 nm and the
emissions were recorded at 485 nm. The results are the mean of three different experiments. (B) The dose–response curve plotting the plateau value of
the ThT fluorescence intensities estimated from the time course of aggregated b-lg sample alone (1) and co-incubated with curcumin (2), DAC (3), IOC
(4) and PY (5) derivatives. The data are the average of at least three independent experiments. Error bars are given within the range of ꢀ2.0. The protein
concentration during the ThT assay was 13.6 mM.
formation of b-lg compared to the other derivatives; the order
of the inhibitory potentials of the compounds was observed to
be PY 4 IOC 4 DAC 4 curcumin. This can be attributed to the
stabilization of low molecular weight (LMW) oligomers or
dimers by the pyrazole form of curcumin; the stabilized species
did not consequently form b-sheet-rich fibrillar species.
Detection of exposed hydrophobic clusters by ANS binding
Fig. 4 (A) Percentage inhibition of aggregation of b-lg by curcumin and
curcumin derivatives as observed by ThT assay. b-lg was co-incubated at
75 1C with curcumin (1, 2), DAC (3, 4), IOC (5, 6) and PY (7, 8) at 1 : 0.5 and
1 : 1 molar concentrations, respectively, for 6 h in 10 mM phosphate buffer
The exposure of the contiguous hydrophobic surface areas
of folding intermediates is detected through 1-anilino-8-
naphthalene sulfonate (ANS) binding, the most commonly
utilized fluorescence probe for detecting the non-polar character
of proteins.52 ANS did not significantly bind to b-lg in its native
state, indicating the absence of exposed hydrophobic pockets.
It was reported that ANS is practically non-fluorescent in
water but produces brilliant fluorescence with an emission
maximum from 510 nm to 480 nm upon binding to hydro-
at pH 7.4 containing 2% ethanol. The percentage of inhibitory effects of
these compounds was calculated using eqn (2). (B) ANS fluorescence of
native b-lg (line a) and b-lg incubated at 75 1C for 6 h in the absence (line b)
and presence of curcumin (line c, line d), DAC (line e, line f), IOC (line g, line h),
and PY (line i, line j) at molar concentration ratios of 1 : 0.5 and 1 : 1,
respectively, in 10 mM phosphate buffer at pH 7.4 containing 2% ethanol.
(inset) Relative fluorescence intensities at 480 nm of native b-lg (1) and
phobic patches of proteins.53 As expected, the spectrum of incubated b-lg at 75 1C for 6 h in the absence (2) and presence of
curcumin (3, 4), DAC (5, 6), IOC (7, 8), PY (9, 10) at molar concentration
native b-lg showed negligible ANS fluorescence (Fig. 4B, profile a).
In the absence of curcumin derivatives, the thermally incubated
b-lg at pH 7.4 showed maximum ANS fluorescence intensity at
ratios of 1 : 0.5 and 1 : 1, respectively in 10 mM phosphate buffer at pH 7.4
containing 2% ethanol. The concentration of b-lg was maintained at
13.6 mM. Fluorescence emissions were monitored in the wavelength range
480 nm with a blue shift of the emission. This enhancement of
fluorescence intensity can be attributed to the increased access
of ANS to the hydrophobic patches present in proteins upon
thermal incubation compared to native b-lg. These hydrophobic
patches increase the protein–protein interactions, leading to
thermal aggregation of b-lg.54 However, in the presence of curcu-
of 460 to 600 nm after excitation at 380 nm. Results are the average of
triplicate measurements. The protein concentration during the ANS
fluorescence studies was 13.6 mM. Standard deviations are within the range
of ꢀ2.0.
min and its potential analogs, during thermal exposure of b-lg, ratios of 1 :0.5 and 1:1 with respect to the same molar concen-
significant decreases of the ANS fluorescence intensity were trations of curcumin (profile c and profile d). A gradual decrease
observed, indicating a loss of ANS binding sites. Fig. 4B suggests of ANS fluorescence intensity was observed during thermal
that a gradual decrease of ANS fluorescence intensity was co-incubation of b-lg with the IOC derivative at molar concen-
observed for DAC (profile e and profile f) at molar concentration tration ratios of 1 : 0.5 and 1 : 1 (Fig. 4B, profile g and profile h).
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In contrast, no significant ANS binding was observed upon remarkable decrease in scattering intensity was noted in the
thermal co-incubation of b-lg with PY derivative at a molar presence of PY derivative at a molar concentration of 1 : 1.
concentration ratio of 1 : 1 (Fig. 4B, profile j). The decreased Control measurements with the aforementioned molar concen-
ANS binding in the presence of the curcumin derivatives suggests trations of the curcumin derivatives were performed and compared.
the formation of fewer hydrophobic patches by b-lg upon thermal Higher concentrations of curcumin derivatives scattered UV light at
stress at 75 1C. Fig. 4B shows the relative fluorescence intensities 350 nm. Hence, the decrease in scattering intensity in the presence
(RFI) at 480 nm of native b-lg and b-lg incubated at 75 1C for 6 h in of PY derivative at a molar concentration ratio of 1 : 1 indicates that
the absence and presence of curcumin and curcumin derivatives. this derivative has an appreciable potential inhibitory effect to
The decreased ANS binding in presence of the curcumin deriva- decrease the self-assembly of b-lg aggregations.
tives is highly consistent with the lower population of partially
folded intermediates; thus, these derivatives are capable of
suppressing b-lg aggregation.
