Journal of Thermal Analysis and Calorimetry (2019) 136:1757–1767

https://doi.org/10.1007/s10973-018-7800-z(0123456789().,-volV)(0123456789().
,- volV)

A comprehensive calorimetric, spectroscopic, and molecular docking

investigation to probe the interaction of colchicine with HEWL
Samima Khatun1 • Riyazuddeen1

Received: 3 February 2018 / Accepted: 7 October 2018 / Published online: 17 October 2018
Ó Akadémiai Kiadó, Budapest, Hungary 2018

Abstract
Colchicine (Col), a naturally occurring alkaloid, has been used as an anti-inflammatory, anti-fibrotic, and anti-tumor drug.
The present study reports in vitro interaction between Col and hen egg white lysozyme (HEWL) by spectroscopy,
isothermal titration calorimetry (ITC), and molecular docking techniques. The fluorescence results indicated that Col
quenches the intrinsic fluorescence of HEWL through static quenching mechanism which is also substantiated by UV–
visible spectroscopy. The average distance (r) between donor (HEWL) and acceptor (Col) was evaluated by Förster’s
resonance energy transfer theory. Thermodynamic parameters obtained from ITC suggested that hydrophobic interaction
plays a major role in the complex formation, and it is an entropically driven process. Col altered the secondary structure of
HEWL revealed by circular dichroism which is further corroborated by synchronous, 3D fluorescence and FTIR tech-
niques. In addition, molecular docking was employed to find out binding site of Col on HEWL and amino acid residues
involved in the binding process. This study is expected to provide insights into the type of interaction and binding
mechanism of Col with HEWL.

Keywords Hen egg white lysozyme  Colchicine  Spectroscopy  Isothermal titration calorimetry  Molecular docking

Introduction degree of structural stability and has unique folding

Hen egg white lysozyme (HEWL), a low molecular
weight (* 14.3 kDa) monomeric protein, is composed of
129 amino acid residues. It contains six tryptophans (Trp),
three tyrosine amino acid residues, and four disulfide
bonds. The six tryptophan residues are located at the
substrate-binding sites, out of which two are in the
hydrophobic matrix box; one is separated from the others.
Trp-62, Trp-63, and Trp-108 are the most dominant flu-
orophores being located at the substrate-binding site [1].
HEWL is abundant in a number of secretions such as
saliva, tears, milk, and mucus and acts as a bacteriolytic
agent to lyse the bacterial cell walls. It possesses higher
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10973-018-7800-z) contains
supplementary material, which is available to authorized
users.
& Riyazuddeen
rz1@rediffmail.com
1
Department of Chemistry, Aligarh Muslim University,
Aligarh, U.P. 202002, India
mechanism and thermodynamic features. HEWL exhibit
many physiological and pharmaceutical functions such as
it acts as antibacterial, antiviral, anti-inflammatory, and
anti-HIV agent [2]. Therefore, the interaction studies
between HEWL and drugs are of importance in view of
realizing disposition, transportation, and metabolism of
drugs as well as efficacy process [3]. Another important
function of HEWL is the ability to carry drugs to their
target receptors. High natural abundance is also one of the
reasons for choosing HEWL as a model protein for
studying protein–ligand interaction.
Colchicine (Col) is obtained from meadow saffron
Colchicum Autumnale and Gloriosa Superba L [4]. It was
the first tubulin-destabilizing agent to be discovered which
binds to tubulin and disrupts mitosis, ending this process at
the metaphase and hence stopping division of the cell’s
nucleus [5]. It is a naturally occurring alkaloid and has
been used as an anti-inflammatory, anti-fibrotic, and anti-
tumor drug in human and veterinary medicines [6]. Col has
received considerable attention in cancer research involv-
ing cell cultures as anti-mitotic agent that has been con-
sidered for chemotherapy applications but is still in

