New insights into binding interaction of novel ester-functionalized m-E2-m gemini surfactants with lysozyme: A detailed multidimensional study

Article abstract of DOI:10.1039/c5ra20576j

In this article fluorescence spectroscopy, UV-visible spectroscopy, circular dichroism (CD), isothermal titration calorimetry (ITC), transmission electron microscopy (TEM) and molecular docking methods have been used to examine the interaction between dicationic ester-bonded gemini surfactants (m-E2-m) and hen egg white lysozyme (HEWL). The fluorescence and UV-visible absorption spectral measurements indicate m-E2-m-HEWL complex formation via static procedure. Binding isotherms reveal mainly cooperative binding of m-E2-m surfactants to HEWL. Circular dichroism, and pyrene fluorescence depict conformational changes in HEWL upon m-E2-m combination. Synchronous fluorescence shows that addition of m-E2-m has a remarkable effect on the micropolarity of aromatic residues (Tyr/Trp) of HEWL. Far-UV CD spectra demonstrate that the α-helical network of HEWL is disrupted and its content decreases from 30.68% to 20.83%/20.40%, respectively, upon 12-E2-12/14-E2-14 combination. ITC confirms the endothermicity of m-E2-m-HEWL interactions while slight exothermicity was observed in the 14-E2-14-HEWL system at higher molar ratios of surfactant. TEM micrographs reveal structural change in HEWL upon m-E2-m addition. Molecular docking illustrates that 14-E2-14 binds principally near to predominant fluorophores of lysozyme viz. Trp-108 and Trp-62 while 12-E2-12 binds in proximity of Trp-123. This study provides an important insight, particularly the contribution of Trp-123 in the fluorescence besides already known predominant fluorophores, Trp-62 and Trp-108. Moreover, this study would be significant in context of protein-surfactant interactions in terms of special m-E2-m molecular structure, which is essential in determining their future use as excipients in pharmaceutical/drug delivery related compilations.

Full text of DOI:10.1039/c5ra20576j

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New insights into binding interaction of novel ester-functionalized m-
E2-m gemini surfactants with lysozyme: A detailed multidimensional
study
Mohd. Akram*, Imtiyaz Ahmad Bhat and KabirꢀudꢀDina
Department of Chemistry, Aligarh Muslim University, Aligarh-202002, India
*Corresponding author
E-mail address: drmohdakram@gmail.com
Tel.: +91 9411040048
aPresent Address: Department of Chemistry, Arba Minch University, Ethiopia
1

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In this article fluorescence spectroscopy, UVꢀvisible spectroscopy, circular dichroism (CD),
isothermal titration calorimetry (ITC), transmission electron microscopy (TEM) and molecular
docking methods have been used to examine the interaction between dicationic esterꢀbonded
gemini surfactants (mꢀE2ꢀm) and hen egg white lysozyme (HEWL). The fluorescence and UV
measurements indicate mꢀE2ꢀmꢀHEWL complex formation via static procedure. Binding
isotherms reveal mainly cooperative binding of mꢀE2ꢀm surfactants to HEWL. Circular
dichroism, and pyrene fluorescence depict conformational changes in HEWL upon mꢀE2ꢀm
combination. Synchronous fluorescence shows that addition of mꢀE2ꢀm has a remarkable effect
on the polarity of the microenvironment in HEWL. FarꢀUV CD spectra demonstrate that the αꢀ
helical network of HEWL is disrupted and its content decreases from 30.68 % to
20.83%/20.40%, respectively, upon 12ꢀE2ꢀ12/14ꢀE2ꢀ14 combination. ITC confirms the
endothermicity of mꢀE2ꢀmꢀHEWL interactions while slight exothermicity was observed in the
14ꢀE2ꢀ14ꢀHEWL system at higher molar ratios of surfactant. TEM micrographs reveal structural
change in HEWL upon mꢀE2ꢀm addition. Molecular docking illustrates that 14ꢀE2ꢀ14 binds
principally to predominant fluorophores of lysozyme viz. Trpꢀ108 and Trpꢀ62 while 12ꢀE2ꢀ12
binds in proximity of Trpꢀ123. This study provides an important insight, particularly the
contribution of Trpꢀ123 in the fluorescence besides already known predominant fluorophores,
Trpꢀ62 and Trpꢀ108. Moreover, this study would be significant in context of proteinꢀsurfactant
interactions in terms of special m-E2-m molecular structure, which is essential in determining
their future use as excipients in pharmaceutical/drug delivery related compilations.
2

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1. Introduction
Owing to diverse implications in cosmetic, industrial, biological, and pharmaceutical
1-8
compilations,
surfactants have been extensively studied from past few decades. Nowꢀaꢀdays,
betterꢀquality and second generation surfactants, gemini surfactants, are of great interest owing
to their superior and outstanding properties than single tail/single head conventional surfactants.
