Molecular interaction of an ester-functionalized biodegradable gemini surfactant with lysozyme: Insights from spectroscopy, calorimetry and molecular docking

Article abstract of DOI:10.1016/j.molliq.2015.10.014

Designing and compilation of novel chemical molecules to optimize the structural characteristics of biomolecules is an interesting and fascinating domain of research at the interface of chemistry and molecular biology. In this context, we have synthesized a green/biocompatible gemini surfactant, ethane-1, 2-diyl bis(N,N-dimethyl-N-hexadecylammoniumacetoxy) dichloride (16-E2-16), and examined its interaction with the model enzyme hen egg white lysozyme (HEWL) utilizing sophisticated spectroscopic, microscopic, calorimetric and molecular modeling techniques. The results obtained through multidimensional approach demonstrate that 16-E2-16 is able to influence the structural aspects of HEWL. The intrinsic fluorescence and UV spectroscopic results reflect HEWL-16-E2-16 complex formation. Synchronous, three-dimensional and pyrene fluorescences show substantial changes in microenviroment around tyrosine and tryptophan residues. CD results demonstrate conformational change in HEWL upon 16-E2-16 combination. ITC suggests the contribution of hydrophobic forces and spontaneous nature of 16-E2-16-HEWL interaction. Molecular modeling confirms the binding of 16-E2-16 gemini surfactant near predominant fluorophores (Trp-62/Trp-108). TEM micrographs infer structural changes in HEWL. This study is thought to have good potential to help scientists to further interpret the surfactant-HEWL interaction at the molecular level, which will be significant to compile surfactant-protein mixtures in general for pharmaceutical and industrial purposes.

Full text of DOI:10.1016/j.molliq.2015.10.014

Journal of Molecular Liquids 212 (2015) 641–649
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Molecular interaction of an ester-functionalized biodegradable gemini
surfactant with lysozyme: Insights from spectroscopy, calorimetry and
molecular docking
1
⁎
Mohd. Akram , Imtiyaz Ahmad Bhat, Sana Anwar, Kabir-ud-Din
Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 August 2015
Received in revised form 3 October 2015
Accepted 6 October 2015
Available online 11 November 2015
Designing and compilation of novel chemical molecules to optimize the structural characteristics of biomolecules
is an interesting and fascinating domain of research at the interface of chemistry and molecular biology. In this
context, we have synthesized a green/biocompatible gemini surfactant, ethane-1, 2-diyl bis(N,N-dimethyl-N-
hexadecylammoniumacetoxy) dichloride (16-E2-16), and examined its interaction with the model enzyme
hen egg white lysozyme (HEWL) utilizing sophisticated spectroscopic, microscopic, calorimetric and molecular
modeling techniques. The results obtained through multidimensional approach demonstrate that 16-E2-16 is
able to influence the structural aspects of HEWL. The intrinsic fluorescence and UV spectroscopic results reflect
HEWL-16-E2-16 complex formation. Synchronous, three-dimensional and pyrene fluorescences show substan-
tial changes in microenviroment around tyrosine and tryptophan residues. CD results demonstrate conforma-
tional change in HEWL upon 16-E2-16 combination. ITC suggests the contribution of hydrophobic forces and
spontaneous nature of 16-E2-16-HEWL interaction. Molecular modeling confirms the binding of 16-E2-16 gem-
ini surfactant near predominant fluorophores (Trp-62/Trp-108). TEM micrographs infer structural changes in
HEWL. This study is thought to have good potential to help scientists to further interpret the surfactant–HEWL
interaction at the molecular level, which will be significant to compile surfactant–protein mixtures in general
for pharmaceutical and industrial purposes.
Keywords:
16-E2-16
Lysozyme
Quenching
ITC
Docking
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
better physicochemical properties than conventional single head/single
tail surfactants. They have lower CMC values, much higher surface activ-
Protein–surfactant interaction is a vital and fascinating concept to
understand the conformational, mechanistic and other relevant func-
tional aspects of proteins. Proteins, being the fundamental constituents
of life, have characteristic aptitude to bind to inorganic/organic moieties
(bilirubin, fatty acids, hematin, metal ions, surfactants, drugs [1–8]). As
protein–surfactant combinations are known to induce stabilization/
destabilization in the former, these studies are always motivating. In ad-
dition, wide scope and significance of protein–surfactant mixtures in
biosciences, foods, cosmetics, detergents and biotechnological process-
es make them even more captivating in the scientific community. In
such systems, however, surfactant design is crucial in governing the
rhythm of interactions.
The literature survey reveals that most studies regarding protein–
surfactant interactions are focused to conventional surfactants [9,10].