Quantifications of amyloid formation employing Congo red
(CR) measurements
To validate the findings of the ThT assay, the inhibition of b-lg
aggregation was also confirmed by the Congo red (CR) binding
assay. Depending on the state of protein aggregation, CR
Monitoring the disaggregation of b-lg utilizing Rayleigh light
scattering (RLS) measurements
Rayleigh light scattering (RLS) is one of the most commonly binding to the extensive b-sheet structures is expected to cause
used experiments in the arena of protein aggregation. The an increase in absorption, with a bathochromic shift in its
utilization of RLS data is based upon the principle that characteristic absorption spectrum (from approximately 490 nm
increased RLS intensity of a protein solution is an indication unbound to 500 nm bound). The CR spectral shift assay poten-
of aggregation. Conventionally, a decrease of RLS intensity is a tially enables quantification of amyloid fibrils, where coloured
useful parameter to support the disaggregation or disassembly compounds interfere with ThT fluorescence.55 Generally, at
of proteins. Thus, Fig. 5A shows the changes in the scattering neutral pH, b-lg showed absorption maxima at 493 nm (Fig. 5B,
intensity measured at 350 nm after exciting heat-treated b-lg profile a) and the thermally incubated b-lg showed absorption
solution at 350 nm in the absence and presence of varying maxima at 500 nm, accompanied with broadening of the areas of
amounts of the aforementioned curcumin derivatives at pH 7.4. absorbance. This is attributed to the confinement of the CR dye in
It was noted that the maximum scattering intensity was the clusters of the b-sheet peptide backbones of the aggregates.
observed for incubated b-lg, suggesting aggregate formation. We observed that the increased peak shift detected in aggregated
Furthermore, when DAC and IOC were used at molar concen- b-lg was not visible in the spectra of b-lg samples containing
tration ratios of 1 : 0.5 and 1 : 1, respectively, significant curcumin and its derivatives, corroborating the results of the ThT
decreases in the scattering intensity were observed with respect binding assay. In the presence of curcumin, DAC and IOC
to curcumin at same concentration level. In particular, a derivatives at molar concentration ratios of 1 : 0.5 and 1 : 1, the
Fig. 5 (A) Rayleigh light scattering data of native b-lg (1) and b-lg incubated at 75 1C for 6 h in the absence (2) and presence of curcumin (3, 4), DAC (5, 6),
IOC (7, 8), and PY (9, 10) at molar concentration ratios of 1 : 0.5 and 1 : 1, respectively, in 10 mM phosphate buffer at pH 7.4 containing 2% ethanol. Control
measurements with curcumin derivatives are shown in circles: [circle a – curcumin (13.6 mM), circle b – curcumin (27 mM), circle c – DAC (13.6 mM), circle
d – DAC (27 mM), circle e – IOC (13.6 mM), circle f – IOC (27 mM), circle g – PY (13.6 mM), and circle h – PY (27 mM)]. The samples were excited and emitted
at 350 nm. (B) Congo red assay of native b-lg (line a) and b-lg incubated at 75 1C for 6 h in the absence (line b) and presence of curcumin (line c, line d),
DAC (line e, line f), IOC (line g, line h), and PY (line i, line j) at molar concentration ratios of 1 : 0.5 and 1 : 1, respectively, in 10 mM phosphate buffer at pH 7.4
containing 2% ethanol. Absorbance was scanned in the wavelength range of 400 nm to 700 nm using a UV-Vis spectrophotometer. Results are the mean
of three different experiments. The protein concentration was maintained at 27.2 mM.
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absorbance decreased with respect to the degree of inhibition of
It should also be pointed out that in presence of DAC at a
amyloid fibril formation of b-lg. The lowest absorbance for PY molar concentration ratio of 1 : 1 (Fig. 6A, profile f), the sizes
derivative at a molar concentration ratio of 1 : 1 may be due to (ranging from 25 nm to 350 nm) of the aggregates decreased
entanglement of the formed fibrils into relatively unordered with respect to curcumin at the same concentration level.
smaller aggregates (Fig. 5B, profile j). It was confirmed that PY However, in the presence of IOC at a molar concentration ratio
derivative at a molar concentration (1:1), stabilizes the dimeric of 1 : 1, the scattering was dominated by smaller particles (size
form of the protein to a greater extent, inhibiting nucleation and ranging from 25 nm to 320 nm) (Fig. 6A, profile h). Specifically,
elongation for the formation of higher aggregates.
a drastic change in the size distribution of large b-lg particles
(aggregates) was observed upon thermal co-incubation with PY
derivative at a molar concentration ratio of 1 : 1 (size ranging
from 25 nm to 260 nm) (Fig. 6A, profile j). The hydrodynamic
radius data suggest the apparent quenching of aggregate
growth which encompasses the association of protein molecules,
probably due to the formation of compact dimers in the
presence of PY derivative. Our DLS data correlates with the
SDS-polyacrylamide gel electrophoresis observations.