Compared to conventional surfactants, gemini surfactants dispatch outstanding properties viz.,
lower critical micelle concentration (CMC), better wetting, foaming and dispersing properties.
Gemini surfactants also offer promising applications in prime research domains like gene
transfection,9 gene delivery10 and compilation of antimicrobial, skin/body care and drug
entrapment/delivery products.11,12 Despite possessing properties of much interest for their
possible endꢀuse, cationic gemini surfactants are found toxic and responsible for skin irritation,
eye infection, and related phenomenon.13-15 Therefore, much efforts are being made to
develop/design attractive featured, easily cleavable, biodegradable, environment friendly and low
toxic esterꢀbonded surfactants (such as m-E2-m, where m is the tail chain length). The
significance with ester functionalized surfactants is that they are easily cleavable and
biodegradable under variant environmental conditions (like pH change, etc16).
Herein, the chosen mꢀE2ꢀm surfactants are esterꢀfunctionalized with a diester linkage (E2) in
the spacer part. This special structural feature enables them with lower CMC, biodegradability
and lower toxicity properties. Moreover, presence of E2 moiety (being hydrophilic) enhances
their selfꢀaggregation capacity in polar and nonꢀpolar solvents via hydrogen bonding. Therefore,
mꢀE2ꢀm surfactants are pertinent and appropriate for study.
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Hen egg white lysozyme (HEWL) is a small globular, monomeric protein with 129 amino
acid residues in which the six tryptophan (Trp) residues are located as: substrate binding residues
(3 Trp), hydrophobic core residues (2 Trp) and one Trp is separate from all other residues. The
dominant fluorophores contributing to fluorescence are Trpꢀ62 and Trpꢀ108 and these are
supposed to be important in binding a substrate or inhibitor and in stabilizing the structure.
Motivation to choose HEWL for the study lies in the fact that it is being generally used as
model system to understand the structure, function, dynamics and folding of proteins.17
Moreover, lysozyme is known to posses various physiological and pharmaceutical (antivirus,
antibacterial and antitumor) properties and have a versatile aptitude to interact with metal ions,
drugs, dyes and surfactants.18, 19
Proteinꢀsurfactant interactions are pertinent and interesting not only for structural
determination/protein separation, but because of their utilization in foods, cosmetics, drug
delivery and other industrial applications.20-22 Surfactants’ structural features (spacer
type/hydrophobic content) primarily control these interactions.23 Further, these interactions are
essential to explore surfactants as denaturants, solubilizing, and denaturizing agents for
proteins.1,2 Binding interaction of surfactants with proteins changes the conformation of the
latter, therefore, it is essential to understand proteinꢀsurfactant interactions for scientific as well
as practical view points. In addition, it has vast perspective to understand the role of surfactants
in diseases such as amyloidosis.24 Amyloidosis or fibril formation is associated and responsible
for various diseases including Alzheimer’s and Parkinson’s diseases.25 Several recent reports
have revealed disintegration/modulation of BSA fibrils in presence of gemini surfactants.25, 26
Therefore, we assume this study will have relevance to address the problems associated with
protein misfolding in future. Also, it is reported that cationic gemini surfactants favour fibril
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formation below the CMC and that the surfactants above their CMC can delay amyloid beta
fibril formation.21 Therefore, we expect mꢀE2ꢀm would be good fibril modulators owing to their
very low CMC values (about 100 times less than the conventional surfactants).
A large number of papers deal with the interaction of proteins with conventional surfactants
(like SDS, CTAB and TritonꢀXꢀ1001,2,27-28), but reports related to interaction of proteins with
gemini surfactants are limited. Recent comparative reports on surfactant – protein interactions
have demonstrated better efficacy of the gemini surfactants.29-38 To the best of our knowledge,
no report relating to interactions of mꢀE2ꢀm and HEWL is available in the literature till date.
Moreover, our previous studies have shown the effect of mꢀE2ꢀm surfactants on globular
xanthine oxidase (XO).39-41 Therefore, in continuation of our previous studies and also keeping
in interest the pharmaceutical, biotechnological and cosmetic relevance, herein, we have
investigated the effect of mꢀE2ꢀm novel biodegradable gemini surfactants on the structural
features of HEWL by employing spectroscopic, calorimetric, microscopic and molecular docking
approach. Fluorescence, CD, UV and ITC measurements were employed to investigate the
binding mechanism, number of binding sites, binding forces, and conformational changes while
computational docking was utilized to confirm the main binding site in HEWL. The results
obtained in this study are quite encouraging and this study would be significant to proteinꢀ
surfactant interaction in terms of special mꢀE2ꢀm molecular structure, which is essential in
determining their future use as excipients in membrane mimitics, pharmaceutical/drug delivery
related compilations etc. Moreover, who could not be optimistic in future to construct surfactant
based systems to disassemble amyloid fibrils into soluble substances.
