Multispectroscopic analysis and molecular modeling to investigate the binding of beta lactoglobulin with curcumin derivatives

Article abstract of DOI:10.1039/C6RA24275H

Bovine beta lactoglobulin (β-lg), the major whey protein, has a great affinity for a wide range of organic compounds like fatty acids, retinol etc. Curcumin, a polyphenolic antioxidant present in turmeric and its isoxazole (IOC) and pyrazole (PY) derivati

Full text of DOI:10.1039/C6RA24275H

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Multispectroscopic analysis and molecular
modeling to investigate the binding of beta
Cite this: RSC Adv., 2016, 6, 112175
lactoglobulin with curcumin derivatives†
Sanhita Maity, Sampa Pal, Subrata Sardar, Nayim Sepay, Hasan Parvej,
*
Jishnu Chakraborty and Umesh Chandra Halder
Bovine beta lactoglobulin (b-lg), the major whey protein, has a great affinity for a wide range of organic
compounds like fatty acids, retinol etc. Curcumin, a polyphenolic antioxidant present in turmeric and its
isoxazole (IOC) and pyrazole (PY) derivatives have been elicited worldwide for their therapeutic activities.
However, the nature of interaction of b-lg with these derivatives remains unexplored. Fluorescence
quenching studies suggest a static quenching mechanism for both the compounds. The average
distances of 7.28 nm and 7.33 nm have been determined for IOC and PY respectively for energy transfer
based on FRET which have application in many biological and biophysical fields. Circular dichroism
spectra (CD) and Fourier transform infrared spectroscopy (FTIR) have been utilized to analyze the
influence on the secondary structure of the protein. Docking simulation reveals a possible mechanism
for different quenching behaviours and modes of binding preferred by the two compounds. Our findings
will be helpful in the design of the drugs and other biologically active molecules that bind more strongly
to b-lg and have the ability to show FRET.
Received 29th September 2016
Accepted 17th November 2016
DOI: 10.1039/c6ra24275h
www.rsc.org/advances
maintain the binding to the sub domain of PKCs, protein kinase
c6.15 Chakraborti et al. showed that both these derivatives were
1 Introduction
equally competent of binding to tubulin and resisting the
tubulin self-assembly formation.16 The proposal for using these
derivatives as an anti-cancer drug also had been under consid-
eration due to their enhanced stability and free radical scav-
enging property.17 Nonetheless, pyrazole (PY) & isoxazole (IOC)
derivatives are used as a potent ligand of brillar Ab-42 aggre-
gates.18 Various attempts have been made to increase the
bioavailability and solubility of curcumin and its analogs either
by nano capping or by nano encapsulation to various biological
macromolecules.19 Bovine b-lg, a protein with hydrophobic core
which consist with eight antiparallel b-strands called b-barrel or
calyx, is one of the mostly used accepted carrier protein for
hydrophobic ligands, having pH dependant opening, encapsu-
lating property and a unique acidic pH resistivity.20 In this way,
bovine beta lactoglobulin (b-lg) performs the well-controlled
drug delivery mechanism indeed.21 This encourages us to
choose b-lg as a model carrier protein. Moreover, R. Narlawar
et al. reported that curcumin derived isoxazoles and pyrazoles
inhibit or modulate APP metabolism by interfering with g-sec-
retase activity.18
Curcumin, a yellow lipid-soluble phenolic b-diketone dietary
spice of turmeric, is found in the powdered root of Curcuma
longa and is well known as a food coloring agent.1 The phar-
macological attributes of curcumin, such as antimalarial,2
antioxidative,3 anti-inammatory,4 anti-angiogenic,5 anti-
amyloid,6 anti-cancer,7 anti-microbial,8 wound healing,9 and
protective activities have been undergoing thorough research
during last few decades due to its nontoxicity and bio-compat-
ibility.10 This non-steroidal phytochemical has a medicinal
value for liver diseases (jaundice), indigestion, urinary track
diseases, rheumatoid arthritis & insect bites.11 The b-diketone
moiety of curcumin is responsible for keto–enol tautomeric
behaviour, exerted enolic –OH (hydroxyl) group, and its insta-
bility in vitro as well as in vivo.12 Anand et al. reported that the
presence of b diketone moiety may play the crucial role in bio-
activities but recent studies also show that the bioactivity of
curcumin derivatives without b-diketones also revolved.13
The pyrazole derivatives of curcumin has been deployed to
study lipoxygenase inhibitory activity,14 endothermal cell prolif-
eration and cytotoxicity.12 Pyrazole and isoxazole derivatives also
In order to go to mechanistic insight, our present work aims
to study the interaction between these derivatives and the
model protein b-lg utilizing different spectroscopic techniques
(Scheme 1). These synthesized IOC and PY derivatives (shown in
Scheme 1) exhibit excellent efficiency of energy transfer (FRET)
from the Trp moiety (W19) of b-lg in bound condition which will
Organic Chemistry Section, Department of Chemistry, Jadavpur University, Kolkata
700032, India. E-mail: uhalder2002@yahoo.com; Fax: +91 33 2413 7902; Tel: +91
33 2414 6223
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra24275h
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Fig. 1 UV-visible spectral changes of IOC (20 mM) (A) and PY (20 mM)
(B) with the gradual addition of b-lg (1–20 mM) and (1–17 mM)
respectively. Double reciprocal plot for 20 mM IOC and 1–20 mM b-lg
[(A) inset] and double reciprocal plot for 20 mM PY and 1–17 mM b-lg [(B)
inset] as per eqn (1). Relative errors have been expressed along with the
corresponding error bars of respective data points (ꢂ0.02).