Recently, gemini surfactants, bearing two hydrophilic heads and two
hydrophobic tails connected covalently through a spacer (at or near
the head groups), are receiving considerable interest owing to their
ity, lower Krafft points, high viscosities at quite low concentrations, etc.
[11–13]. They are used as promising surfactants in detergency, gene
transfection, ion exchange, vesicle formation, environmental protection,
antimicrobial activity, skin/body care products [14], food industry [15],
gene delivery [16], drug entrapment/release [17], antimicrobial prod-
ucts [18],etc. Despite these outstanding properties, most of them are
non-biodegradable and hence pose environmental concerns. Therefore,
cleavable surfactants (conventional as well as gemini) are of keen inter-
est in the scientific community.
The defined cleavable surfactant (16-E2-16) is essential owing to the
presence of weak diester (E2) linkage in the spacer. This ester structure
makes it biodegradable, cleavable and lowers its CMC (1000 times less
than the conventional ones). Thus, 16-E2-16 surfactant is a significant
entity to be taken up for further studies.
The monomeric globular protein hen egg white lysozyme (HEWL)
contains 129 amino acid residues with four disulfide bonds in its ter-
tiary structure. Its structural architecture involves two domains viz.,
α-domain (1–39 and 89–129 amino acid residues) and β-domain
(40–88 amino acid residues [19,20]). It possesses a rigid structure [21]
and has six Trp residues arranged, respectively, at active site (3 resi-
dues), hydrophobic core (two residues) and separate (one residue).
⁎
Corresponding author.
E-mail address: drmohdakram@rediff.com (M. Akram).
Present address: Department of Chemistry, Arba Minch University, Ethiopia.
1
http://dx.doi.org/10.1016/j.molliq.2015.10.014
0167-7322/© 2015 Elsevier B.V. All rights reserved.

642
M. Akram et al. / Journal of Molecular Liquids 212 (2015) 641–649
The predominant fluorescence of lysozyme is mainly due to the Trp 62
and Trp 108 fluorophores [22].
the literature procedure [26–29]. The synthesis protocol is shown in
Scheme 1. Working solution concentrations were: [HEWL] = 20 ×
10−6 M, [PB] = 12 × 10−3 M, pH = 7.4.
In this work we have examined the molecular interaction of ester-
functionalized green/biocomapatible gemini surfactant, ethane-1, 2-
diyl bis(N,N-dimethyl-N-hexadecylammoniumacetoxy) dichloride
(16-E2-16) with HEWL. Lysozyme was chosen owing to its physiologi-
cal and pharmaceutical properties. Moreover, in our recent studies,
16-E2-16 surfactant was observed to be quite effective in modulating
BSA fibrils [23], Therefore, we believe that HEWL-16-E2-16 interaction
too will serve the means to further interpret heredity-systematic amy-
loidosis disease (in vitro) by taking HEWL as model. Other reason
being the structural homology of HEWL with human lysozyme and
both form quite similar fibrils [24,25].
2.2. Methods
2.2.1. Spectroscopic measurements
The steady-state fluorescence spectra were obtained using a Hitachi
F-2700 fluorescence spectrophotometer (Japan), equipped with a PC.
Prior to experiment, the instrument parameters were set as: excitation
wavelength (280 nm), emission wavelength range (300–450 nm), and
slit width (5 nm). Three-dimensional fluorescence was recorded in
the range of 250–600 nm, keeping other parameters relevant to
steady-state fluorescence. Synchronous fluorescence spectra were ob-
served from the same instrument keeping Δλ between the excitation
and emission wavelengths equal to 20 and 60 nm, respectively. For ob-
servation of extrinsic fluorescence spectra, excitation wavelength was
kept at 337 nm and emission spectra were recorded in the range of
350–450 nm. For UV absorption spectra recording (200–330 nm),
Perkin Elmer Lambda-25 (Singapore) was used. CD measurements
were performed on Jasco-J-815 spectropolarimeter with instrument
parameters as: temperature (298 K), scan range (200–250 nm), scan
speed (100 nm/min), and response time (1 s).
Keeping the above facts in consideration, we can conclude that this
study can be significant in biomedical, pharmaceutical and industrial
(cosmetics, food and feed) realms at molecular level.
2. Materials and methods
2.1. Materials
Chloroacetyl chloride (98%, Loba-Chemie, India), ethylene glycol
(99%, Sigma Aldrich, USA), N,N-dimethylhexadecylamine (95%, Sigma
Aldrich, USA), hen egg white lysozyme (HEWL, Sigma Aldrich, USA),
and pyrene (98%, Acros Organics, Belgium) were used as received. The
gemini surfactant, ethane-1,2-diyl bis(N,N-dimethyl-N-hexadecyl-
ammoniumacetoxy) dichloride (16-E2-16), was synthesized following
2.2.2. ITC measurements
Isothermal titration calorimetry was performed using a Microcal
ITC-200 Calorimeter (USA). Gemini surfactant titrants were dissolved
Scheme 1. Protocol for 16-E2-16 gemini surfactant synthesis.