123
1758
preclinical cancer research due to its toxicity. Furthermore,
it is used for alleviation of inflammatory process during
podagra. It has been proposed as a drug to reduce chronic
lung inflammation and liver diseases in cystic fibrosis
patients [7]. The chemical structure of Col is shown in
Fig. 1.
Some researchers have reported binding studies of Col
and its analogues with proteins like human serum albumin
(HSA), bovine serum albumin (BSA), and tubulin through
different techniques [8–16]. To the best of our knowledge,
no report on binding studies of HEWL-Col system has
been mentioned in the literature. The present study is
focused to explore the number of binding sites and binding
mechanism by steady-state fluorescence and to characterize
thermodynamic parameters of HEWL-Col complex by
isothermal titration calorimetry (ITC) technique. The con-
formational changes in HEWL in the presence of Col have
been proved through UV–Vis, synchronous, 3D fluores-
cence, circular dichroism (CD) and Fourier transform
infrared (FTIR) spectroscopy. Förster’s resonance energy
transfer (FRET) theory has been applied to calculate the
molecular distance (r) between HEWL and Col. Recogni-
tion of binding site of Col on HEWL and amino acid
residues involved in the interactions have been unrevealed
by molecular docking study. The outcome of this study is
expected to provide an insight into the understanding of
pharmacological and structural changes underlying the
binding process of Col with HEWL.
O
O
H3CO
HN
H3CO
H3CO
OCH3
Fig. 1 Chemical structure of Col
123
S. Khatun, Riyazuddeen
Experimental
Materials and sample preparation
Hen egg white lysozyme (L6876, mass fraction C 90%),
Colchicine (C9754, mass fraction C 95%) were purchased
from Sigma-Aldrich. All other reagents used were of ana-
lytical grade. Double-distilled water, free from any fluo-
rescent contaminants, was used. The stock solution of
HEWL and Col were prepared by dissolving in sodium
phosphate buffer (20 mM, pH = 7.4). The concentration of
HEWL was determined by UV–visible spectrophotometer
(Perkin Elmer, k-850) using E282 1%
nm = 26.4. All drug
solutions were prepared by weight/volume (w/v) method.
The details of the chemicals used in the present work are
given in Table 1.
UV–visible measurements
UV–visible absorption spectra of HEWL in the absence
and presence of Col were recorded in the range
of 200–400 nm on UV–visible spectrophotometer
(Perkin Elmer, k-850). The spectrophotometer was
attached with Peltier temperature programmer-1 (PTP-1) to
maintain temperature at 298.15 K throughout the experi-
ments in a one-centimeter path length cuvette of 3 mL.
HEWL concentration was fixed at 5 lM, whereas that of
Col was varied from 0 to 60 lM. All absorbance spectra
for HEWL-Col system were corrected with respective
blank which consists of Col in buffer solution at same
concentration under same experimental setup.
Fluorescence spectroscopy
The steady-state fluorescence measurements were recorded
on F-2700 fluorescence spectrophotometer (Hitachi).
HEWL solution (5 lM) was titrated with varying concen-
trations of Col (0–50 lM) solution using an excitation
wavelength of 295 nm which is owing to tryptophan (Trp)
residue. The fluorescence emission spectra were collected
between 300 and 420 nm wavelength. The excitation and
emission slits were set at 5 nm and 10 nm, respectively.
The fluorescence quenching data were analyzed by the
Stern–Volmer equation [17]
Fo
¼ 1 þ Ksv ½Q ð1Þ
F
where Fo and F are the steady-state fluorescence intensities
in the absence and presence of quencher. Ksv and [Q] are
the Stern–Volmer quenching constant and the concentra-
tion of quencher (Col), respectively. The bimolecular rate
constant (kq) is evaluated using the following equation
A comprehensive calorimetric, spectroscopic, and molecular docking investigation to probe... 1759

Table 1 Compounds used in this study with their molecular weight, mass fraction purity, and sources
Sample Provenance Mass fraction puritya Molecular weight

Hen egg white lysozyme (HEWL) Sigma-Aldrich 0.95 14,000

Colchicine Sigma-Aldrich 0.95 399.44
Di-sodium hydrogen orthophosphate dihydrate (Na2HPO42H2O) Merck 0.98 177.99
Sodium dihydrogen orthophosphate dihydrate (NaH2PO42H2O) Merck 0.98 141.96
a
Purity as stated by the supplier

kq ¼ Ksv =so ð2Þ excitation) were recorded in the wavelength range of

where so is the average integral fluorescence lifetime of
tryptophan which is 5.7 9 10-9 s. The values of stoi-
chiometry, n, and binding constant, Kb, are determined
from the following equation [18]
 