Scheme 1 The synthesis of IOC and PY derivatives and their respective
interaction with b-lg.
that particular wavelength. The intensity of the bands at 335 nm
and 325 nm are indicative of a greater hydrophobic/nonpolar
environment.23 Addition of b-lg enhances the polarity due to
its charged nature, which also may be the reason for the
decreased intensity at 335 and 325 nm. When IOC (20 mM) was
used, a sharp peak at 335 nm, a shoulder at 291 nm, and a weak
absorption band at 285 nm are visible in the absorption spec-
trum (for IOC) and when of PY (20 mM) was used, a sharp peak at
325 nm, a shoulder at 298 nm, and a weak absorption band at
285 nm are visible in the absorption spectrum.
The intensity of the absorption maxima of IOC at 335 nm
decreases, and the absorption peak at 285 nm increases,
simultaneously with increasing protein concentration 1 mM to
20 mM (Fig. 1(A)). In case of PY, same type of change of
absorption maxima was observed at 325 nm and 285 nm
(Fig. 1(B)) with increasing protein concentration 1 mM to 15 mM.
The association constant (Ka) can be calculated from the
following equation
be a benecial and staple technique for structural elucidation of
biological molecules and their interactions in vitro assays, in
vivo monitoring in cellular research, nucleic acid analysis,
signal transduction, light harvesting and metallic nano-
material.22 The conformational aspects of b-lg were also inves-
tigated through CD and FTIR studies. The modes of binding
and encapsulation were also investigated through molecular
docking method.
2 Results and discussion
2.1. UV-visible spectroscopy
Curcumin consists of a 1,3-diketone moiety that enables an
extended conjugation of the p electron system between the two
feruloyl groups.23 The extended conjugation between two fer-
uloyl moieties was disrupted due to lack of planarity by incor-
poration of isoxazole and pyrazole ring and the absorption
maxima of these derivatives shied at 335 and 325 nm respec-
tively.24 In these cases the lmax also shis to the near UV region.
Fig. 1(A) and (B) show the absorption spectra of IOC and PY
derivatives in the absence and presence of varying concentra-
tions of b-lg. The absorption spectrum of IOC and PY derivative
show a prominent peak at 335 nm and 325 nm respectively and
a weak shoulder at 291 nm for IOC and 298 nm for PY derivative
respectively. These peaks appear due to n to p* and p to p*
transitions in the IOC and PY derivatives of curcumin.
1/DA ¼ 1/(3b ꢀ 3f)LT + 1/(3b ꢀ 3f)LTKaM
(1)
where 3 is the molar extinction coefficient and the subscripts b,
f, and T refers to the bound, free, and total ligand concentra-
tion; L refers to the ligands (IOC, PY) and M refers to the
macromolecule (b-lg, 1 to 20 mM or 1 to 15 mM). The equilibrium
constant can be calculated by measuring the absorbance at
a xed wavelength. A double-reciprocal plot is obtained by
plotting 1/(A0 ꢀ A) against 1/[protein] [Fig. 1(A) inset and
Fig. 1(B) inset]. The ratio of intercept and the slope gives the Ka
value, which is found to be 1.83 ꢁ 105 Mꢀ1 for b-lg–IOC and 2.83
ꢁ 104 Mꢀ1 for b-lg–PY (Table S1) (ESI†). These values are well in
agreement with the Ka values reported for strong interactions.25
The forbidden n to p* transition occurs in the high-energy
ultraviolet region of the spectrum (291 nm for IOC and
298 nm for PY), while the p to p* transitions occur in the lower
energy end of the spectrum (335 nm for IOC and 325 nm for PY
2.2. Fluorescence measurements
derivative). The intensity of the absorption bands at 335 and Fluorescence measurement is
a convenient approach to
291 nm for IOC and at 325 and 298 nm for PY gradually understand the interaction between ligands and proteins
decreases on successive addition of protein, and the isosbestic because the uorophore is responsive to the polarity of its
points were observed at 303 nm for IOC and at 304 nm for PY. surrounding environment.26 In general, uorescence intensity
The formation of isosbestic point conrms the existence of enhances as this polarity reduces.