M. Akram et al. / Journal of Molecular Liquids 212 (2015) 641–649
643
in the 12 mM PB (pH 7.4). 20 μM HEWL samples were placed in the
3. Results and discussion
reaction cell, and the reference cell was filled with the buffer. All titra-
tions were performed at 298 K. After temperature equilibration, the
titrant was injected sequentially in 2 μl aliquots increments at 120 s
interval with stirring at 500 rpm to ensure a complete equilibration.
The resulting heats of reaction were measured upto 19 consecutive
injections.
3.1. Fluorescence quenching of HEWL by 16-E2-16 and the binding
mechanism analysis
Steady-state fluorescence is an effective method to study interac-
tions between small molecules and proteins. Fig. 1 (a) shows the fluo-
rescence spectra of HEWL with increasing concentration of 16-E2-16
gemini surfactant which reveals quenching of fluorescence intensity of
HEWL with slight blue shift (~3 nm). This behavior may be attributed
to the HEWL-16-E2-16 complex formation and an exposure of aromatic
residues (mainly Trp/Tyr) to a non-polar environment.
Fluorescence quenching usually proceeds through two mechanisms:
static and the dynamic. The static mechanism involves the formation of
ground state complex between fluorophore and quencher while dy-
namic quenching proceeds through the formation of excited state com-
plex. A better understanding of the quenching mechanism can be
reached from quantitative analysis of the experimental data. One such
method involves the application of Stern–Volmer Eq. (1)
2.2.3. Molecular modeling measurements
Molecular modeling was performed by using molecular graphics
program HEX 6.1 [30]. The PDB (1 DPX) of HEWL was downloaded
from the protein data bank and PDB files of 16-E2-16 surfactant
were developed by using Chimera 1.9 and visualization of docked
conformation was achieved by using Pymol [31]. The docking exper-
iment was run on computer using Windows 7 as an operating
system.
2.2.4. TEM measurements
Transmission electron microscopy was performed on a JEM-2100
(JEOL, Japan) electron microscope. The samples of HEWL and
HEWL + 16-E2-16 were prepared by negative staining method [32,
33]. Then TEM photos were pictured after thorough drying of the
samples.
F0
F
¼ 1 þ KSV½Qꢀ
ð1Þ
where F0 and F represent the fluorescence intensities in the absence and
presence of quencher, [Q] and KSV, respectively, indicate the quencher
Fig. 1. The fluorescence quenching results of the lysozyme HEWL/16-E2-16 gemini surfactant system:(a) quenching profiles with varying concentrations of the surfactant, (b) the Stern–
Volmer plot, and (c) the modified Stern–Volmer plot.

644
M. Akram et al. / Journal of Molecular Liquids 212 (2015) 641–649
concentration and the Stern–Volmer quenching constant. The KSV
(8.8 × 104 L mol−1), obtained from slope of Fig. 1 (b), reveals substantial
quenching of the emission spectra of HEWL by 16-E2-16. The KSV value
was then utilized to obtain quenching rate constant (kq) from the fol-
lowing equation
slope (n) of plot (Fig. 1 (c)) were used to compute the binding constant
and number of binding sites, respectively
logfðF0−F=FÞg ¼ log Kb þ n log½16‐E2‐16ꢀ
ð3Þ
The obtained value of binding constant (Kb = 6.3 × 104 L mol−1
)
KSV
τ0
is significant which discloses convincing binding of 16-E2-16 to
HEWL and the value of n = 1.48 infers the single pattern of binding
sites on HEWL. Further, binding process was observed to be spontane-
ous and thermodynamically favored as the ΔGob value (obtained by
employing equation ΔGob = −RT lnKb) was found to be negative
(−6.55 kcal mol−1).
Further authentication to HEWL-16-E2-16 complex formation was
provided by three-dimensional fluorescence. It gives reliable informa-
tion about the microenvironment changes around aromatic residues.
Fig. 2 (a–b) represents the relevant spectra of HEWL in the absence
and presence of 16-E2-16. It can be seen that HEWL displays two
kq
¼
ð2Þ
Here, τ0 is the life time of the fluorophore and is 10−8 s for biomol-
ecules [34]. Magnitude of the evaluated kq (8.8 × 1012 L mol−1 s−1) is
greater than the scatter constant (2 × 1010 L mol−1 s−1 [35]). Therefore,
it can be concluded that the quenching proceeds by means of static
procedure rather than dynamic and involves the ground state complex
formation between 16-E2-16 and HEWL. Moreover, to compute the
binding constant (Kb) and number of binding sites, the modified
Stern–Volmer Eq. (3) was used. The antilog of intercept (logKb) and
Fig. 2. Three-dimensional fluorescence spectra of lysozyme (HEWL) in the absence (a), and presence (b) of 16-E2-16 gemini surfactant.