Dynamic light scattering (DLS) study to investigate amyloid
fibrillation of b-lg
Dynamic light scattering (DLS) studies are frequently employed
to measure the hydrodynamic radii of spherical particles in
solution, and the intensity of scattered light is dominated by
the large particles present in the solution.56 Although the b-lg
aggregates are heterogeneous, either spherical or rod-like
fibrous shapes, DLS data analysis can provide quantitative
estimations of the size distributions in the presence of curcumin
derivatives. Fig. 6A shows that the hydrodynamic radius of native
b-lg ranges from 25 nm to 100 nm (profile a). During thermal
incubation at 75 1C, the monomers of b-lg aggregated in an
oligomeric state and formed a new population of particles,
increasing the particle size by 5-fold (sizes ranging from 25 nm
to 500 nm) (Fig. 6A, profile b). The data were plotted as the
scattered light intensity versus the radius in nm. Notably, the
presence of higher ordered aggregates indicates that the aggre-
gates are formed from b-lg dimers (possibly in equilibrium with
monomers) with involvement of multimers, such as tetramers
and pentamers. From the experimental point of view, when b-lg
was co-incubated with curcumin at 75 1C for 6 h at a molar
concentration ratio of 1 : 1 (Fig. 6A, profile d), the sizes (ranging
from 25 nm to 360 nm) of the aggregates decreased to a greater
extent than at a molar concentration ratio of 1 : 0.5 (sizes ranging
from 25 nm to 475 nm) (Fig. 6A, profile c).
CD spectroscopy to monitor the secondary structural changes
of b-lg
Far-UV CD spectroscopy was exploited to monitor the secondary
structural changes of b-lg in the presence of curcumin deriva-
tives at molar concentration ratios of 1 : 0.5 and 1 : 1 at pH 7.4.
The native b-lg shows a negative signal around 216 nm, which
is characteristic of the b-sheet structure of b-lg, with a very
small minimum at 207 nm, which also indicates some a-helical
structures (Fig. 6B, profile a).57 As shown in Fig. 6B, circular
dichroism (CD) experiments revealed the expected increase in
the random coil/b-sheet conformation transition of b-lg after
incubation at 75 1C for 6 h (Fig. 6B, profile b). This transition of
the conformation is accompanied by a decrease in the a-helical
content of the protein structure, indicating the possibility of
formation of crosslinked b-sheet structures. However, the thermal
exposure of the protein in the presence of curcumin at a molar
concentration ratio of 1 : 0.5 displays a significant decrease in
intensity, with shifts in the band positions similar to those of
the native protein (Fig. 6B, profile c). However, in the presence
of curcumin at a molar concentration ratio of 1 : 1, the peak
positions shifted to 214 nm and 209 nm (Fig. 6B, profile d). The
shifting of the peak positions signifies that a conformational
transition of b-lg from a b-sheet state to a physiological unfolded
random coil state occurs in the presence of curcumin. In contrast,
during thermal co-incubation of b-lg with DAC at a molar
concentration ratio of 1 : 1, minima at 209 and 219 nm were
observed (Fig. 6B, profile f). On the other hand, during thermal
co-incubation of b-lg with IOC derivative at a molar concentration
ratio of 1 : 1, the b-sheet to random coil conformation transition
became dominant, indicating a loss of the gross b-sheet structure
(Fig. 6B, profile h). In particular, in the presence of PY derivative at
a 1 : 1 molar concentration ratio, there was a concomitant increase
in the a-helical structure, as evidenced by increases in ellipticity at
208 nm and 220 nm, respectively. It is of interest to note that at a
molar concentration ratio of 1 : 1, PY showed a more pronounced
helical conformation compared to the other curcumin derivatives
(Fig. 6B, profile j). In this light, it can be assumed that a selective
b-lg intermediate species was stabilized through interactions
with this particular curcumin derivative. This may occur through
Fig. 6 (A) Number particle size distribution spectra in DLS studies of
native b-lg (profile a) and b-lg incubated at 75 1C for 6 h in the absence
(profile b) and presence of curcumin (profile c, profile d), DAC (profile e,
profile f), IOC (profile g, profile h), and PY (profile i, profile j) at molar
concentration ratios of 1 : 0.5 and 1 : 1, respectively in 10 mM phosphate
buffer at pH 7.4 containing 2% ethanol. Each spectrum is an average of
48 scans. The protein concentration was maintained at 54.3 mM. (B) Far
UV-CD spectra of native b-lg (profile a) and b-lg incubated at 75 1C for 6 h
in the absence (profile b) and presence of curcumin (profile c, profile d),
DAC (profile e, profile f), IOC (profile g, profile h), and PY (profile i, profile j)
at 1 : 0.5 and 1 : 1 molar concentration ratios, respectively in 10 mM
phosphate buffer at pH 7.4 containing 2% ethanol. The concentrations
of the b-lg samples in the far UV-CD measurements were 13.6 mM.