interactions between IOC and PY derivative and b-lg and both
Fluorescence quenching measurement of protein has been
the interacting species exist in equilibrium with one another at widely used to explore the binding properties of small molecule
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with protein in solution. At excitation wavelength of 280 nm, lifetime of the uorophore (10ꢀ8 s), for different concentrations
both tryptophan (Trp) and tyrosine (Tyr) residues show uo- of the quencher, which remains unchanged during static
rescence emission, but at 295 nm, only the Trp residue show quenching (interaction takes place only in the ground state).29
uorescence emission.26 Bovine b-lg contains two Trp residues
in positions 19 and 61. Trp19 is located at the bottom of calyx,
a strong hydrophobic region, while Trp61 is situated near the
aperture of the barrel which compels this residue exposed to the
hydrophilic environment.27 On the other hand; Trp61 has close
proximity of Cys66–Cys160 disulphide bridge which is a strong
quencher of the uorescence. So the uorescence of b-lg at
For dynamic quenching the following relation can be used
Ksv ¼ kqs0
(3)
where Ksv is Stern–Volmer quenching constant, kq is the bimo-
lecular quenching constant and s0 lifetime of the uorophore
(10ꢀ8 s).
The values of kq of IOC and PY derivatives are 1.4 ꢁ 1013 Mꢀ1
Sꢀ1 and 1.3 ꢁ 1013 Mꢀ1 Sꢀ1 respectively (Table S1) (ESI†) and
their kq values are much higher than the maximum dynamic
excitation wavelength of 295 nm is solely contributed by Trp19
.
Fig. 2(A) and (B) represents the change of uorescence inten-
sities of b-lg in the presence of IOC and PY derivatives with an
excitation wavelength at 295 nm. The steady-state uorescence
quenching patterns can be explained with the Stern–Volmer
equation
quenching constant (2.0
ꢁ
1010 Mꢀ1 Sꢀ1
) in aqueous
medium.26,29 Thus the result clearly shows that quenching is not
due to collision factors but rather as a result of association
between the uorophore and the quencher in the ground state.
So, in our case, mechanism of quenching is static in nature.
For the static quenching, the binding constant (Ks) and the
F0/F ¼ 1 + Ksv[Q] ¼ 1 + kqs0[Q]
(2)
where F and F0 corresponds to the uorescence intensity of b-lg binding sites (n) can be calculated employing the following
in the presence and absence of two ligands respectively, [Q] is equation
the concentration of the ligands (quencher) and Ksv represents
log[(F0 ꢀ F)/F] ¼ log Ks + n log[Q]
(4)
the Stern–Volmer quenching constant. s0 is the lifetime of the
uorophore without quencher (s0 ¼ 10ꢀ8 s).28 kq is the bimo-
lecular quenching rate constant.
where F0, F and [Q] are the same as the parameters involved in
Stern–Volmer equation. The linear plot of log[(F0 ꢀ F)/F] as
a function of log[IOC] and log[PY] are shown in Fig. 3(A) and (C).
From the slope and intercept of the plot, the values of n
(number of binding sites) and Ks can obtained, which were
shown in the Table S1.† The values of n are about 1 for both the
IOC and PY derivatives. Thus it can be assumed that IOC–b-lg
and PY–b-lg complexes were formed by the combination of one
molecule of protein with one molecule of IOC and PY derivative
separately.
The plot of F0/F against the concentration of IOC and PY
derivative [Fig. 2(A) and (B) inset] predicts to nd the Stern–
Volmer quenching constant Ksv, which were found to be 1.4 ꢁ
105 Mꢀ1 for IOC and 1.3 ꢁ 105 Mꢀ1 for PY (Table S1) (ESI†).
Fluorescence quenching can be either static or dynamic. Static
quenching arises when there is a complex formation between
the uorophore and the quencher in the ground state. The
numbers of the uorophore locating in the excited state thus
were diminished. Hence, the uorescence intensity is reduced
in the presence of quencher. Dynamic or collision quenching
occurs when the interaction between the uorophore and the
quencher appears in the excited state. The fundamental
difference between the static and dynamic quenching lies in the
Fig. 2 (A) Fluorescence emission spectrum of b-lg (10 mM) in the
absence and presence of IOC (1–21 mM). (B) Fluorescence emission
spectrum of b-lg (10 mM) in the absence and presence of PY (1–15 mM).
Inset: (A) plot of F0/F versus [IOC] and (B) plot of F0/F versus [PY] as per
Stern–Volmer eqn (2). Excitations were done at 295 nm and emissions Fig. 3 (A) Plots of log[(F0 ꢀ F)/F] vs. log[IOC] and (C) plots of log[(F0
ꢀ
were observed at 329 nm for both IOC and PY derivatives. Slit width F)/F] vs. log[PY] as per eqn (4). (B) Plots of F0/(F0 ꢀ F) vs. [IOC]ꢀ1 ꢁ 106
was 3 for excitation and 5 for emission for both the compounds. Mꢀ1 and (D) plots of F0/(F0 ꢀ F) vs. [PY]ꢀ1 ꢁ 106 Mꢀ1 as per modified
Relative errors have been expressed along with the corresponding Stern–Volmer eqn (5). Relative errors have been expressed along with
error bars of respective data points (ꢂ0.02).
the corresponding error bars of respective data points (ꢂ0.02).