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2. Experimental
2.1 Materials
Chloroacetyl chloride (98%, LobaꢀChemie, India), ethylene glycol (99%, Sigma Aldrich, USA),
N,Nꢀdimethyldodecylamine (95%, Sigma Aldrich, USA), N,Nꢀdimethyltetradecylamine (95%,
Sigma Aldrich, USA), hen egg white lysozyme (HEWL, Sigma Aldrich, USA), and pyrene
(98%, Acros Organics, Belgium) were used as received. The gemini surfactants, ethaneꢀ1, 2ꢀdiyl
bis (N,NꢀdimethylꢀNꢀalkylꢀammoniumacetoxy) dichloride (mꢀE2ꢀm; where m = number of
carbon atoms in the tail and E2 represents diester spacer), were synthesized following the
literature procedure39-41 The synthesis protocol and structure of gemini surfactants are shown in
Scheme 1 and Scheme 2. Working solution concentrations were: [HEWL] = 20 × 10ꢀ6 M, [PBS]
= 12 × 10ꢀ3 M, pH= 7.4.
Scheme 1 Synthesis protocol of mꢀE2ꢀm gemini surfactants (m=12, 14, is the number of
carbon atoms in the tail of surfactant).
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(a)
(b)
Scheme 2 Structures of mꢀE2ꢀm gemini surfactants; (a) 12ꢀE2ꢀ12 and (b) 14ꢀE2ꢀ14.
2.2 Methods
2.2.1 Fluorescence measurements. The intrinsic fluorescence measurements were carried
out using a Hitachiꢀ2700 fluorescence spectrophotometer (Japan). The intrinsic fluorescence of
HEWL was monitored at excitation wavelength of 280 nm and the emission spectra were
recorded in the range of 300–450 nm. Both the slit widths (excitation as well as emission) were
set at 5 nm. Synchronous fluorescence spectra of HEWL were recorded using the same
spectrophotometer at a wavelength interval (ꢀλ) of 20 and 60 nm to probe, respectively, the
microenvironment changes around the tyrosine (Tyr) and tryptophan (Trp) residues42-44. Threeꢀ
dimensional fluorescence spectra were recorded in the range of 200 to 800 nm. For extrinsic
pyrene fluorescence, the excitation was kept at 237 nm and emission was observed in the range
of 350ꢀ450 nm.
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2.2.2 UV measurements. The absorption spectra were recorded on Perkin Elmer Lambdaꢀ25
spectrophotometer in the wavelength range of 200ꢀ345 nm. The concentration of mꢀE2ꢀm gemini
surfactants were varied keeping HEWL concentration constant (20 µM).
2.2.3 CD measurements. Circular dichroism measurements were performed on JASCOꢀJ815
CD spectropolarimeter equipped with a microꢀcomputer. Prior to an experiment the instrument
was calibrated with dꢀ10 camphorsulfonic acid and parameters were set as: temperature, 298 K;
average number of scans, 3; scan speed, 100 nm/min; scan range, 200ꢀ250 nm; and response
time, 1s. Requisite solutions of HEWL and HEWL + mꢀE2ꢀm (12ꢀE2ꢀ12, 14ꢀE2ꢀ14) were taken
in a 0.1 cm path length cell and then the farꢀUV CD spectra were recorded. A reference signal of
mꢀE2ꢀm + buffer was subtracted from the CD signal for all measurements.
2.2.4 ITC measurements. The calorimetric measurements were performed with a MicroCal
ITCꢀ200 (USA) at 298 K. The sample cell was loaded with solution of HEWL (20 µM), and the
titrant syringe was filled with 40 µL of mꢀE2ꢀm solution and 2 µL of the solution was injected
into the sample cell.
2.2.5 Molecular docking analysis. Computational molecular modeling was performed using
molecular graphics program Hex 6.1.45 The essential requirements were made available as:
HEWL PDB (1 DPX, http://www.rcsb.org./pdb), mol files of mꢀE2ꢀm (CHEMSKETCH,
http://www.acdlabs.com), PDB files of mꢀE2ꢀm (Chimera 1.9, www.cgl.ucsf.edu/chimera) and
the final visual scenes of docked structures were observed utilizing graphics program, Pymol46
(http://pymol.sourceforge.net). The docking experiment was run on computer with Windows 7 as
an operating system.