F0
log  1 ¼ log Kb þ n log½Q ð3Þ
F Fo Ro þ r
Synchronous fluorescence
Synchronous fluorescence measurements were performed
to elucidate the molecular microenvironment changes in
the vicinity of the intrinsic fluorophores, tryptophan (Trp),
and tyrosine (Tyr) [19]. The spectra of HEWL in the
presence of Col were recorded by simultaneous scanning
the excitation and emission monochromator with Dk = 20
nm and Dk = 60 nm for monitoring the microenvironment
of tyrosine and tryptophan in HEWL, respectively. The
protein concentration was kept constant at 5 lM, and
concentration of Col was varied from 0 to 50 lM. The
excitation and emission slits were set at 5 nm and 10 nm,
respectively.
300–450 nm. The overlap of the UV absorption spectrum
of Col with the fluorescence emission spectrum of HEWL
was used to calculate the energy transfer as per the För-
ster’s theory [20]. The efficiency of energy transfer (EFRET )
was calculated using the following relation
F R6
EFRET ¼ 1  ¼ 6 o 6 ð4Þ
where Fo and F are the fluorescence intensities of HEWL
in the absence and presence of Col, respectively; r is the
distance between donor and acceptor, and Ro is the critical
distance at which transfer efficiency equals to 50%, which
can be calculated from the following equation [21]
R6o ¼ 8:79  1025 K 2 n4 u J ð5Þ
where K2 is the orientation factor related to the geometry of
the donor and acceptor of dipoles; n is the refractive index
of the medium; u is the fluorescence quantum yield of the
donor in the absence of acceptor and J expresses the degree
of spectral overlap between the donor emission and the
acceptor absorption which can be evaluated by integrating
the overlap spectral area from 300 to 450 nm from fol-
lowing equation [22]

Three-dimensional fluorescence Z1
F ðkÞeðkÞk4 dk
J¼ ð6Þ
F ðkÞdk
Three-dimensional fluorescence spectroscopy has been 0
demonstrated to be a specific method to comprehensively where F(k) is the fluorescence intensity of the donor at
exhibit the fluorescence information of the chromophore wavelength range k which is dimensionless, and e(k) is the
and to investigate the characteristic conformational chan- molar absorptivity (extinction coefficient) of the acceptor
ges of protein. Three-dimensional fluorescence spectra of at wavelength k in M-1 cm-1.
HEWL (5 lM) in the absence and presence of Col (25 lM)
were measured under at the emission wavelength range of Isothermal titration calorimetry (ITC)
220 and 550 nm. The initial excitation wavelength was set measurements
at 220 nm with an increment of 10 nm up to 350 nm, and
the excitation and emission slits both were set at 5 nm. The thermodynamic parameters for the interaction of Col
with HEWL were determined using ITC200
Fluorescence resonance energy transfer (FRET) microcalorimeter (Micro Cal Inc., Northampton, MA) at
T = 303.15 K. The sample cell was loaded with HEWL
The absorption spectrum of Col and the emission spectrum which was dissolved in 20 mM sodium phosphate buffer
of HEWL at kex = 295 nm (specific for tryptophan (pH = 7.4). The HEWL solution (0.100 mM) was titrated