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In addition, analogous results were observed by some
workers studying interactions of other phenolic compound with
b-lg or other proteins. For the interaction of b-lg with resveratrol
the values of kq and n were 3.6 ꢁ 1013 Mꢀ1 Sꢀ1 and 1.2.26 It also
demonstrated that one molecule of b-lg combined with one
molecule of resveratrol to form 1 : 1 complex and the mecha-
nism of quenching is static.
The quenching constant Ka and the number of accessible
uorophore can be quantitatively determined following
a modied Stern–Volmer equation,
2.3. Fluorescence resonance energy transfer (FRET)
FRET is an electrodynamic phenomenon that happens between
a donor molecule in the excited state and an acceptor molecule
in the ground state.30 FRET study was considered to be an
important parameter for study of energy transfer efficiency in
between the donor and acceptor moiety, here b-lg acts as
a donor and derivatives act as acceptors accordingly. The
distance between ligands and Trp moieties in protein can be
achieved by this method. In both the cases the FRET pattern
shows the concomitant decrease in uorescence intensity of Trp
(W19) in b-lg with gradual addition of IOC and PY derivatives
accompanied by an increase in emission of extrinsic uo-
rophore ligands at their characteristic emission region, which
signies the efficient transfer of excited electronic energy from
the donor site to the acceptor site [Fig. 2(A) and (B)]. According
F0/(F0 ꢀ F) ¼ 1/faKaQ + 1/fa
(5)
where F0 and F denote the uorescence intensities in the
absence and presence of two ligands respectively. Ka and fa
denote the quenching constant and the fraction of accessible
uorophore respectively. From the modied Stern–Volmer
equation, plots of F0/F0 ꢀ F against 1/[ligand] were shown for
IOC and PY [Fig. 3(B) and (D)]. The quenching constant Ka was
calculated from the ratio of the intercept to the slope, and the
fraction of the accessible uorophore (fa) was calculated from
the inverse of the intercept, which were found to be 1.04 ꢁ 105
Mꢀ1 and 1.203 for IOC and 6.83 ꢁ 104 Mꢀ1 and 1.39 for PY. The
linearity of the plot depicts that both the ligands bind with b-lg
to form 1 : 1 complexes.27
¨
to the Forster's theory, the efficiency of the energy transfer, E, is
established with the help of the following equations
E ¼ 1 ꢀ F/F0 ¼ R0 /(R0 + r6)
(6)
6
6
where, F0 and F are the uorescence intensities in the absence
and presence of equimolar amounts of ligands respectively, r is
the distance from the ligands bound on b-lg to the Trp residue,
¨
and R0 is the Forster's critical distance which can be dened as
the distance at which 50% uorescence energy transfer is
In our study, steady state uorescence data shows a sharp
peak of IOC-derivative and PY-derivative at 437 nm and 391 nm
upon excitation of the uorophore at 335 nm and 325 nm
maintaining the concentration at 10 mM for IOC and PY
derivative respectively [Fig. 4(A) and (B)]. There was a gradual
increase in uorescence intensity of IOC derivative upon
concomitant addition of b-lg solution up to 20 mM accompanied
by a distinct blue shi in emission maxima about 17–20 nm. It
signies the encapsulation of IOC derivative into a more non
polar microenvironment in presence of b-lg [Fig. 4(A)]. On the
other hand, the increase in emission maxima in case of PY
derivative was also observed without any signicant change in
emission maxima and was merely a blue shi of 6–8 nm
[Fig. 4(B)], which signies the accessibility of less hydrophobic
environment as compared to the IOC derivative. This may
indicate the different binding sites as well as the alteration in b-
lg tertiary structure in presence of extrinsic uorophore.