2.2.6 Transmission electron microscopy (TEM) measurements. Transmission electron
microscopy was performed on JEMꢀ2100 (JEOL, Japan) electron microscope. Negative staining
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method was used to prepare the HEWL and HEWL+ mꢀE2-m systems. Then TEM micrographs
were taken after proper drying of samples in air for 1 day.
3. Results and discussion
3.1 HEWL−m-E2-m complex formation
In proteins, fluorescence emission of intrinsic fluorophores (tryptophan (Trp), tyrosine (Tyr) and
phenylalanine (Phe)) are usually used to probe ligandꢀprotein binding, protein conformation and
intermolecular interactions.39, 47 In case of HEWL fluorescence, emission is mainly due to Trpꢀ
108 and Trpꢀ62. The crystal structure of HEWL delineates that these dominant fluorophores are
located near the substrate binding site and are essential in HEWL structure stabilization and
ligandꢀinduced conformational change around binding site.47 HEWLꢀsurfactant interactions can
be well investigated by exciting aromatic residues at wavelength 280 nm, as both residues
(Tyr/Trp) are known to get excited at this particular wavelength.
The fluorescence emission spectra of HEWL in the absence and presence of mꢀE2ꢀm gemini
surfactants are shown in Fig.1. It can be seen that, with increasing surfactant concentrations, a
decrease in fluorescence, accompanied by slight blue shift, is observed. This decrease in
fluorescence intensity infers the nonꢀfluorescent complex formation between HEWL and mꢀE2ꢀ
m gemini surfactants. The small blue shift observed can be attributed to unfolding of HEWL
upon mꢀE2ꢀm addition.35 It is also evident from Fig. 1 that 14ꢀE2ꢀ14 causes larger diminution in
fluorescence intensity than 12ꢀE2ꢀ12, this being due to the higher hydrophobicity of the former.
At pH 7.4, HEWL bears a net negative charge and offers well accountability to interact with the
positively charged head groups of mꢀE2ꢀm surfactants. As a result, mꢀE2ꢀm aggregation on the
protein surface increases, favoring the hydrophobic interactions; thus diminution with higher
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chain length surfactant is justified. Similar results on fluorescence intensity of BSA were
observed by Ge et al.38 Moreover, Martin et al. also attributed higher quenching in BSA to a
higher hydrophobicity exerted by the dimeric surfactants than the conventional one.37 Thus, our
results fit well to the recent literature reports.
(a)
500
400
300
200
100
0
Intensity = 432 at zero [12ꢀE2ꢀ12]
[12ꢀE2ꢀ12] (mM)
0.00
0.15
0.52
0.85
1.13
1.40
1.56
1.75
Intensity = 205
at [12ꢀE2ꢀ12] = 1.75 (mM)
300
350
400
450
Wavelength (nm)
(b)
500
Intensity = 432 at zero [14ꢀE2ꢀ14]
[14ꢀE2ꢀ14] (mM)
400
300
200
100
0
0.00
0.15
0.52
0.85
1.13
1.40
1.56
1.75
Intensity = 185
at [14ꢀE2ꢀ14] = 1.75 (mM)
Intensity = 185
at [14ꢀE2ꢀ14] = 1.75 (mM)
300
350
400
450
Wavelength (nm)
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Fig. 1 Fluorescence emission spectra of HEWL in the absence and presence of 12ꢀE2ꢀ
12 (a), and 14ꢀE2ꢀ14 (b) gemini surfactants at 298K and pH 7.4.
3.2 Binding mechanism, binding parameters, and binding isotherm
As is evident from Fig. 1, binding of mꢀE2ꢀm gemini surfactants quenches the fluorescence
intensity of HEWL. This quenching of fluorescence intensity may be static or dynamic. To
confirm the exact quenching mechanism, the fluorescence data were analyzed by SternꢀVolmer
equation (1) 48
F0
=1+ KSV [m ꢀ E2 ꢀ m]
(1)
F
where F0 and F are the fluorescence intensities of HEWL in the absence and presence of the
quencher, Ksv is the Stern−Volmer quenching constant (which reflects the extent of quenching),
and [mꢀE2ꢀm] is the concentration of the quencher. Fig. 2 gives the Stern−Volmer plots of
HEWL−mꢀE2ꢀm systems whose slopes were used to calculate the SternꢀVolmer constant.
(a)
2.0
(b)
2.2
2.0
1.8
1.6
1.4
1.2
1.0
1.8
1.6
1.4
1.2
1.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
[12ꢀE2ꢀ12] (mM)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
[14ꢀE2ꢀ14] (mM)
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Fig. 2 Stern−Volmer plots of fluorescence quenching of HEWL by 12–E2ꢀ12 (a), and 14ꢀE2ꢀ14
(b) gemini surfactants.