123
1760
with Col (5 mM) using a rotating stirrer syringe keeping
stirring speed fixed at 600 rpm. Each experiment consisted
of 19 consecutive injections of 2 lL Col solution each into
the sample cell solution. Each injection had duration of 4 s,
and the time interval between the consecutive injections
was kept at 120 s [23]. The reference power was set at
0.025 mW. The data were analyzed using the Origin 7.0
software, provided by Micro Cal. Heats of dilution for the
ligand were determined in control experiments and were
subtracted from the integrated data before curve fitting.
The data were fitted to obtain the binding constant (Kb),
change in standard enthalpy (DH°) and change in standard
entropy (DS°) values. The change in standard Gibbs free
energy (DG°) was calculated using the following equation
[24].
DG ¼ DH   TDS ð7Þ
The instrument was periodically calibrated and verified
with (water ? water) dilution experiments as per the
requirement of the manufacturer. The combined standard
uncertainty, uc (x) (level of confidence = 0.95, k = 2) for
the calorimetric data, was calculated using equation
h i1=2
uc ðxÞ ¼ ðu1 ð xÞÞ2 þðu2 ð xÞÞ2 þðu3 ð xÞÞ2 þ    þ etc:
ð8Þ
Circular dichroism (CD) measurements
The far-UV CD spectra of HEWL in the absence and
presence of Col were measured using CD spectrometer
(JASCO, J-815) equipped with Peltier temperature con-
troller to keep the temperature fixed at 298.15 K. The
calibration of the instrument was done with d-10-cam-
phorsulfonic acid. The scan speed, data pitch, and response
time were set at 100 nm min-1, 1 nm, and 1 s, respec-
tively. Each spectrum obtained was the average of two
scans. The path length of the cell was 0.1 cm for the far-
UV CD (200–250 nm) measurements. The HEWL con-
centration was kept at 5 lM. The observed ellipticity is
converted to mean residue ellipticity (MRE) using the
following equation [25]
Hobs ðm Þ
MRE ¼ ð9Þ
10  n  C  l
where Hobs (mdeg) is observed ellipticity; n is the total
number of amino acid residues (129) in HEWL; C is the
molar concentration of HEWL and l (cm) is the path
length. The a-helical content of the protein was calculated
using the following equation [26]
 