possible from the donor site to the acceptor and was expressed
as
6
R0 ¼ [8.79 ꢁ 10ꢀ25 ꢁ K2Nꢀ4FJ]
(7)
here, the K2 is a factor narrating the relative orientation in space
for the transition dipoles of the donor and acceptor. K2 is
usually assumed to be equal to 2/3.31 N is the refractive index
which is typically assumed to be 1.33 for bio molecules in
aqueous solution. F is the quantum yield of the donor in the
absence of acceptor and was considered to be 0.45.32 J is the
overlap integral between the normalized emission spectra of the
donor and absorption spectra of the acceptor. J is expressed by
the following equation
P
P
J ¼ F(l)3(l)l4dl/ F(l)dl
(8)
In this equation, F(l) is the uorescence intensity of the
donor at wavelength l and is dimensionless. 3(l) is the molar
absorption coefficient of the acceptor at wavelength l. In the
UV-absorption study, the concentrations of IOC and PY deriv-
atives and the concentration of b-lg in uorescence study kept
constant [Fig. 5(A) and (B)]. J values calculated by integrating the
overlap area of the spectra from 305 to 455 nm and were found
to be 5.0046 ꢁ 10ꢀ12 for Mꢀ1 cm3 IOC and 3.7497 ꢁ 10ꢀ12 Mꢀ1
cm3 for PY. The efficiency of energy transfer and Forster
¨
distance can be calculated from the eqn (6) and (7). Using the
calculated value of R0 and E, the value for r can be obtained. The
R0 values of IOC and PY derivatives were 8.66 nm and 8.25 nm
respectively. The E values for IOC and PY derivatives were 0.74
and 0.51 respectively. The calculated r value is the average
distance between ligand and two tryptophan residues (W19 and
Fig. 4 (A) Fluorescence emission spectrum of IOC (10 mM) in the
absence and presence of b-lg (1–20 mM). (B) Fluorescence emission
spectrum of PY (10 mM) in the absence and presence of b-lg (1–20 mM).
Excitations were done at 335 nm (IOC) and 325 nm (PY) and emissions
were observed at 437 nm (IOC) and 391 nm (PY) respectively. Slit width
was 3 for excitation and 5 for emission for both of the compounds.
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Fig. 5 (A) Spectral overlap of absorption spectra of IOC (b) and fluo-
rescence spectra of b-lg (a). (B) Spectral overlap of absorption spectra
of PY (c) and fluorescence spectra of b-lg (a). c(b-lg) ¼ c(IOC) ¼ c(PY)
¼ 1 ꢁ 10ꢀ5 mol Lꢀ1
.
W61) of b-lg. The r values were 7.28 nm for IOC and 7.33 nm for
PY. The donor-to-acceptor distance lies within the range of
0.5R0 to 2R0 in each case, suggesting efficient energy transfer
from b-lg to both of the ligands.33 This suggests that one or both
Trp residues are in close proximity to IOC and PY derivatives.
Bovine b-lg contains three binding sites, which are situated
at the calyx, at the surface cle and at the monomer/monomer
Fig. 6 (A) Time resolved fluorescence intensity decay profile of (a) Trp
of native b-lg; (b) Trp in b-lg–IOC complex (1 : 1), (c) Trp in b-lg–PY
complex (1 : 1) and (d) the sharp profile on the left (black) signifies for
prompt. Excitation wavelength was chosen as 300 nm and fluores-
cence was collected at 350 nm. (B) Time resolved fluorescence
interface.34 The interactions of IOC and PY derivatives with b-lg intensity decay profile of (a) free IOC, (b) IOC in presence of b-lg (1 : 1)
and (c) the sharp profile on the left (black) signifies for prompt. Exci-
tation wavelength was chosen as 300 nm and fluorescence was
collected at 410 nm. (C) Time resolved fluorescence intensity decay
profile of (a) free PY, (b) in presence of b-lg (1 : 1), (c) the sharp profile
on the left (black) signifies for prompt. Excitation wavelength was
are dominated by hydrophobic interaction as shown by FTIR
study.
2.4. Time-correlated single-photon counting (TCSPC) study
chosen as 300 nm and fluorescence was collected at 410 nm.
We also extended our study for better understanding of steady
state uorescence results to time resolved uorescence experi-
or PY and thus causing the change in micro-heterogeneity
either by more solvent accessibility towards Trp moiety or due
to proximity of substantial quencher moieties present in b-lg
structure due to allosterism.
ment by using SDS as prompt. The bi-exponential curve tting
programme, ran into the decay proles, shows that the average
life time, hti of Trp in b-lg is 1.41 ns. Whereas, in presence of
IOC, hti of Trp is 1.38 ns and in presence of PY, hti of Trp is 0.967
ns, this signies that Trp was facing rather more polar envi-
ronment in presence of IOC and PY at a higher extent resulting
in decrease of average life time of Trp19, and showing in Table 1
accompanied by the decrease in both longer and shorter life
time components of Trp19 present in the b-lg molecule. None-
theless, the intrinsic time resolved uorescence data shows
decrease in average life time parameters, either cause by the
change in micro polarity or by the change in micro conforma-
tion [Fig. 6(A)]. It also enlightens that the change is more
pronounced in case of PY derivative than the IOC derivative. On
the basis of this study it may be inferred that the micro polarity
of the intrinsic uorophore changed due to introduction of IOC
The extrinsic TCSPC uorescence study shows that increase
in average lifetime of both IOC and PY in presence of b-lg, which
was accompanied by both the increase in longer and shorter
lifetime parameters under bi exponential curve tting pro-
gramme, [Fig. 6(B) and (C)] it strongly signies the anchoring of
the IOC and or PY to a less polar or more hydrophobic micro
environment in b-lg matrix (Table 1) either they found the
hydrophobic pockets of b-lg or they get conned into more
hydrophobic regions within the b-lg matrix under altered
conformation. The relative connements of the derivatives are
more pronounced in case of PY, which depicts the better
binding of PY, in b-lg hydrophobic calyx. It may also be
Table 1 Time-correlated single-photon counting (TCSPC) parameters
Compounds
a1
s1 (ns)
a2
s2 (ns)
c2
sav (ns)
Trp in native b-lg
Trp in IOC + b-lg (1 : 1)
Trp in PY + b-lg (1 : 1)
IOC (free)
IOC in presence of b-lg
PY (free)
0.925
0.69
0.67
0.971
0.942
0.250
0.766
1.28
1.05
0.67
0.15
0.241
0.142
0.498
0.074
0.309
0.33
0.028
0.0577
0.025
0.2339
3.06
2.14
1.57
1.005
1.132
0.991
1.709
0.9432
1.0263
1.0494
1.0881
1.0838
1.0656
1.0129
1.41
1.38
0.967
0.173
0.3
0.163
0.781
PY in presence of b-lg
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proposed that the presence of more hydrophobic aromatic I–VII. Of these, the amide I and II bands are the most important
moieties enhances the binding with PY, either by p–p type of bands. They are sensitive to the protein secondary structure.