Significant Ksv values (Table 1) indicate substantial quenching of fluorescence spectra by the mꢀ
E2ꢀm surfactants. These Ksv values were then used to evaluate bimolecular quenching rate
constants (kq) from equation (2)
KSV
(2)
kq =
τ0
τ
(
being the average lifetime of the fluorophore in the absence of quencher with a value of
0
10−8 s for most of the biomolecules ).49,50 It can be observed from the Table 1 that 14ꢀE2ꢀ14 has
higher value of quenching rate constant than 12ꢀE2ꢀ12, indicating higher quenching efficiency of
14ꢀE2ꢀ14 than 12ꢀE2ꢀ12. Further, quenching rate constant was found two orders of magnitude
than the scattering collision quenching rate constant (2×1010 L mol−1 s−1)49, suggesting that the
mechanism of quenching proceeds through static procedure and, therefore, is assumed to initiate
via ground state complex formation between mꢀE2ꢀm and HEWL.49
To obtain binding parameters, the fluorescence data were further analyzed by equation (3)
(3)
log {(F ꢀ F)/F} = log K b + n log [m ꢀ E2 ꢀ m]
0
where F0 and F are the same as in eq.(1), Kb and n represent the binding constant and number of
binding sites, respectively, and are obtainable from the intercept and slope of log{(FoꢀF)/F}
versus log[mꢀE2ꢀm] plots (Fig. 3). The obtained Kb and n values are given in Table 1.
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(a)
0.4
0.2
(b)
0.3
0.0
ꢀ0.3
ꢀ0.6
ꢀ0.9
ꢀ1.2
ꢀ1.5
ꢀ1.8
0.0
ꢀ0.2
ꢀ0.4
ꢀ0.6
ꢀ0.8
ꢀ1.0
ꢀ1.2
ꢀ1.4
ꢀ0.8
ꢀ0.6
ꢀ0.4
ꢀ0.2
0.0
0.2
0.4
ꢀ0.8
ꢀ0.6
ꢀ0.4
ꢀ0.2
0.0
0.2
0.4
log[14ꢀE2ꢀ14]
log[12ꢀE2ꢀ12]
Fig. 3 Plots of log {(F0ꢀF)/F} versus log [mꢀE2ꢀm] at 298K and pH 7.4.
A higher Kb value of 14ꢀE2ꢀ14 depicts that it binds more effectively than 12ꢀE2ꢀ12. Further, the
values of n, found to be 1.35 and 1.67, respectively, for 12ꢀE2ꢀ12 and 14ꢀE2ꢀ14, confirm single
pattern of binding sites on HEWL for the mꢀE2ꢀm gemini surfactants.
Table 1 Binding parameters for the interaction of mꢀE2ꢀm surfactants with HEWL at pH
7.4 and 298K
(L mol─1 s─1)
SV (M─1)
(M─1)
Kb
ꢁGob (kJ mol─1)
System
R2
R2
n
kq
K
12ꢀE2ꢀ12+HEWL 5.3×104
14ꢀE2ꢀ14+HEWL 6.5×104
5.3×1012
6.5×1012
0.98 4.5×104
0.98 4.9×104
0.99
0.99
1.35
1.67
−26.55
−26.76
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The average number of mꢀE2ꢀm surfactant molecules (υ), bound to HEWL, were also
computed by using the following equation
υ = α (C mꢀE2ꢀm/C HEWL
)
(4)
where C represents the molar surfactant concentration of the indicated species. α (the fraction
of mꢀE2ꢀm molecules bound to HEWL) was estimated from equation (5)29
Fobs − Fo
(5)
α =
Fmin − Fo
where Fobs is the HEWL fluorescence intensity at any surfactant concentration, Fo is the HEWL
fluorescence intensity in the absence of surfactant, and Fmin is the minimal HEWL fluorescence
intensity observed in the presence of surfactant. It is clearly seen (Fig. 4) that there are two
regions of binding in the chosen concentration range of the surfactants. The first region (lower
sloppy region), at lower concentrations of mꢀE2ꢀm gemini surfactants, corresponds to nonꢀ
cooperative binding, and the second region reveals abrupt increase in binding, which can be
attributed to cooperative binding of mꢀE2ꢀm surfactants to HEWL. Thus, from the binding
isotherms, we can conclude that binding of mꢀE2ꢀm surfactants is mainly governed by
cooperative interactions. The reason for these cooperative interactions can be attributed to the
higher aggregation of mꢀE2ꢀm microstructures at higher surfactant concentrations.