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charge–charge interactions between PY derivative and b-lg; indicating that there were no significant structural changes of
these interactions may force the protein to attain a specific b-lg (Fig. 7, profile c). However, in the presence of curcumin at a
conformation, thus altering its aggregation properties rather than molar concentration ratio of 1 : 1, the amide I band shifted to
disrupting the helix to force aggregation of b-lg. To further assess B1639 cmꢁ1, indicating a b-sheet to random coil structural
the curcumin derivative-induced structural transitions, the indi- transition of b-lg (Fig. 7, profile d). However, b-lg acquired a
vidual secondary structural elements were analyzed with CDNN random coil-like structure in the presence of DAC at a molar
2.1 software, and the results are shown in Table S1 (ESI†).
concentration ratio of 1 : 0.5, where the amide-I band was
centered at B1640 cmꢁ1 (Fig. 7, profile e). In addition, in the
presence of DAC at a molar concentration ratio of 1 : 1, the amide
I band shifted to B1643 cmꢁ1, indicating increased accessibility
of random coil structures a with consequent small increase in
the a-helical content of b-lg (Fig. 7, profile f). Specifically, more
random coil structures were obtained when the protein was
incubated with IOC derivative at molar concentration ratios of
1:0.5 and 1:1, respectively (Fig. 7, profile g and profile h).
Interestingly, shifting of the amide-I band to 1653 cmꢁ1, which
corresponds to the preferable a-helical conformation, was
obtained by b-lg when incubated with PY derivative at a molar
concentration ratio of 1 : 1 (Fig. 7, profile j). The FTIR data
adequately support the results from the CD measurements.
This led us to speculate that more inhibitory ability is exerted
by PY derivative with respect to the aforementioned curcumin
derivatives.
FTIR study to assess secondary structural changes during b-lg
fibrillation in the presence of curcumin derivatives
Fourier transform IR (FTIR) spectroscopy is one of the most
powerful spectroscopic tools for determining the secondary
structures of proteins in solution and has been utilized as an
independent technique for decades to confirm or refute CD
measurements.37 The main advantage of this approach in
comparison with CD is that FTIR is much more sensitive to
b-structures.37 Information about the secondary structures
of proteins can be obtained from the study of the amide I
(1500–1700 cmꢁ1) band in the ATR-FTIR spectra. Fig. 7 shows
the FTIR spectra of b-lg samples (native and thermally incu-
bated in the absence and presence of curcumin derivatives) in
D2O-buffer. As a b-sheet rich protein, b-lg has a characteristic
peak at around 1634 cmꢁ1 in its amide I contour, which
corresponds to intramolecular b-sheet structures.58 At elevated
temperature, the peak shifts from 1634 cmꢁ1 to 1624 cmꢁ1
(Fig. 7, profile b). As a blue sheet rich protein, b-lg has a
characteristic peak at around 1634 cmꢁ1 in its amide I contour,
which corresponds to intramolecular shifting of the b-sheet
band in the protein FTIR spectra, consistent with the presence
of intermolecular (rather than intramolecular) b-sheet struc-
tures, and is the hallmark of amyloid-like fibril formation.37
In the presence of curcumin at a molar concentration ratio of
The effects of curcumin derivatives on fibril morphology
To gain more insight, atomic force microscopy was also
employed; it has been proved to be a powerful tool in the study
of fibril formation.59 AFM analysis was performed to visualize
the extent of disruption of b-lg samples aggregated alone or
with curcumin derivatives at molar concentration ratios of 1 : 1.