interactions or by H-bonding, which has been thoroughly The amide-I peak position occurs in the region of 1700–1600
studied in docking experiment.
cmꢀ1 (mainly C]O stretch), amide-II band in the region of
1600–1500 cmꢀ1 (C–N stretch coupled with N–H bending
mode). Amide I band is useful and more sensitive to the change
of protein secondary structure than amide II band.36 In our
study amide I band was monitored to investigate the change of
secondary structure of b-lg in the presence and absence of the
ligands IOC and PY. Native b-lg molecule in absence of any
derivatives shows the amide-I band at around 1632 cmꢀ1 which
is the characteristic feature for the globular protein like b-lg
having predominant b-sheet structure [Fig. 7(C) spectra a].37
Thus the shiing of amide-I band of native b-lg occurs from
1632 cmꢀ1 to 1650 cmꢀ1 (bathochromic shi) in presence of two
derivatives. Our experimental results clearly indicate a sharp
secondary structural transition of b-lg from predominant b-
sheet structure to a-helical structural contents upon binding
with IOC and PY derivatives. Furthermore, the PY derivative is
more effective than IOC derivative to incorporate such struc-
tural transition during their binding with b-lg. These observa-
tions were noted during the study of far UV-CD spectra.
2.5. CD results
The change in secondary structure of b-lg in presence of ligands
was investigated by circular dichroism spectroscopy (CD). Far
UV-CD spectra analysis shows insignicant change in the
minima at 215 nm, characteristic b-sheet signal for b-lg struc-
ture.35 It clearly shows that the secondary structure of b-lg
remains almost intact during binding with IOC [Fig. 7(A)]. In
the presence of PY, a distinctive change of b-lg conformation
occurs, resulting from the enhanced signals at 208 and 222 nm
and thus indicating more a-helical structure upon binding with
b-lg [Fig. 7(B)]. It clearly indicates an evidence of allosterism in
b-lg conformation when it binds with PY derivative. The
appearance of a strong signal at 208 nm at higher concentration
of PY derivative primarily indicates the cumulative change
arising from the alteration of tertiary structure and hence the
change in micro environment and it was also supported by
steady state uorescence study. The calculation of secondary
structural changes of b-lg in presence of IOC and PY derivative
was done by CDNN soware and shown in Table S3 (ESI†).
2.7. Docking study
The protein b-lg is a small globular protein with the molecular
mass 18 400 Da. There are 162 amino acid residues construct-
ing eight antiparallel b-strands forming the b-barrel or calyx,
and one a-helix at the outer surface of the barrel in the mono-
meric form of b-lg. Three binding pockets for ligands: the calyx,
a small space between a-helix and the barrel and the region near
Trp19–Arg124, are found in the tertiary structure of b-lg. Between
the two tryptophan residues, Trp61 and Trp19, the Trp19 of the
protein is mainly responsible for it's the uorescence nature. If
a uorophore bind with the protein in such a way that it comes
2.6. FT-IR spectra
Infrared (IR) spectroscopy is one of the well-established exper-
imental techniques for the analysis of secondary structure of
proteins. The vibrations of a protein structure give rise to nine
characteristic IR absorption bands, namely, amide A, B, and
¨
into the Forster distance of any of the Trp residues, then that
Trp residue can transfer its energy efficiently to the uorophore
aer excitation. The phenomenon is popularly known as FRET.