MRE222 nm  2340
%a  helix ¼  100 ð10Þ
30300
123
S. Khatun, Riyazuddeen
Fourier transforms infrared (FTIR) measurements
Fourier transforms infrared (FTIR) spectra of protein–drug
solutions were recorded on KBr pellets at T = 298.15 K
and the spectra were collected in the range of
2000–1000 cm-1 using FTIR/FIR spectrometer (Perkin
Elmer) equipped with a attenuated total reflection (ATR)
accessory with resolution 4 cm-1 and 40 scans [27]. FTIR
spectrum of bound HEWL was obtained by subtracting the
spectrum of Col from the respective mixtures spectrum.
The concentrations of both HEWL and Col were kept at
75 lM.
Molecular docking study
The three-dimensional structure of HEWL (PDB ID:
2LYZ) was downloaded from the RCSB protein data bank
(http://www.rcsb.org/pdb) [28, 29]. The structure of Col
[PubChem, CID: 10281] was constructed using Chem
Draw 12.0. The energy minimization of ligand file was
done using MM-2 method implemented in Chem. 3D
Pro12.0. The docking studies were performed with Auto
Dock Vina program. To avoid hindrance while docking, all
water molecules were removed and hydrogen atoms were
added. The Lamarckian genetic algorithm (LGA) was
applied for minimization using default parameter to deal
with the HEWL–drug interaction. Kollman charges were
merged to the protein, and Gasteiger charges were added to
the ligand using Auto Dock Tools (ADT), and then pre-
pared file was saved in PDBQT format [30]. During the
docking of Col to HEWL, the protein structure was fixed at
the initial input and set to be rigid, while all torsional bonds
of the ligand were set to be flexible. The ligand-centered
maps were generated by the program Auto-Grid with grid
center x = - 1.691, y = 14.716, z = 21.682 and the grid
was set to 32 9 38 9 50 Å points to cover all the active
site residues. The post-modeling analysis and the visual-
ization of the complex was done using Accelrys Discovery
Studio 4.5 [31].
Results and discussion
UV–Vis absorption measurements
UV–Vis absorption measurement method is applied to
know the complex formation between protein and drug and
feasible quenching mechanism. Figure S1(a) (Supplemen-
tary material) shows UV–Vis absorption spectra of HEWL
in the absence and presence of Col. On gradual addition of
Col to HEWL solution, the intensity of the peak at 281 nm
increases which suggest complex formation between Col
A comprehensive calorimetric, spectroscopic, and molecular docking investigation to probe...
and HEWL. There was emergence of two new peaks at
246 nm and 353 nm which are characteristic absorbance of
Col as shown in Fig. S1(b) (Supplementary material)
[32, 33]. The possible quenching mechanism (either
dynamic or static) can be distinguished by UV–Vis spec-
troscopy. In dynamic quenching, only the excited state
fluorophore is affected by the quencher, and so the
absorption spectra do not show any change, whereas in the
case of static quenching, complex formation takes place
between ground-state fluorophore and quencher [34]. Since
the absorption spectrum of HEWL gradually increases after
each addition of Col, the quenching mechanism is identi-
fied to be static in nature.
Col-induced fluorescence quenching of HEWL
Fluorescence spectroscopy is a sensitive tool employed to
study the interaction of small molecules with biological
macromolecules such as protein and also to determine the
protein conformation and structure changes on drug bind-
ing. Tryptophan, tyrosine, and phenylalanine are the three
aromatic fluorophores responsible for quenching. However,
among them, the contribution of tryptophan is maximum.
Native HEWL shows strong emission peak at 340 nm on
excitation at 295 nm which decreases upon increasing the
concentration of Col from 0 lM to 50 lM with blue shift
in wavelength maximum as shown in Fig. 2 suggesting an
increased hydrophobicity (less polar) of the region sur-
rounding the tryptophan residue. The Stern–Volmer and
modified Stern–Volmer plots for HEWL-Col system are
given in Fig. 3a, b, respectively, and all calculated
parameters are summarized in Table 2. The binding con-
stant and Gibbs free energy change values were found to be
5.45 9 104 M-1 and - 27.13 kJ mol-1, respectively. The
800
600
Fluorescence intensity
400
200
0 shown in Fig. 4, peak 1 (kex = 280 nm, kem = 340 nm)
300 330 360 390
Wavelength/nm
Fig. 2 Fluorescence quenching spectra of HEWL in the presence of
Col (0–50) lM at T = 298.15 K and pH = 7.4
1761
negative value of DGo suggests that the binding of Col to
HEWL is a spontaneous process. Several processes such as
excited state reaction, molecular rearrangement, energy
transfer, collisional quenching, and the ground-state com-
plex formation are account for the fluorescence quenching
phenomenon [35]. Fluorescence quenching is either static
(by complex formation) or dynamic (by collision pro-
cesses) in nature. To know about the type of quenching
mechanism of HEWL by Col, we calculated kq from
Eq. (2) which lies in the order of 1012 which was found to
be larger than the maximum dynamic quenching constant
value (2 9 1010 M-1 s-1) [36]. Therefore, it was con-
cluded that Col-induced quenching was not due to dynamic
diffusion rather resulted from complex formation.
Conformational investigation by synchronous
fluorescence
Synchronous fluorescence provides information about
microenvironment perturbation around the fluorophore
(Tyr and Trp) present in protein upon binding with drug.
Any shift in maximum wavelength describes changes in
polarity around the fluorophore [37]. Figure S2 (a) and
(b) (Supplementary material) shows the synchronous flu-
orescence spectra of HEWL in the presence of Col at
Dk = 60 nm and Dk = 20 nm, respectively. It is evident
from Fig. S2 (a) and (b) that when Dk was set at 60 nm, the
fluorescence intensity decreases with increase in the Col
concentration with a slight blueshift in wavelength maxi-
mum clearly indicating that the polarity around the tryp-
tophan residue decreases which causes an increase in
hydrophobicity. On the other hand, when Dk was set at
20 nm, there is a decrease in fluorescence intensity, but the
position of the wavelength maximum does not change
which implies that the microenvironment around Tyr
residue does not change. The effect of Col near Trp residue
is more than Tyr as it is seen from Fig. S2(c) (Supplemen-
tary material).
3D fluorescence spectra analysis
The three-dimensional fluorescence spectra of HEWL in
the absence and presence of Col are shown in Fig. 4, and
the corresponding parameters are listed in Table 3. The
useful information about the conformational and microen-
vironmental changes of HEWL is obtained by comparing
the spectral changes in HEWL with and without Col. As
420 mainly reveals the spectral behaviors of tryptophan and
tyrosine residues. The maximum emission wavelength and
the fluorescence intensity of the residues are sensitive to
the polarity of their microenvironment. Apart from peak 1,
123
1762 S. Khatun, Riyazuddeen

(a) (b)
3

0.4

log(Fo/F–1)
Fo/F 0.0

1 – 0.4

– 0.8
0
0 15 30 45 – 5.2 – 4.8 – 4.4
[Col]/μΜ log[Col]

Fig. 3 a Stern–Volmer plot for quenching of tryptophan by Col in native HEWL, b the log(Fo/F - 1) vs log[Col] plot for the interaction of Col
with HEWL