It has been proposed that only short and curly aggregates
are formed at high pH (higher than the isoelectric point, e.g.,
pH 4 7).26 The AFM image of fibrils (Fig. 8, image b) grown
at pH 7.4 and 75 1C is different from those at lower protein
concentrations. With higher concentrations, more numerous
and mature fibrils can be obtained; therefore, in our studies, the
concentration of b-lg was maintained at 543 mM. To investigate
the formation of b-lg fibrils, time-dependent transformation into
fibrils was monitored by incubating b-lg samples at 75 1C for 6 h
and 24 h. During incubation for 6 h at 75 1C, the monomers of
b-lg swelled and deformed, forming aggregates. At this stage, the
aggregates consisted of thicker filaments, and a protofibril-like
appearance was observed (Fig. 8, image a). These protofibrils were
assumed to be precursors for the formation of longer fibrils. Upon
prolonged incubation at 24 h, subunits of well-developed fibrils
are formed. However, filaments were no longer observed, and
elongated fibrillar aggregates with apparent diameters of 140
to 150 nm formed (Fig. 8, image c). When a b-lg sample was
co-incubated at 75 1C for 24 h in the presence of curcumin at a
molar concentration ratio of 1 : 1, the number and sizes of the
fibrils (diameter B120 to 130 nm) decreased on the mica surface,
accompanied by the formation of smaller globular particles
with fibrillar morphologies (Fig. 8, image e). Morphologically
fragmented worm-like fibrils with diameters B100 to 110 nm
formed when a b-lg sample was co-incubated in the presence of
DAC (Fig. 8, image g). Furthermore, the matured fibrils were split
1 : 0.5, Fig. 7 shows a shifting of the amide I band to B1636 cmꢁ1
,
Fig. 7 FTIR spectra of native b-lg (profile a) and b-lg incubated at 75 1C for
6 h in the absence (profile b) and presence of curcumin (profile c, profile d),
DAC (profile e, profile f), IOC (profile g, profile h), PY (profile i, profile j) at
molar concentration ratios of 1 : 0.5 and 1 : 1, respectively in 10 mM
phosphate buffer at pH 7.4 containing 2% ethanol. Protein concentrations
were 1087 mM. FTIR spectra were taken in the amide-I region of 1500 to
1700 cmꢁ1. Each spectrum is an average of 32 scans in D2O solvent
at 25 1C.
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Fig. 8 AFM images representing the aggregate morphologies of b-lg in the presence of curcumin derivatives. b-lg (543 mM) was co-incubated with
equimolar concentrations of curcumin, DAC, IOC and PY for 24 h. Representative AFM images of b-lg aggregates (a) alone for 6 h [(b) zoomed-in image
of (a)] and (c) for 24 h [(d) zoomed-in image of (c)] and co-incubated with (e) curcumin [(f) zoomed-in image of (e)], (g) DAC [(h) zoomed in-image of (g)],
(i) IOC [(j) zoomed-in image of (i)] and (k) PY [(l) zoomed-in image of (k)] for 24 h.
into smaller fragments, and the extent of fibril formation was
diminished in the presence of DAC. However, in the case of IOC,
higher order oligomeric structures were incorporated with small
oligomers (Fig. 8, image i). Interestingly, PY promoted complete
disruption of b-lg fibrils into smaller oligomers with unique
morphologies (Fig. 8, image k).
Docking study
A molecular docking study was performed to obtain insight into
the types of interactions involved between b-lg and curcumin-
stacking and p-interactions with Trp19 and Tyr20, Tyr20 and
Glu44 and Glu158, respectively (Fig. 9, image c). PY binds
derivatives, which is responsible for the inhibition of aggregation.
When the 1,3-dicarbonyl moiety was retained in DAC and was
substituted by isoxazole and pyrazole, alterations in the inhibitory
effects of curcumin were observed; DAC exhibited fewer inhibitory
effects than curcumin, and PY showed superior inhibitory effects
compared to IOC and DAC. The anti-amyloidogenic activity
of curcumin can be attributed to its phenolic and polycyclic
structure, which results in the formation of one hydrogen bond
Fig. 9 The structures of the most stable (a) b-lg-curcumin, (b) b-lg-DAC,
(c) b-lg-IOC and (d) b-lg-PY complexes obtained by molecular docking,
and possible interactions contributing to the binding (PDB ID of b-lg:
1BSY).
of the carbonyl of the b-diketone moiety of curcumin with b-lg potential. The compound IOC prefers surface binding through
accompanied by weak forces (Fig. 9, image a). The molecular H-bonding, p-stacking and p-interactions with Trp19 and Tyr20
,
docking results indicated that weak electrostatic interactions are Tyr20, and Glu44 and Glu158, respectively (Fig. 9, image c).
favored by DAC (Fig. 9, image b). However, substitution in the Meanwhile, PY binds inside the calyx and interacts with the
b-diketone moiety of curcumin increased the anti-aggregation amino acids Val43, Ile56, Ile84, Asn90, Val92, Met107, Phe105 and
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concentrations of protein samples were prepared by dissolving
b-lg samples in Milli-Q water and then measuring the O.D. at
280 nm.
Preparation of solutions of curcumin derivatives
The concentrations of curcumin and the IOC, PY and DAC
derivatives were determined spectroscopically. The solutions of
the derivatives were obtained by dissolving them in absolute
ethanol to give concentrations of 1 ꢂ 10ꢁ3 M.