M. Akram et al. / Journal of Molecular Liquids 212 (2015) 641–649
645
peaks. Peak 1 corresponds to Rayleigh scattering and peak 2 is a contri-
bution from the aromatic residues (Tyr/Trp). Fig. 2(b) shows that the
16-E2-16 addition leads to decrease in the fluorescence intensity of
the aromatic residue peak, which infers distinct change in the molecular
microenvironment in the vicinity of Tyr/Trp-residues. The reason for
this quenching can be attributed to the higher chain length of 16-E2-
16. Higher chain length lets 16-E2-16 to form micelles quite readily,
consequently leading to an extended structure of HEWL with exposed
hydrophobic patches. The quenching of fluorescence peak can also be
explained by assuming HEWL anion with considerable negative sites.
This negatively charged surface of HEWL interacts with the positively
charged head groups of 16-E2-16 gemini surfactant, which favors miti-
gation of the electrostatic repulsions; this enhances the aggregation of
16-E2-16 moieties on to the protein surface and hence produce
quenching.
3.2. Synchronous fluorescence
Microenvironment fluctuation around fluorophores (Tyr/Trp) was
also monitored by synchronous fluorescence. As compared to conven-
tional fluorescence, synchronous fluorescence is considered suitable
due to its specificity towards aromatic residues. It also mitigates spectral
overlaps by thinning spectral bands and simplifies spectra by using an
appropriate wavelength [36]. Keeping the Δλ between the
excitation and emission wavelengths at 20 and 60 nm, respectively,
microenvironment changes around tyrosine and tryptophan residues
can be probed [36]. The relevant synchronous fluorescence spectra
(Fig. 3(a–b)) reveal that addition of 16-E2-16 gemini surfactant pro-
duces quenching around both the fluorophores (Tyr or Trp), indicating
the interaction and complexation of 16-E2-16 with HEWL. This
quenching in fluorescence is due to an exposure of an aromatic residue
to hydrophobic tail of 16-E2-16. Interactions are more effectively
evident in Fig. 3(b) than in Fig. 3(a); the reason being a higher affinity
of 16-E2-16 molecules towards tryptophan (Trp) than tyrosine (Tyr)
residue. This fact is in coherence with the results obtained in steady–
state fluorescence.
3.3. Pyrene fluorescence and micropolarity assay
To add further confirmation to the fact that there is a structural
change in HEWL upon 16-E2-16 gemini surfactant combination, pyrene,
an extrinsic fluorescent probe, was utilized. Pyrene excites at 337 nm
and has characteristic five peak vibronic spectra (Fig. 4 (a)) in the
scan range of 350–450 nm. It is interesting to note that in the absence
of gemini surfactant, the fluorescence peak is having low intensity
(Fig. 4 (a)), suggesting the occurrence of pyrene in the hydrophobic do-
mains of HEWL. The addition of 16-E2-16 gemini surfactant leads to an
increase in the fluorescence intensity (Fig. 4 (a)), depicting an exposure
of buried hydrophobic patches of HEWL to a non-polar environment
exerted by the surfactant. After few additions, the fluorescence intensity
starts diminishing (Fig. 4 (a)), which indicates migration of the probe
molecules into the 16-E2-16 microstructures. Moreover, the local polar-
ity index (F1/F3 ratio) of the protein solution was also monitored and
calculated from the first and third vibronic peaks of pyrene [32]. The re-
sults are shown in Fig. 4 (b). A careful observation of Fig. 4 (b) reveals
that the HEWL solution has a lower F1/F3 value, which again confirms
the localization of pyrene in the hydrophobic network. Subsequent ad-
ditions of 16-E2-16 lead to an increase in the F1/F3 ratio, followed by
near constancy at higher concentrations of the surfactant. The increas-
ing trend can be attributed to the enzyme unfolding, and the unchang-
ing micropolarity at higher concentrations can be assigned to abundant
aggregate microstructures formed by the 16-E2-16 gemini surfactant.
Thus, from the micropolarity assay, we can conclude that there are sub-
stantial structural changes in the HEWL solution upon gemini surfactant
combination.
Fig. 3. Synchronous spectra of HEWL in the presence of varying concentrations of 16-E2-
16 at 298 K (a) Δλ = 20 nm (represents contributions due to tyrosine), and (b) Δλ =
60 nm (represents contributions due to tryptophan).