Table 2 Binding parameters

obtained from fluorescence
experiment for the binding of
Col with HEWL at
T = 298.15 K, P = 101.13 kPa
and pH = 7.4
Parameter Value
Stern–Volmer constant (Ksv) (4.29 ± 0.11) 9 104 M-1
Quenching Constant (kq) (7.52 ± 0.19) 9 1012 M-1 s-1
Binding Constant (Kb) (5.45 ± 0.14) 9 104 M-1
Stoichiometry (n) 1.03 ± 0.03
Gibbs Free Energy change (DGo) - 27.13 ± 0.68 kJ mol-1
The standard uncertainties, u are u (P) = 10 kPa and u (T) = 0.01 K
The standard uncertainty u in the molarity of the buffer is u (m) = 0.04 9 10-3 mol kg-1
The reported uncertainties are combined expanded uncertainties at 0.95 level of confidence (k = 2)

there is another strong peak 2 (kex = 230 nm,
kem = 330 nm) representing the fluorescence spectral
behavior of polypeptide backbone structure [38]. With the
addition of Col, the fluorescence intensities of both peak 1
and peak 2 decrease which indicates that the binding of Col
to HEWL induces conformational changes in HEWL.
and 3.45 nm, respectively. The average distance between
HEWL and Col is within the range of 2–7 nm, and the
reliability of Ro and r values is evident from the satisfying
the required criteria of 0.5Ro \ r \ 1.5Ro [40]. These
results concluded that the energy transfer from HEWL to
Col occurs with high probability.

Energy transfer between HEWL and Col
In the process of Forster’s resonance energy transfer, donor
fluorophore (HEWL) in its excited state transfers energy to
an acceptor molecule (Col) through non-radiative dipole–
dipole coupling [39]. The efficacy of this energy transfer
has been used to estimate the distance (r) between Col and
HEWL. Energy transfer process mainly depends on mag-
nitude of overlap between emission spectrum of donor and
absorption spectrum of acceptor. As it can be seen in Fig. 5
that the emission spectrum of HEWL is well overlapped
with absorption spectrum of Col which indicates that there
is possibility of energy transfer from excited state of
HEWL to Col. The values of J, EFRET , Ro and r were
evaluated according to Eqs. (4–6) and using n = 1.336 and
u = 0.14 for the HEWL-Col system and are listed in
Table 4. The values of Ro and r were found to be 2.76 nm
Mode of binding and thermodynamic parameters
by ITC
In order to measure accurate and detailed thermodynamic
profile of binding of Col with HEWL, we performed ITC
experiment at T = 303.15 K and at pH = 7.4. The upper
panel of Fig. 6 shows the raw data for sequential injections
of the drug into the protein solution, while the lower panel
depicted the positive heat deflection in the plot of the
amount of heat liberated per injection as a function of the
[Col]/[HEWL] ratio. The heat of dilution (Col solution into
phosphate buffer solution) is subtracted from HEWL-Col
titration data. The association constant (Ka), standard
enthalpy change (DH°), and standard entropy change (DS°)
have been directly obtained after the best nonlinear least-
squares fitting for the integrated heats by using two site
sequential binding model [41], and the thermodynamic

123
A comprehensive calorimetric, spectroscopic, and molecular docking investigation to probe... 1763

Fig. 4 Three-dimensional (a)

fluorescence spectra of HEWL LYS 10
350
in a absence and b presence of
Col at pH = 7.4,
[HEWL] = 10 lM,
[Col] = 25 lM 1000
300