The molecular docking study has drawn much attention to both
the theoretical and experimental researchers. It is frequently used
to determine the binding behavior of small molecules into the
target specic protein environment where they bind mainly
through a non-covalent interaction like hydrogen bonding, elec-
trostatic and steric effects, van der Waal's forces and stacking
interactions. A molecular docking study has been carried out with
IOC and PY on b-lg (PDB ID: 3NQ3) to nd protein environment
where they interacts. The energetically most favourable docking
positions were calculated. It has been observed that both the
molecule stabilized into the binding site through hydrogen
bonding, p-stacking, p-alkyl, p-anion and p-sigma interaction. The
change of Gibbs free energy has been calculated for IOC and PY
Fig. 7 (A) CD spectra of (a) b-lg (13.6 mM) in the presence of (b) 5 mM, and was found to be ꢀ5.45 and ꢀ6.55 kcal molꢀ1 respectively. This
(c) 10 mM, (d) 15 mM, (e) 20 mM, (f) 30 mM, (g) 40 mM, (h) 50 mM, (i) 100
result indicates that PY binds with b-lg more strongly than that of
mM, (j) 200 mM IOC. (B) CD spectra of (a) b-lg (13.6 mM) in the presence
of (b) 5 mM, (c) 10 mM, (d) 15 mM, (e) 20 mM, (f) 30 mM, (g) 40 mM, (h) 50
mM, (i) 100 mM, (j) 200 mM PY. (C) FTIR spectra of (a) free b-lg (500 mM),
(b) complex of b-lg with IOC (1 : 1) and (c) complex of b-lg with PY
IOC. The compound IOC prefers surface binding at the region near
Trp19–Arg124 through H-bonding with Trp19 and Tyr20, p-stacking
with Tyr20 and other p-interaction with Glu44 and Glu158 [Fig. 8(A)–
(1 : 1).
(C)]. However, PY binds inside the calyx [Fig. 9(A) and (B)] and
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¨
is under the range of Forster distance (range of 0.5R0 to 2R0,
ꢀ
experimentally determined R0 value is 8.66 A, so the range
ꢀ
ꢀ
become 4.33 A to 17.32 A). However, in the case of compound PY
ꢀ
¨
the Trp61 is 10.4 A apart from it. This is also under the Forster
distance range of Trp61 (for this compound experimentally
ꢀ
ꢀ
ꢀ
determined R0 value is 8.25 A and the range is 4.12 A to 16.50 A).
Docking study successfully predict the ability to show FRET
with b-lg for both the compound.
3 Experimental
3.1. Materials and methods
Bovine b-lactoglobulin (b-lg) was isolated and puried from cow
milk described earlier.38 Pure curcumin was purchased from
Sigma-Aldrich, India. IOC and PY were synthesized according to
the methods.39 IOC and PY were further characterised by 1H-
NMR, 13C-NMR and ESI-MS (all spectra and data were given in
the ESI†). Protein stock solutions were prepared using phos-
phate buffer containing 2% ethanol pH-7.4 while IOC and PY
stock solutions prepared in absolute ethanol because of its poor
solubility in aqueous buffer. Concentrations of IOC, PY and b-lg
were determined spectroscopically using extinction co-efficient
of b-lg 0.96 mgꢀ1 mlꢀ1 cmꢀ1 at 280 nm and molar extinction co-
efficient 40 268 Mꢀ1 cmꢀ1 at 335 nm for IOC and 42 246 Mꢀ1
cmꢀ1 at 325 nm for PY.24
Fig. 8 Schematic representation of docked conformation of IOC with
interacting residues of b-lg. Docking pose shows IOC binds at the
surface of the protein.
Val43, Ile56, Ile84, Asn90, Val92, Phe105, Met107 and Ans109, residues of
the protein are in the vicinity of the molecule [Fig. 9(C)]. In this
environment of the protein, PY molecule is stabilized by H-
bonding with Asn109, p-stacking with Phe105 and other p-interac-
tion with Asn90, Ile84, Ile56, Val43, Val92 and Met107 residues.
Docking results also shows that IOC and PY are inside the
3.2. Instrumentation
3.2.1. UV-visible spectroscopy. Absorbance measurements
for the determination of binding affinity and binding constants
were performed utilizing UV-Visible SHIMADZU Spectropho-
ꢃ
¨
region of the Forster distance of Trp19 and Trp61 respectively.
tometer model no. TCC-240A at room temperature (25 C). UV
ꢀ
The distance between compound IOC and Trp19 is 11.3 A which
titrations were performed in a 1 cm quartz cuvette with the
concentration of IOC (20 mM) and PY (20 mM) as constant and by
the successive addition of increasing concentrations of b-lg (1–
20 mM) for IOC and (1–17 mM) for PY. The respective spectra
were recorded over the wavelength range 200–600 nm.
3.2.2. Spectrouorimetry. Fluorescence quenching studies
were carried out using Shimadzu RF-5301 PC at room temper-
ature with the bandwidths of excitation and emission slits at 3
and 5 nm respectively. Both the extrinsic uorescence titration
was performed, rstly keeping the ligand concentration xed at
10 mM and varying protein concentration and vice versa. The
concentration of b-lg (10 mM) was kept constant and then IOC
and PY added successively from 1 to 21 mM and 1 to 15 mM. The
excitation wave length was 335 nm and 325 nm for IOC and PY
derivative and emission was recorded from 350–550 nm. For
FRET study purpose, the excitation wavelength of b-lg (10 mM,
with phosphate buffer, pH-7.4, with 2% ethanol) was 295 nm
and emission scans were recorded in the range 310–550 nm at
room temperature.