Thermal aggregation assay of b-lg
Bovine b-lg readily undergoes aggregation upon thermal expo-
sure at 75 1C when 163 mM (3 mg mLꢁ1) of b-lg is used.39,40
Thus, all the experiments were carried out upon thermal
co-incubation of b-lg at 75 1C in the presence of curcumin
and its derivatives (IOC, PY and DAC) at molar concentration
ratios of 1 : 0.5 and 1 : 1 for 6 h. Each sample was freshly
prepared and filtered through Sartorius filter paper (pore size
0.20 mm).
Fig. 10 Schematic representation of this work.
Asn109 through p-interactions, p-stacking and H-bonding,
respectively (Fig. 9, image d). The main interaction forces
between b-lg and PY are hydrophobic interactions and aromatic
stacking; these contribute to the binding of this compound to
the intermediate structure of b-lg, stabilizing it and finally
suppressing amyloid formation. Moreover, it was demonstrated
that aliphatic residues such as alanine and valine present in the
central region of protein facilitated the binding of more than
one derivative to the protein, preventing interactions between
monomers and retarding the overall aggregation process.18
Thus, amino acids with aliphatic side chains in b-lg can also serve
as binding sites for the aforementioned curcumin derivatives;
this may be a mechanism by which these compounds decrease
aggregation of b-lg (Fig. 10).
Methods
Electrophoresis measurements
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed under non-reducing conditions
using 12% acrylamide resolving gel according to Laemmli’s
method.41 Samples of b-lg solution (163 mM) in 10 mM sodium
phosphate buffer, pH 7.4, were incubated at 75 1C for 6 h in the
absence and presence of curcumin and its derivatives (IOC, PY
and DAC) separately at molar concentration ratios of 1 : 0.5 and
1 : 1. The resulting solutions were filtered with a syringe filter
with a 0.2 mM membrane filter. The sample solutions were
mixed with equal amounts of SDS-gel loading solution and
boiled at 95 1C to 100 1C before the SDS-PAGE analysis. Aliquots
(20 mL) of thermally incubated b-lg solution in the presence and
absence of curcumin derivatives were loaded in the wells.
Similarly, 20 mL of native b-lg solution (108 mM) were loaded
in another well. Electrophoretic separations were performed by
applying a maximum voltage of 100 volts for 1 h. The gel was
stained with Coomassie Brilliant Blue R-250 and destained by
the usual method using a solution containing methanol and
acetic acid.
Experimental
Reagents and chemicals required
Bovine b-lg was isolated and purified from cow milk as
described earlier.36 The final product was lyophilized and
stored at 4 1C. Sodium dihydrogen phosphate was purchased
from Merck (Mumbai, India). Curcumin and different fluores-
cent probes, viz., 8-anilinonaphthalene-1-sulfonic acid ammo-
nium salt (ANS), Congo red (CR) and Thioflavin T (ThT), were
obtained from Sigma Chemical Co. (St. Louis, USA) and used
as received without further purification. IOC and PY were
synthesized according to previously described methods.37
DAC was synthesized according to a previously described
Intrinsic fluorescence
1
method38 and further characterized by H-NMR spectroscopy,
Intrinsic fluorescence measurements of all the above-described
protein solutions were performed in Na-phosphate buffer,
pH 7.4, containing 2% ethanol on a Shimadzu spectrofluori-
meter (Shimadzu 5301 PC). The temperature was maintained at
25 1C. The fluorescence spectra were measured using a 1 cm
path-length rectangular quartz cell while maintaining a b-lg
13C-NMR spectroscopy and ESI-MS (all spectral data are given in
SI3–SI5, ESI†). The other chemicals used were of the highest
purity available.
Preparation of b-lg solution
For spectroscopic sample preparation, b-lg was weighed and concentration of 13.6 mM. To monitor the intrinsic fluorescence
dissolved in 0.01 M pH 7.4 phosphate buffer solution containing of b-lg, the samples were excited at 295 nm and the spectra were
2% ethanol. Protein stock solutions were prepared using recorded between 310 nm and 400 nm. The excitation and
phosphate buffer at pH 7.4. Because the extinction coefficient emission slits were set at 3 nm and 5 nm, respectively. All the
of b-lg (0.96 mgꢁ1 mLꢁ1 cmꢁ1 at 280 nm) is known, different experiments were performed at least three times.
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ANS fluorescence study
phosphate buffer at pH 7.4 containing 2% ethanol in a Shimadzu
spectrofluorimeter (Shimadzu 5301 PC). The fluorescence spectra
were collected at 25 1C using a 1 cm path length cell and a protein
concentration of 13.6 mM. The excitation and emission slits were
set at 3 nm.