EX/nm
350

EX/nm
250 300
250
0
300 400 500
300 400 500
EX/nm
EX/nm

(b)
LYS 10+COL25
350

1000
300
EX/nm

350

EX/nm
300
250
250 0
300 400 500
EX/nm
300 400 500
EX/nm

Table 3 Three-dimensional fluorescence characteristic parameters of also decipher valuable information about the different
HEWL and HEWL–Col system at T = 298.15 K, P = 101.13 kPa and binding forces involved. The positive standard enthalpy
pH 7.4, [HEWL] = 10 lM, [Col] = 25 lM change (DH°) suggested that the binding is an endothermic
Systems Peak no. Peak position Peak intensity process. The positive DH° and TDS° values indicated that
[kex/kem (nm/nm)] hydrophobic interactions play a major role in stabilizing
HEWL 1 280/340 641.8
the HEWL-Col complex [42, 43]. The condition
(1: 0) 2 230/330 184.2
|TDS°| [ |DH°| for both sites shows that the binding of Col
HEWL ? Col 1 280/330 361.9
with HEWL is entropically driven process. The negative
standard Gibbs free change (DG°) calculated from Eq. 7
(1: 2.5) 2 230/330 40.90
suggests that the binding of Col with HEWL is a sponta-
The standard uncertainties, u are u (P) = 10 kPa and u (T) = 0.01 K neous process. The order of the overall binding constant K0
The standard uncertainty u in the molarity of the buffer is (= K1 K2 = 7.54 9 104 M-1) obtained from ITC is con-
u (m) = 0.04 9 10-3 mol kg-1
sistent well with the order of Kb obtained from our fluo-
The reported uncertainties are combined expanded uncertainties at
rescence study [44].
0.95 level of confidence (k = 2)

Secondary structure analysis by CD spectroscopy

CD is a sensitive spectroscopic technique to study protein

parameters obtained from this fitting are listed in Table S1 secondary and tertiary structures. Proteins rich in alpha
(Supplementary material). We have found that the binding helices show two negative bands at 208 nm (p–p* transi-
constants values, K1 = 1.19 9 103 M-1 and tion) and 222 nm (n–p* transition) in the CD spectra when
-1
K2 = 63.4 M , are referred as high and low affinity sites, recorded in far-UV range [45]. Upon binding with ligand,
respectively. The values of the thermodynamic parameters the intermolecular forces responsible for sustaining the
not only give the energies associated with the reaction but secondary and tertiary structure may get rearranged,

123
1764 S. Khatun, Riyazuddeen

Fig. 5 Spectral overlaps of 1.2 1.2

a fluorescence spectrum of
HEWL with, b absorption
spectrum of Col
1 1
(a)

Fluorescence intensity
0.8 0.8

Absorbance
0.6 (b) 0.6

0.4 0.4

0.2 0.2

0 0
320 340 360 380 400 420 440
Wavelength/nm

Table 4 FRET parameters from steady-state measurements per- Time/min

formed at T = 298.15 K, P = 101.13 kPa and pH 7.4 0 20 40 60
4.00
Parameter Value

J/cm-3 M-1 (1.73 ± 0.043) 9 10-14

3.00
Ro/nm 2.764 ± 0.07
r/nm 3.456 ± 0.09
EFRET 0.207 ± 0.005 2.00
µW

The standard uncertainties, u are u (P) = 10 kPa and u (T) = 0.01 K

The standard uncertainty u in the molarity of the buffer is 1.00
u (m) = 0.04 9 10-3 mol kg-1
The reported uncertainties are combined expanded uncertainties at
0.95 level of confidence (k = 2) 0.00

16.0
resulting in the conformational alteration in the protein.
kJ mol of injectant

Figure S3 (Supplementary material) shows the far-UV CD

spectra of HEWL in the absence and presence of Col (1:0,
12.0
1:2 and 1:4). The Col-induced alterations in secondary
structures of HEWL are calculated from Eqs. 9–10 and
–1

summarized in Table S2 (Supplementary material). The

data revealed that on increasing the concentration of Col, 8.0
the % a-helical content decreases which illustrated that the
secondary structure of HEWL destabilized by Col binding.
0 2 4
FTIR characterization of HEWL-Col system
Molar ratio
FTIR spectroscopy was employed to extract information on Fig. 6 ITC isotherm for the interaction of Col with HEWL. The upper
the secondary structures of HEWL in the presence of Col. panel shows calorimetric response as successive injections of Col into
FTIR spectra of proteins exhibit number of amide bands the sample cell containing HEWL. The bottom panel represents
integrated heats of interaction as a function of [Col]/[HEWL] molar
(amide I, II and III) which represent different vibrations of ratio