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80
60
40
20
0
12ꢀE2ꢀ12
14ꢀE2ꢀ14
ꢀ3.8
ꢀ3.6
ꢀ3.4
ꢀ3.2
ꢀ3.0
ꢀ2.8
log [mꢀE2ꢀm, M]
Fig. 4 The binding curves of the mꢀE2ꢀm gemini surfactants with HEWL at pH 7.4 and
298 K.
3.3 Three-dimensional fluorescence spectroscopy
Threeꢀdimensional fluorescence spectroscopy is an essential analytical technique to
comprehensively exhibit the fluorescence information and conformational changes of proteins in
a more suitable and realistic way.51 Fig. 5 shows the threeꢀdimensional fluorescence spectra of
HEWL in the absence and presence of mꢀE2ꢀm surfactants. Two peaks are seen in the spectra;
peak 1 is the Rayleigh scattering peak and peak 2 reveals the behaviour of aromatic residues
(Tyr/Trp) and is correlated to the micropolarity around these residues. The fluorescence intensity
of peak 2 decreases in the presence of mꢀE2-m surfactants, suggesting that micropolarity of these
residues changes (Fig. 5b and Fig. 5c) due to the exposure of aromatic residues to an outer
environment. Moreover, this quenching of peak 2 is also attributed to the formation of HEWLꢀ
mꢀE2ꢀm complex formation. Thus, the results are consistent with the above mentioned
techniques.
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(a)
(b)
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(c)
Fig. 5 Threeꢀdimensional fluorescence spectra of native HEWL (a), HEWL + 12ꢀE2ꢀ12
(b), and HEWL + 14ꢀE2ꢀ14 (c) at 298K and pH 7.4; [HEWL] = 20 ꢁM, [12ꢀE2ꢀ12] and
[14ꢀE2ꢀ14] = 1.13mM.
3.4 Synchronous fluorescence spectroscopy
For obtaining specific micropolarity information around the tyrosine (Tyr) and tryptophan (Trp)
residues, synchronous fluorescence spectroscopy is considered an appropriate technique.52, 53 Its
specialty is also owing to its important features such as spectral bandwidth reduction, spectral
simplification and avoiding different perturbing effects. Fig. 6 shows the synchronous
fluorescence spectra of HEWL in the absence and presence of mꢀE2ꢀm surfactants. Quite
interestingly, it can be seen from the synchronous spectra that subsequent additions of mꢀE2ꢀm
quenches the fluorescence intensity around both the residues, indicating the binding of mꢀE2ꢀm
gemini surfactants to HEWL. The nonꢀpolar environment generated by the mꢀE2ꢀm gemini
surfactant around aromatic residues can be accountable for the same. It is also evident that the
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Trp residues contribute more to the intrinsic fluorescence of HEWL than the Tyr residues,
because the fluorescence intensity at 60 nm is higher than that at 20 nm (Fig. 6b and Fig. 6d)). A
comparative analysis of the spectra reveals that 14ꢀE2ꢀ14 interacts strongly with both the
residues than 12ꢀE2ꢀ12; again higher hydrophobic contribution of the former are being
attributable.
(a) 120
100
80
(b)
ꢀλ = 20 nm (for tyrosine)
ꢀλ = 60 nm (for tryptophan)
500
[12ꢀE2ꢀ12] (mM)
[12ꢀE2ꢀ12] (mM)
0.00
0.15
1.13
1.75
400
300
200
100
0
0.00
0.15
1.13
1.75
60
40
20
0
240
260
280
300
320
340
240
260
280
300
320
340
Wavelength (nm)
Wavelength (nm)
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120
100
80
60
40
20
0
500
(d)
(c)
ꢀλ = 60 nm (for tryptophan)
ꢀλ = 20 nm (for tyrosine)
400
300
200
100
0
[14ꢀE2ꢀ14] (mM)
[14ꢀE2ꢀ14] (mM)
0.00
0.15
1.13
1.75
0.00
0.15
1.13
1.75
240
260
280
300
320
340
240
260
280
300
320
340
Wavelength (nm)
Wavelength (nm)
Fig. 6 Synchronous fluorescence spectra of HEWL in the absence and presence of
various concentrations of mꢀE2ꢀm gemini surfactants.
Moreover, the synchronous fluorescence spectroscopy was further employed to confirm the
quenching mechanism54. The data were fitted in equation (6) and the obtained plots of reduction
in synchronous fluorescence intensity with concentration of mꢀE2ꢀm gemini surfactants are
shown in Fig. 7.
F0
=1+ (KSV + kq τ0 )[m ꢀ E2 ꢀ m] + KSV kq τ0 [m ꢀ E2 ꢀ m]2
(6)
F
It can be clearly seen that all plots are linear, devoid of upward curvature (which is possibly in
both dynamic and static situations), inferring that complex formation proceeds through static
quenching rather than dynamic one. Further, absence of upward curvature depicts the
independency on the square part of equation (6).