3.4. Far-UV circular dichroism analysis
To further validate and support the above findings concerning the
effect of 16-E2-16 gemini surfactant on the secondary structure of
HEWL, circular dichroism (CD) studies were performed. CD is regarded
as a very consistent technique to authenticate the conformational
changes in proteins [37,38]. The CD spectra of HEWL in presence of dif-
ferent concentrations of 16-E2-16 gemini surfactant are given in Fig. 5.
The spectra show two characterized α-helix negative peaks, respective-
ly, at 220 and 208 nm. Subsequent addition of 16-E2-16 leads to de-
crease in the negative ellipticity, inferring the structural change in
HEWL. The quantitative estimation of percentage of α-helicity was ob-
tained by converting the obtained CD data into mean residue ellipticity
(MRE), by employing the following equation
Observed CD ðm degreeÞ
MRE ¼
ð4Þ
Cpnl ꢁ 10

646
M. Akram et al. / Journal of Molecular Liquids 212 (2015) 641–649
where Cp, n and l, respectively, represent the molar concentration of
protein, number of amino acid residues (129 in HEWL) and path-
length (1 mm).The α-helical content of free and combined systems of
HEWL from MRE values (at θ = 208 nm) were then calculated by
employing the following equation [39] and the results obtained are
listed in Table 1
MRE208−4000
33; 000−4000
α‐Helix ð%Þ ¼
ꢁ 100:
ð5Þ
It can be observed from Table 1 that the addition of gemini surfactant
leads to a reduction in the negative helicity, indicating that the binding
of 16-E2-16 gemini surfactant to HEWL induces change in the second-
ary structure. At low surfactant concentration, there is little change in
the helicity but, at higher surfactant concentration, helicity decreases
appreciably. The reason for this structural change can be attributed to
preferential adsorption (at high surfactant concentration) of 16-E2-16
molecules on the HEWL surface which leads to a sort of bulging of the
enzyme; this, in turn, exposes the hydrophobic residues and ultimately
α-helices are broken to give a more open disordered structure. Thus, we
conclude that gemini surfactant at higher concentration disrupts the α-
helical network and leads to a more open, random, solvent-exposed
protein structure. This indicates significant perturbation of the surface
tryptophan and tyrosyl residues. Thus, 16-E2-16 gemini surfactant
perturbs the structure of the protein surface, mediating the water
layer and microenvironment around the superficial aromatic residues.
This finding also supports the fluorescence quenching of tryptophan
and tyrosyl residues upon their exposure to the outer environment.
Thus, our fluorescence results are in corroboration with the CD results.
3.5. UV–visible study
UV–visible absorption studies are widely applied to analyze
the structural changes and complex formation between ligands and
proteins. The absorbance at 280 nm is due to the aromatic residues
(Trp or Tyr) of HEWL and is a sensitive parameter to confirm the
micropolarity changes as well as complex formation between ligand
and enzyme. This absorbance is mainly attributed to π–π* transitions
of aromatic residues. Fig. 6 shows the absorption spectra of HEWL
with increasing concentration of 16-E2-16 gemini surfactant. Clearly,
the absorbance at 280 nm decreases with an increase in the concen-
tration of 16-E2-16. This decrease in absorbance infers the change in
the microenvironment around aromatic residues [40] and reflects the
inducement of structural change thereupon. A careful observation of
the spectra also indicates the presence of slight blue shift (~5 nm), infer-
ring an exposure of hydrophobic residues to the outer environment. In
other words, we can conclude that binding of the 16-E2-16 gemini sur-
factant to HEWL induces unfolding in the latter.
Fig. 4. (a) Pyrene emission spectra of lysozyme (HEWL) in the presence of varying
amounts of 16-E2-16 surfactant, and (b) variation of F1/F3 ratio with the 16-E2-16 surfac-
tant concentration.
3.6. Isothermal titration calorimetry (ITC)
ITC is an interesting and novel approach to validate interactions be-
tween proteins and ligands. Its multidimensional approach makes
it even more attractive and appropriate for protein/drug/surfactant
interactions. The ITC titration profile of HEWL with different molar ra-
tios of 16-E2-16 is shown in Fig. 7. Each peak in the thermogram
Table 1
Variation of α-helical content of HEWL as function of 16-E2-16
concentration.
[16-E2-16] (mM)
α-Helix (%)
0
30.68
29.79
25.11
20.90
15.86
0.15
0.85
1.40
1.75
Fig. 5. Far UV-CD spectra of lysozyme (HEWL) in the presence of varying amounts of 16-
E2-16 surfactant.

M. Akram et al. / Journal of Molecular Liquids 212 (2015) 641–649
647
Fig. 6. Absorption spectra of lysozyme (HEWL) with varying amounts of 16-E2-16
surfactant.