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A comprehensive calorimetric, spectroscopic, and molecular docking investigation to probe...
Absorbance
1800 1500
Wavenumber/cm
–1
Fig. 7 FTIR spectra of HEWL in the absence and presence of Col.
The concentrations of HEWL and Col were fixed at 75 lM
the peptide moiety. The amide I band appears at
1700–1600 cm-1 principally attributed to the C=O
stretching vibration of the backbone amide groups while
amide II band near 1600–1500 cm-1 is associated with C–
Fig. 8 Most stable docked pose
of Col on the active site of
HEWL: a Cartoon
representation (Col as ball–stick
while HEWL by ribbon model),
b molecular surface
representation, c close view of
Col with nearby amino acid of
HEWL and d 2D diagram by
discovery studio 4.5 with amino
acid residues interacting with
Col
1765
N stretch and N–H bending modes. Both bands are sensi-
1:0
tive to the changes in the protein secondary structure
1:1
[46, 47] and amide I is more sensitive to the changes in
secondary structure of protein compared to amide II. The
FTIR spectra of HEWL in the absence and presence of Col
are shown in Fig. 7. The peak intensities of amide I and
amide II change along with the shifting in peak position
from 1640 to 1642 cm-1 for amide I and from 1566 to
1574 cm-1 for amide II band upon the addition of Col to
HEWL solution. This substantiates that the secondary
structure of HEWL is perturbed due to the interaction with
Col.
Molecular docking study of HEWL-Col interaction
1200
The most favored binding mode of Col to its binding site on
HEWL was predicted by molecular docking analysis. X-ray
studies on HEWL and its complexes revealed that there are six
Trp residues in HEWL and three of them (Trp 62, Trp 63, and
Trp 108) are located in the active sites [48]. Auto Dock Vina
program resulted in nine conformations with increasing Gibbs
free energy change and the best-fit conformation is that of
with lowest binding energy. The best docked conformation of
Col with HEWL is shown in Fig. 8a, b. Figure 8(c) shows that
the active site of HEWL is made up of the residues: Gln 57, Ile
123
1766
58, Asn 59, Arg 61, Trp 62, Ile 98, Asn 106, Ala 107, and Trp
108 are mainly responsible for hydrophobic and van der
Waals interactions. Col also forms hydrogen bonds with Trp
63 and Arg 112 residues in the active site of HEWL are shown
in Fig. 8d. Similar kinds of results were also reported in lit-
erature for the binding of flavokawain B and vandetanib with
lysozyme [49, 50]. Col was found to interact with HEWL with
binding free energy of - 28.56 kJ mol-1 which is also con-
sistent with the experimental results (- 28.01 kJ mol-1
obtained by ITC and - 27.132 kJ mol-1 by fluorescence
quenching, respectively).
Conclusions
We studied the binding of Col with HEWL at molecular
level under physiological condition (pH = 7.4) using UV–
Vis, fluorescence emission, synchronous, 3D fluorescence,
FRET, ITC, CD, FTIR, and molecular docking methods.
The results of UV–Vis absorption and fluorescence experi-
ments manifest the formation of ground-state HEWL–Col
complex and observed quenching of fluorescence intensity
of HEWL is static in nature. The distance (r = 3.45 nm)
obtained from FRET method indicates that donor (HEWL)
and acceptor (Col) are in close proximity to each other.
Thermodynamic analysis by ITC for the binding process
between Col and HEWL yielded positive DH° and TDS°
values suggesting involvement of hydrophobic interactions
in stabilizing the HEWL–Col complex. The order of the
overall binding constant (104) obtained from ITC is same as
obtained from fluorescence measurements. Evaluation of
secondary structure by far-UV CD and the FTIR results
clearly indicated that Col altered the secondary structure of
HEWL. The molecular docking result showed that the Col
enters the hydrophobic cleft of active site of HEWL near
Trp-62, Trp-63 and Trp-108 amino acid residues and also
form specific hydrogen bonds with Trp-63 and Arg-112
residues. The information obtained in this study could help
to shed light on the interaction mechanism between Col and
HEWL. It could also guide the design of new drugs for the
treatment of variety of diseases associated with Col.
Acknowledgements Authors are thankful to the Chairman, Depart-
ment of Chemistry, A.M.U, Aligarh, for providing the necessary
facilities to carry out this research work. The PURSE and FIST grants
from DST and SAP (DRS-II) grant from UGC, New Delhi are
gratefully acknowledged. One of the authors (S.K.) gratefully
acknowledge to UGC, New Delhi, for awarding Maulana Azad
National Fellowship (MANF).
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