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1.3
1.2
1.1
1.0
ꢀλ= 20 nm (for Tyr)
1.3
ꢀλ= 20 nm (for Tyr)
1.2
1.1
1.0
0.0
0.3
0.6
0.9
1.2
1.5
1.8
0.0
0.3
0.6
0.9
1.2
1.5
1.8
[12ꢀE2ꢀ12] (mM)
[14ꢀE2ꢀ14] (mM)
1.3
1.3
1.2
1.1
1.0
ꢀλ=60 nm (for Trp)
ꢀλ= 60nm (for Trp)
1.2
1.1
1.0
0.0
0.3
0.6
0.9
1.2
1.5
1.8
0.0
0.3
0.6
0.9
1.2
1.5
1.8
[12ꢀE2ꢀ12] (mM)
[14ꢀE2ꢀ14] (mM)
Fig. 7 Reduction in synchronous fluorescence intensity of HEWL by mꢀE2ꢀm gemini
surfactants at ∆λ= 20 nm (for tyrosine) and ∆λ = 60 nm (for tryptophan).
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3.5 Micropolarity assay
In order to further investigate the micropolarity changes around the aromatic residues, pyrene
fluorescence was used55. Pyrene serves as probe in mꢀE2ꢀm–HEWL system and is sensitive to
micropolarity changes. The fiveꢀpeaked pyrene emission spectra of HEWL in the absence and
presence of different concentrations of mꢀE2ꢀm surfactants are shown Fig. 8. In the absence of
surfactants, the fluorescence intensity is low, indicating that pyrene is localized in the Trp/Tyr
residues. Additions of gemini surfactant increases the fluorescence intensity, which implies the
exposure of aromatic residues. At higher concentrations of mꢀE2ꢀm, on the other hand, decrease
in fluorescence intensity is observed, most probably due to incorporation of probe molecules
(pyrene) into the mꢀE2ꢀm aggregates.
(a)
(b)
500
450
400
350
300
250
200
150
100
50
400
350
300
250
200
150
100
50
[12ꢀE2ꢀ12] (mM)
[14ꢀE2ꢀ14] (mM)
0.00
0.15
0.52
0.85
1.13
1.40
1.56
1.75
1.94
0.00
0.15
0.52
0.85
1.13
1.40
1.56
1.75
0
0
ꢀ50
340
ꢀ50
340
360
380
400
420
440
460
360
380
400
420
440
460
Wavelength (nm)
Wavelength (nm)
Fig. 8 Pyrene emission fluorescence spectra of HEWL in the absence and presence
of varying concentrations of 12ꢀE2ꢀ12 (a) and 14ꢀE2ꢀ14 (b) at 298K and pH 7.4.
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Moreover, the F1/F3 ratios, computed on the basis of first and third vibrational peaks55 and
depicted in Fig. 9, show that the ratio of native HEWL is low, indicating pyrene to be in the
hydrophobic network. On subsequent additions of mꢀE2ꢀm gemini surfactants, the ratio slightly
increases, revealing its exposure to outer environment. Constancy and decrease at higher
concentrations can be attributed to numerous aggregated structures formed by mꢀE2ꢀm at higher
concentrations. Lower F1/F3 ratios, observed for HEWLꢀ14ꢀE2ꢀ14 surfactant, indicate higher
potential of 14ꢀE2ꢀ14 to influence the microstructure of HEWL.
(a)
1.42
1.41
1.40
1.39
1.38
1.37
1.36
1.35
1.34
(b) 1.40
1.39
1.38
1.37
1.36
1.35
1.34
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
[12ꢀE2ꢀ12] (mM)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
[14ꢀE2ꢀ14] (mM)
Fig. 9 Variation of F1/F3 ratio as a function of mꢀE2ꢀm concentration.
3.6 UV-vis absorption spectroscopy
UVꢀVis spectroscopy is extensively used to investigate structural changes of proteins and to
explore ligand–protein complex formations. It is known as a straightꢀforward but efficient
technique in detecting complex formation.56,57 The UVꢀvis spectra of HEWL at different gemini
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concentrations (Fig. 10) show maximum absorption band at 279 nm, related to electron transition
(π−π*) of bonds of aromatic residues. The relevant spectra show
π
hyposchromism/hypsochromism effects upon the addition of mꢀE2ꢀm surfactants, signifying
mꢀE2ꢀm–HEWL complex formation and the observed small blue shift is indicative of unfolding
of HEWL upon mꢀE2ꢀm combination. The blue shift being more prominent in 14ꢀE2ꢀ14ꢀHEWL
combination is due to the higher efficiency of 14ꢀE2ꢀ14 towards HEWL (than 12ꢀE2ꢀ12) to
change the protein structure.