(upper panel) represents a single injection of 16-E2-16 into the HEWL
solution. All the peaks are upward, indicating that reactions mainly
proceed through endothermic event. The integrated and normalized
patterns are shown in Fig. 7 (lower panel). It is evident from Fig. 7
(lower panel) that the consecutive additions of 16-E2-16 gemini surfac-
tant in to HEWL solution induce reduction in the endothermicity which
evidences the interaction of 16-E2-16 with HEWL. Careful observation
of the Fig. 7 (lower panel) reveals that all the observed enthalpy values
are positive, suggesting rupture of hydrogen bonding [41] during the
HEWL and 16-E2-16 interactions. The reason for this structural reorien-
tation are hydrophobic interactions [42], which are supposed to induce
random arrangement of solvent molecules during the interaction.
Moreover, the obtained calorimetric data are given in Table 2. It is inter-
esting to note from Table 2 that ΔH° and ΔS° are positive, indicating that
van der Walls forces are not operating in the 16-E2-16-HEWL system
and predominance of hydrophobic interactions. Further, ΔG°b value are
found to be negative, indicating interaction of 16-E2-16 with HEWL is
spontaneous. This fact is in coherence with our fluorescence results, in
which we have also obtained negative value of ΔG°b. However, on com-
paring the magnitude of calorimetric parameters with fluorescence
binding it is clear that in ITC Kb as well ΔG°b are found higher in magni-
tude than the fluorescence, reason being ITC measures the global
change (i.e. enthalpy change of the binding reaction as well as the en-
thalpy of all possible concomitant reactions which may accompany
the binding reactions but not directly influence the Kb) while as fluores-
cence is specific to aromatic residues [43]. Further the obtained binding
stoichiometry in ITC suggests the possibility of two binding sites which
is different from one binding site approximation of fluorescence. This
may be due to absence of tryptophan residue in the close vicinity of
binding event captured by fluorescence. Other reason for higher values
of binding parameters obtained in ITC may be attributed to long chain
length of 16-E2-16, as it is known that longer chains (with 16–22 carbon
number) can alter Kb values strongly [44].
Fig. 7. ITC profiles for binding of 16-E2-16 gemini surfactant to lysozyme (HEWL) at 298 K.
Arg-6, Gln-57, Glu-35, Asn-44, Thr-44, Ser-36, Asp-52, amino acid resi-
dues. It can be seen (Fig. 8 (b)) that 16-E2-16 gemini surfactant accom-
modates itself in the locality of Trp-108, Trp-62 and Trp-63 residues.
This observation supports the fluorescence quenching. Interestingly,
3.7. Computational molecular docking
Table 2
ITC derived biophysical parameters for the binding of 16-E2-16 with HEWL
at 298 K and pH 7.4.
Molecular docking is an attractive computational tool to ascertain
and validate the probable interactions of small molecules with
biomacromolecules [45], which has rational significance in molecular
recognition and drug design related phenomenon. Therefore, computa-
tional molecular docking was performed to examine the binding loci
of 16-E2-16 gemini surfactant in the HEWL. The stereo docked pose of
16-E2-16 + HEWL (Fig. 8(a)) reveals that 16-E2-16 binds in the vicinity
of Trp-111, Val-109, Trp-108, Ala-107, Trp-62, Trp-63, Ser-60, Cys-64,
16-E2-16-HEWL
n
Kb
2.6
5.6 × 1014 M−1
350.2 cal/mol
68.7 cal/mol/deg
−20.13 kcal/mol
ΔH°
ΔS°
ΔG°b

648
M. Akram et al. / Journal of Molecular Liquids 212 (2015) 641–649
Fig. 8. Docked pose of 16-E2-16 (a) in the binding site of lysozyme HEWL, and (b) shown in the close vicinity of Trp 62, Trp-63, Trp-108 and Tyr-53 aromatic residues.
the presence of 16-E2-16 gemini in hydrophobic residues infers that
hydrophobic forces mainly govern the binding process. Further, possi-
bility of hydrogen bonding interactions in the glutamine (Gln-57) and
the oxygen of the ester moiety of 16-E2-16 gemini is also evident
in docking results. The significant binding interaction energy (−320.
44 kJ mol−1) also unveils considerable interaction between 16-E2-16
and HEWL. Here, it is also interesting to note that magnitude of binding
interaction energy is different from fluorescence and ITC energy magni-
tudes; it is due to exclusion of solvent during docking experiment.
such forms were observed in untreated native HEWL (Fig. 9(a)). The
predominance of electrostatic repulsion at higher surfactant concentra-
tions were found to be attributive. Moreover, our overall results further
get support from the literature [46].