Exposure of hydrophobic patches in the protein during the
aggregation process was monitored using the polarity-sensitive
fluorescent probe 1-anilinonapthalene-8-sulfonate (ANS).42
A stock solution of ANS was added to each aliquot of b-lg
solution (both in the absence and presence of curcumin and
curcumin derivatives) so that the final ANS concentration in
Congo red assay
each aliquot was 30 mM. Typically, the ANS concentration was The formation of aggregates by thermally exposed b-lg in the
50 molar in excess of the protein concentration. The ANS- absence and presence of curcumin and curcumin derivatives
fluorescence intensities were measured using a Shimadzu was investigated by measuring the shifts in the absorbance of
RF-5301 PC instrument with excitation at 370 nm and by Congo red in the region of 400 to 700 nm. Congo red stock
scanning the emission wavelength from 400 nm to 650 nm.43 solution (10 mM) was prepared by dissolving the dye in 10 mM
Slit widths were set at 3 nm for excitation and 5 nm for sodium phosphate buffer (pH 7.0) containing 150 mM NaCl
emission, respectively. Each spectrum was blank corrected. under continuous stirring. The solution was then filtered with a
Data points were the average of triplicate measurements.
0.2 mm Millipore filter. A fresh working solution was prepared
by diluting the stock solution 100 times.45 For this study,
250 mL (27.2 mM) aliquots of the protein solutions (containing
curcumin and curcumin derivatives) were withdrawn and
mixed with 250 mL of a solution containing 40 mM Congo red
solution. The final volume (2 mL) was adjusted with 10 mM
sodium phosphate buffer, pH 7.4, containing 2% ethanol.
Thioflavin T (ThT) binding assay
ThT is a dye which shows enhanced fluorescence at around
485 nm when bound to amyloid fibrils.44 Thus, to investigate
and compare the aggregates formed by thermally incubated
b-lg in the absence and presence of curcumin and curcumin
derivatives, the following assays were employed. A 5 mM stock
solution of ThT was prepared in 20 mM sodium phosphate
buffer, pH 7.5, and filtered through a 0.2 mm polyether sulfone
filter. A solution containing 50 mL of 163 mM b-lg sample
[incubated in the absence and presence of curcumin derivatives
for 6 h at molar concentration ratios of 1 : 1] was diluted in
950 mL of 50 mM ThT (dissolved in 0.01 M pH 7.4 phosphate
buffer solution containing 2% ethanol) and incubated for
30 min at 25 1C in the dark. The resulting ThT fluorescence
intensities of the samples were measured at each time point
at an emission wavelength of 485 nm using an excitation
wavelength of 440 nm. The percent change in fluorescence
intensity (Fp) was calculated using eqn (1):
Analysis of secondary structures by CD spectroscopy
To trace the structural or conformational changes that curcumin
and curcumin derivatives impose on b-lg, circular dichroism
measurements (CD) were carried out on a Jasco spectropolari-
meter (J-815) at 20 1C in the far-UV region (200 to 260 nm) using
rectangular cells with 2 mm path lengths. Thermally incubated
b-lg solutions (in the presence and absence of curcumin and
curcumin derivatives) with concentrations of 13.6 mM were used
for the far-UV CD measurements. All the spectra are the average of
three scans. The final spectrum was obtained after subtraction of
the corresponding solvent spectrum. The far UV-CD curves were
fitted with the curve-fitting software program CDNN 2.1 to
determine the percentages of secondary structures present in
b-lg under different conditions.
ꢀ
ꢁ
Ft ꢁ F0
Fp ¼
ꢂ 100
(1)
F0
Dynamic light scattering (DLS) measurements
where Ft is the fluorescence at a specific time point and F0 is the
fluorescence at the starting time of incubation. All experiments
were performed in triplicate. Percent inhibition was calculated
using eqn (2):
The diffusion of particulates in solution induces fluctuations
in the intensity of the scattered light. DLS detects these
fluctuations using an auto-correlator on a microsecond time
scale and is used to analyze the distribution of molecules
and supramolecular aggregates because it is very sensitive to
particle size.46 Different sizes of molecules in the solution can
be observed at different peaks if their sizes vary sufficiently.
In our experiments, DLS measurements were performed with
thermally incubated b-lg in the presence and absence of
curcumin and curcumin derivatives employing a Zetasizer
Nanos (Malvern Instrument, U.K.) equipped with a 633 nm
laser and using a 2 mL rectangular cuvette (path length
10 mm). Measurements were performed at 20 1C with 250 mL
Fx = 100 ꢁ (100Fs C Fc)
(2)
where Fx is the percent inhibition, Fs is the percent increase in
fluorescence intensity in the presence of the compounds and
Fc is the percent increase in the fluorescence intensity of b-lg
alone. The slit widths for both the excitation and emission were
maintained at 5 nm. Three replicates were performed, and the
data were averaged.
Rayleigh light scattering
The effects of curcumin and curcumin derivatives on the aggrega- of coincubated b-lg sample in 1.75 mL Na-phosphate buffer.
tion of thermally incubated b-lg were monitored by Rayleigh light Then, the solutions were maintained for 30 minutes. Similar
scattering (RLS) measurements by observing the emission at measurements were performed separately with the incubated
350 nm after exciting the solution at 350 nm using 10 mM b-lg in the absence of curcumin and curcumin derivatives.