3.2.3. Time resolved uorescence study. Lifetime
measurements were acquired using the time-correlated single-
photon counting (TCSPC) apparatus using a picoseconds
diode laser at 295 nm (IBH, UK, nanoLED-295) as the light
source and TBX-04 as detector. The full width at half-maximum
(FWHM) of the instrument response function was 1 ns. All the
Fig. 9 Schematic representation of docked conformation of PY with
interacting residues of b-lg. Docking pose shows PY binds inside the
calyx of the protein.
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¨
measurements were done in a 45 ns time window with a reso- remain inside the region of the Forster distance of Trp19 and
lution of 100 ps per channel. A 1 cm path length quartz cuvette Trp61 of b-lg to exhibit FRET as evidenced by docking experi-
was used for all the time-resolved measurements. Fluorescence ment. The change in micro polarity as a result of the change in
decays were collected at the magic angle (polarization of 54.7ꢃ) micro conformation was more pronounced in case of PY
with respect to the vertical excitation light at 295 nm. Excitation derivative than that of IOC derivative as revealed from time
at 295 nm exclusively for tryptophan residues and prevent resolved uorescence data. FTIR data also supports the
background uorescence from other aromatic amino acids like conformational changes of b-lg with these compounds. The
phenyl alanine and tyrosine. In another experiment beta lacto- studies of interaction of IOC and PY with b-lg suggest an
globulin mixed with IOC and PY derivative of curcumin with effective association with PY compared to IOC. The compound
1 : 1 molar ratio. In both cases the excitation wavelength was IOC favours surface binding at the region near Trp19–Arg124
300 nm and life time decay traces were obtained from 400 to through H-bonding and other weak forces. However, PY prefers
450 nm. The decays were analyzed using IBH DAS-6 decay to bind inside the calyx and stabilization was achieved by H-
analysis soware.
bonding with Asn109, p-stacking with Phe105 and other p-
3.2.4. CD spectroscopy. CD spectra of b-lg and IOC and PY interaction with Asn90, Ile84, Ile56, Val43, Val92 and Met107 resi-
derivative of curcumin complexes were recorded on a Jasco dues. This preliminary study and useful information about the
spectropolarimeter (J-815). For measurements in the far-UV interaction of curcumin derivatives furnishes the promise in
(200–260 nm) region, quartz cell in the path length 0.2 cm designing the drugs and other biologically active molecules that
was used in the nitrogen atmosphere. Concentration of b lg was bind more strongly to b-lg and have ability to show FRET.
kept constant (13.6 mM) while varying concentrations (5 mM,
10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 200 mM) of
IOC and PY derivatives. Scan speed 50 nm per minute was
Conflict of interest
performed and data were collected for each nm from 260 to
The authors declare no competing nancial interest.
200 nm. Sample temperature was maintained at 25 ꢃC using
a Neslab RTE-111 circulating water bath connected to the water
jacketed quartz cuvettes. The secondary structures of b-lg were Acknowledgements
calculated from CDNN 2.1 soware.
Financial support of University Grant Commission UGC-CAS-II
and DST-PURSE-II (Govt. of India) Program of Department of
3.2.5. FT-IR absorption spectra. The infra-red absorption
spectra of the compounds and b-lg solution were measured at
Chemistry, Jadavpur University, Kolkata are greatly acknowl-
room temperature. FT-IR scans were recorded in the range of
edged. Sanhita Maity, SRF, is a recipient of UGC-NET Research
Fellowship in Chemistry.
1550–1750 cmꢀ1 at a resolution of 2 cmꢀ1 in N2 environment
using a Spectrum 100 FT-IR spectrometer (Perkin Elmer) with
ATR accessory. The concentration of b-lg was 500 mM.
3.2.6. Molecular modeling study. The crystal structure of
b-lg was obtained from RCSB Protein Data Bank website (PDB
References
ID: 3NQ3), the structure of the biopolymer was analysed and
prepared for the docking experiment. The structure of IOC and
PY were optimized to lower energy using Density Functional
Theory (DFT) B3LYP/6-31G level of theory. These optimized
structures were used for docking with b-lg protein to nd the
probable site and mode of binding using a grid-based docking
program Auto Dock 4.2. For the docking study, ligand present in
the protein (3NQ3) and water molecules were removed from the
protein, atom Kollman charges were assigned aer the addition
of polar hydrogen for the protein. The default parameters of
Auto Dock and generic algorithm were used for this calculation.
The visualization effects were prepared by Discovery Studio 4.1
Client and Chimera 1.10.1rc.
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