4. Conclusion
In this research article we have investigated the interaction of an
ester-functionalized green/biocompatible gemini surfactant, ethane-1,
2-diyl bis(N,N-dimethyl-N-hexadecylammoniumacetoxy) dichloride
(16-E2-16), with HEWL by employing fluorescence, absorption spec-
troscopy (UV), circular dichroism, ITC, TEM and molecular modeling.
The results obtained through various spectroscopic approaches delin-
eate that the biodegradable gemini surfactant effectively interacts
with the HEWL and causes structural alteration in the latter. Various
3.8. Transmission electron microscopy
Further validation to our overall results were added by transmis-
sion electron microscopy (TEM). The aggregated globular structures
(Fig. 9(b)) were observed in mixed systems (16-E2-16 + HEWL). No

M. Akram et al. / Journal of Molecular Liquids 212 (2015) 641–649
649
also thankful to the USIF (University Sophisticated Instruments Facility),
Aligarh Muslim University, for providing TEM (Transmission Electron
Microscopy) facility.
References
[1] A. Sułkowska, B. Bojko, J. Równicka, D. Pentak, W. Sułkowski, J. Mol. Struct. 651–653
(2003) 237–243.
[2] S. De, A. Girigoswami, S. Das, J. Colloid Interface Sci. 285 (2005) 562–573.
[3] D. Kelley, D.J. McClements, J. Food Hydrocoll. 17 (2003) 73–85.
[4] M. Vasilescu, D. Angelescu, M. Almgren, A. Valstar, Langmuir 15 (1999) 2635–2643.
[5] B.P. Kamat, J. Seetharamappa, J. Pharm. Biomed. Anal. 35 (2004) 655–664.
[6] H. Bai, X. Liu, Z. Zhang, S. Dong, Spectrochim. Acta A 60 (2004) 155–160.
[7] B. Farruggia, B. Nerli, H.D. Nuci, R. Rigatusso, G. Picó, Int. J. Biol. Macromol. 26 (1999)
23–33.
[8] A. Sulkowska, J. Mol. Struct. 614 (2002) 227–232.
[9] N.J. Turro, X.G. Lei, K.P. Ananthapadmanabhan, M. Aronson, Langmuir 11 (1995)
2525–2533.
[10] S. Ghosh, A. Banerjee, Biomacromolecules 3 (2002) 9–16.
[11] F.M. Menger, C.A. Littau, J. Am. Chem. Soc. 113 (1991) 1451–1452.
[12] R. Zana, J. Colloid Interface Sci. 248 (2002) 203–220.
[13] M.J. Rosen, Surfactants and Interfacial Phenomenon, 3rd ed. John Wiley and Sons,
New York, 2004.
[14] M. Macián, J. Seguer, M.R. Infante, C. Selve, M.P. Vinardell, Toxicology 106 (1996)
1–9.
[15] R. Zana, J. Xia, Gemini Surfactants: Synthesis, Interfacial and Solution-Phase Behav-
ior and Applications Surfactant Science Series, vol. 117, Marcel Dekker, Inc., New
York, 2004.
[16] A. Bajaj, B. Paul, S.S. Indi, P. Kondaiah, S. Bhattacharya, Bioconjug. Chem. 18 (2007)
2144–2158.
[17] C. Bombelli, G. Caracciolo, P.D. Profio, M. Diociaiuti, P. Luciani, G. Mancini, C.
Mazzuca, M. Marra, A. Molinari, D. Monti, L. Toccacieli, M. Venanzi, J. Med. Chem.
48 (2005) 4882–4891.
[18] L. Caillier, E.T.D. Givenchy, R. Levy, Y. Vandenberghe, S. Geribaldi, F. Guittard, J.
Colloid Interface Sci. 332 (2009) 201–207.
[19] M.C. Vaney, S. Maignan, M.R. Kautt, A. Ducriux, Acta Crystallogr. Sect. D: Biol.
Crystallogr. 52 (1996) 505–517.
[20] M.B. Pepys, P.N. Hawkins, D.R. Booth, D.M. Vigushin, G.A. Tennent, A.K. Soutar, N.
Totty, O. Nguyen, C.C.F. Blake, C.J. Terry, T.G. Feest, A.M. Zalin, J.J. Hsuan, Nature
362 (1993) 553–557.
[21] C.C.F. Blake, D.F. Koenig, G.A. Mair, A.C.T. North, D.C. Phillips, V.R. Sarma, Nature 206
(1965) 757–761.
[22] T. Imoto, L.S. Foster, J.A. Ruoley, F. Tanaka, Proc. Natl. Acad. Sci. U. S. A. 69 (1972)
1151–1155.