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The time-dependent auto correlation functions were acquired and imaging tools, including SDS-PAGE, ThT fluorescence,
with twelve acquisitions for each run.
ANS binding, CD measurements, DLS, RLS, FTIR and AFM.
In summary, these four curcuminoids can prevent aggregation
of the protein and its toxic intermediates via different moieties
of the curcuminoid framework, such as the b-diketone moiety,
phenolic OH groups, acetyl groups, hepta-diene moiety, sub-
stitutions on the benzene nucleus and substitutions on the
b-diketone moiety. Alternatively, it can be suggested that
curcumin analogs also directly bind to fibrillar assemblies in
a similar fashion, but more efficiently than curcumin, and
disrupt preformed fibrils. Overall, our data revealed the efficacy
of the compounds in the order of PY 4 IOC 4 DAC 4 curcumin.
The adoption of a helical intermediate and the formation of
nonfibrillar aggregates in the presence of PY may be a feature
to exploit for structural investigations in molecular docking
studies. The hypothesis of protein association into dimers aided
by the PY derivative was further supported by SDS-PAGE studies
and DLS measurements of protein solutions. Simultaneously,
these measurements excluded the possibility of formation of
higher rank linear aggregates. Hydrophobic interactions and
hydrogen bonding between b-lg and PY are key forces implicated
to play dominant roles in the inhibited activity. Curcumin pyrazole
and its derivative N-(3-nitrophenylpyrazole) exhibited remarkable
potency in arresting fibrillization and disrupting preformed
fibrils of a-synuclein.18 The future projections of this study are
to investigate the modulatory role of these molecules in drug
formulations against disease progression in systemic amyloidosis
and Alzheimer’s disease.
Monitoring the secondary structural changes of b-lg during
thermal incubation by FTIR spectroscopy
For the FTIR measurements, native and incubated b-lg solutions
(50 mL) (in the presence and absence of curcumin and curcumin
derivatives) with concentrations of 20 mg mLꢁ1 were taken in a
Microcon filter device and diluted with 200 mL of D2O. The
mixture was then quickly centrifuged at 4000 ꢂ g for 8 min until
the volume reached B50 mL. After that, 200 mL of D2O was
added again, and the mixture was centrifuged for another 8 to
10 minutes. This process of D2O exchange was repeated 3 to
4 times.47 Finally, the D2O-exchanged b-lg samples were placed
between two CaF2 windows separated by a 50 mM thick Teflon
spacer. FTIR scans were collected in the range of 1500 to
1700 cmꢁ1 at a resolution of 2 cmꢁ1 in a N2 environment using
a Spectrum 100 FT-IR spectrometer (Perkin Elmer). The spectrum
of D2O at pD 7.5 was collected and subtracted from the sample
spectrum.
Atomic force microscopy (AFM)
AFM was used to monitor the effects of curcumin and curcumin
derivatives on b-lg fibril formation. For AFM analysis, the
protein samples were diluted to 0.2 mg mLꢁ1 in buffer, and
10 mL of each sample was placed on a piece of mica (1 cm ꢂ 1 cm
pieces). The AFM samples were dried at room temperature and
then used to acquire the AFM images. The AFM images were
recorded using a VEECO DICP II autoprobe (Model AP 0100)
instrument.
Conflicts of interest
Molecular docking study of b-lg-curcumin derivative interactions
The authors declare no competing financial interest.
The molecular docking study was performed using Autodock
4.2 and Autodock Tools (ADT) using a Lamarckian genetic
algorithm. The crystal structure of b-lg (PDB ID: 1BSY) was
obtained from the RCSB Protein Data Bank. The structures of
curcumin, DAC, IOC and PY were optimized to lower energies
using density functional theory (DFT) at the B3LYP/6-31G level
of theory. These optimized structures were used for docking
with b-lg to predict the binding sites and modes of binding. For
the docking study, ligands present in the protein (1BSY) and
water molecules were removed from the protein, and atom
Kollman charges were assigned after the addition of polar
hydrogen to the protein. The protein was set to be rigid, and
there was no consideration of solvent molecules on docking.
Discovery Studio 4.1 Client and Chimera 1.10.1rc were used for
visualization effects and to identify the residues involved in
binding.
Acknowledgements
Financial support of University Grant Commission UGC-CAS-II
and DST-PURSE-II (Govt of India) Program of Department of
Chemistry, Jadavpur University, Kolkata are greatly acknowledged.
The authors wish to acknowledge Prof. S. C. Bhattacharya, Depart-
ment of Chemistry, Jadavpur University for providing the DLS
instrumental facility. Sanhita Maity, SRF, is a recipient of UGC-
NET Research Fellowship in Chemistry. AP acknowledges DST for
funding through the INSPIRE program.
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