[23] Z. Yaseen, S. Rehman, M. Tabish, A.H. Shalla, Kabir-ud-Din, RSC Adv. 5 (2015)
58616–58624.
[24] L.N. Arnaudov, R. de Vries, Biophys. J. 88 (2005) 515–526.
[25] L.A. Morozova-Roche, J. Zurdo, A. Spencer, W. Noppe, V. Receveur, D.B. Archer, M.
Joniau, C.M. Dobson, J. Struct. Biol. 130 (2000) 339–351.
[26] G. Zhinong, T. Shuxin, Z. Qi, Z. Yu, L. Bo, G. Yushu, H. Li, T. Xiaoyan, Wuhan Univ. J.
Nat. Sci. 13 (2008) 227–231.
Fig. 9. TEM micrographs of (a) HEWL, and (b) HEWL + 16-E2-16.
[27] M. Akram, I.A. Bhat, Z. Yaseen, Kabir-ud-Din, Colloids Surf. A 444 (2014) 209–216.
[28] M. Akram, I.A. Bhat, Kabir-ud-Din, J. Phys. Chem. B 119 (2015) 3499–3509.
[29] M. Akram, I.A. Bhat, W.F. Bhat, Kabir-ud-Din, Spectrochim. Acta A 150 (2015)
440–450.
[30] D. Mustard, D.W. Ritchie, Proteins Struct. Funct. Bioinf. 60 (2005) 269–274.
[31] W.L. DeLano, The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos,
CA, USA, 2002.
biophysical binding parameters infer that quenching of fluorescence
spectra proceeds through static pathway rather than dynamic one. CD
results depict conformational alteration upon gemini combination.
Appearance of globular aggregated structures in transmission electron
microscopy results (TEM) validates the structural change in HEWL
and 16-E2-16–HEWL complex formation. Molecular docking provides
insight that 16-E2-16 binds near to predominant fluorophores, Trp-
108 and Trp-62. This accounts for predominant hydrophobic forces
governing the interaction, which is in well coherence with ITC results
in which a single endothermic event was also observed. Positive values
of ΔS° and ΔH° infer the predominance of hydrophobic interactions dur-
ing 16-E2-16 and HEWL interaction. Moreover, negative ΔGb° value ob-
tained in ITC suggests the spontaneous nature of concerned interactions
as was indicated by fluorescence and docking studies.
[32] P. Khullar, V. Singh, A. Mahal, H. Kumar, G. Kaur, M. Bakshi, J. Phys. Chem. B 117
(2013) 3028–3039.
[33] N.A. Fazili, A. Naeem, Cell Biochem. Biophys. 66 (2013) 265–275.
[34] M. Amiri, K. Jankeje, J.R. Albani, J. Fluoresc. 20 (2010) 651–656.
[35] Y. Ni, S. Wang, S. Kokot, Anal. Chim. Acta 663 (2010) 139–146.
[36] V.I. Martín, A. Rodríguez, A. Maestre, M.L. Moya, Langmuir 29 (2013) 7629–7641.
[37] M. Dockal, D.C. Carter, F. Rüker, J. Biol. Chem. 275 (2000) 3042–3050.
[38] Y.H. Chen, J.T. Yang, H.M. Martinez, Biochemistry 11 (1972) 4120–4131.
[39] X.L. Han, P. Mei, Y. Liu, Q. Xiao, F.L. Jiang, R. Li, Spectrochim. Acta A 74 (2009) 781–787.
[40] X. Pan, R. Liu, P. Qin, L. Wang, X. Zhao, J. Lumin. 130 (2010) 611–617.
[41] P.D. Ross, S. Subramanian, Biochemistry 20 (1981) 3096–3102.
[42] J. Tian, J. Liu, Z. Hu, X. Chen, Bioorg. Med. Chem. 13 (2005) 4124–4129.
[43] N.J. Faergeman, B.W. Sigurskjold, B.B. Kragelund, K.V. Andersen, J. Knudsen, Bio-
chemistry 35 (1996) 14118–14126.
[44] J. Rosendal, P. Ertbjerg, J. Knudsen, Biochem. J. 290 (1993) 316–321.
[45] M. Akram, I.A. Bhat, Kabir-ud-Din, Int. J. Biol. Macromol. 78 (2015) 62–71.
[46] P. Khullar, V. Singh, A. Mahal, P.N. Dave, S. Thakur, G. Kaur, J. Singh, S.S. Kamboj, M.S.
Bakshi, J. Phys. Chem. C 116 (2012) 8834–8843.
This study will be significant in understanding the protein–
surfactant interaction at the molecular level and assumes importance
in the biomedical and pharmaceutical world.
Acknowledgments
IAB gratefully acknowledges the University Grants Commission,
New Delhi, India for BSR (Basic Science Research) fellowship. We are