As explained in fluorescence spectroscopic results, the quenching mechanism is static; this is
further supported by changes in the UVꢀvisible spectrum of HEWL. As per literature, 58-60 in
case a complex of protein and ligand is formed through static quenching, then there will be some
changes in the UVꢀvis spectrum of the protein, whereas dynamic quenching produces no such
change. In our case, the UVꢀvis spectra show substantial change upon geminiꢀsurfactant
combination; thus we can safely conclude that the quenching is primarily caused by complex
formation between mꢀE2ꢀm and HEWL. Obviously, our results are in mutual complement with
the fluorescence results. Moreover, similar quenching validation by UVꢀvis spectra was also
reported by another research group.4
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(a)
(b)
0.5
0.4
0.3
0.2
0.1
0.0
0.5
0.4
0.3
0.2
0.1
0.0
[12ꢀE2ꢀ12] (mM)
[14ꢀE2ꢀ14] (mM)
0.00
0.85
1.40
1.75
0.00
085
1.40
1.75
210
240
270
300
330
210
240
270
300
330
Wavelength (nm)
Wavelength (nm)
Fig. 10 UV spectra of HEWL in the absence and presence of 12ꢀE2ꢀ12 (a) and 14ꢀE2ꢀ
14 (b) gemini surfactants.
Thus, by analyzing different spectroscopic techniques it is found that all the techniques are in
well corroboration with each other. Intrinsic fluorescence spectroscopy depicts substantial
binding of mꢀE2ꢀm gemini surfactants to HEWL which, in threeꢀdimensional fluorescence is
indicated by distinct quenching at higher surfactant concentrations. In case of synchronous
fluorescence spectroscopy, mixed systems are found to have lower fluorescence intensity than
native HEWL which again is in well coherence with pyrene fluorescence in which we obtained
lower F1/F3 ratios. Hypochromic/hypsochromic effects observed in UVꢀvis spectroscopy further
advocate the microenvironmental change around aromatic residues of HEWL upon gemini
combination.
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3.7 Conformational analysis (circular dichroism spectroscopy)
The CD spectroscopy can be exploited to explore fluctuations in the secondary structure of
proteins during binding process. The CD spectra of the native HEWL at different concentrations
of mꢀE2ꢀm gemini surfactants are shown in Fig. 11. Two negative bands are exhibited in the farꢀ
UV region at around 208 and 222 nm, which are characteristic of the αꢀhelical structure and
attributed to nꢀπ* transitions.61 Alterations in ellipticity at 208 nm are useful for visualizing
variations in the αꢀhelical content. Fig. 11 shows that the addition of surfactants results in a
decrease of negative helicity, suggesting unfolding of HEWL.
(a)
(b)
5
0
5
0
ꢀ5
ꢀ5
ꢀ10
ꢀ15
ꢀ20
ꢀ25
ꢀ30
ꢀ35
ꢀ10
ꢀ15
ꢀ20
ꢀ25
ꢀ30
ꢀ35
[12ꢀE2ꢀ12] (mM)
[14ꢀE2ꢀ14] (mM)
0.00
0.15
0.85
1.40
1.75
0.00
0.15
0.85
1.40
1.75
200
210
220
230
240
250
200
210
220
230
240
250
Wavelength (nm)
Wavelength (nm)
Fig. 11 FarꢀUV CD spectra of HEWL in the absence and presence of 12ꢀE2ꢀ12 (a), and
14ꢀE2ꢀ14 (b) gemini surfactants.
As per equation (7), the CD results are expressed in terms of mean residue ellipticity (MRE) in
degree cm2 dmolꢀ1
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ObservedCD (m degree)
MRE =
(7)
CHEWLn l ×10
where CHEWL is the molar concentration of the protein, n is the number of amino acid residues
(129), and l is the path length (0.1 cm). Then, using equation (8), the αꢀhelical content
MRE 208 − 4000
α − Helix (%) =
×100
33,000 ꢀ 4000
(8)
of HEWL was evaluated. It can be seen (Table 2) that the αꢀhelicity decreases, inferring that
binding of mꢀE2ꢀm to HEWL may induce some conformational changes.65 The trend to decrease
αꢀhelicity among the geminis is 14ꢀE2ꢀ14 > 12ꢀE2ꢀ12. The reason might lie in the fact that more
hydrophobic nature of 14ꢀE2ꢀ14 causes more extended polypeptide structure of HEWL which
leads to an exposure of the hydrophobic cavities and hence αꢀhelical content decreases
significantly.62 It is also interesting to note that the obtained CD signals are of similar shape,
indicating that the secondary structure (αꢀcontent) remains predominant even after binding of mꢀ
E2ꢀm to the protein.61
Table 2 Variation of αꢀhelical content of HEWL as function of mꢀE2ꢀm (12ꢀE2ꢀ12, 14ꢀ
E2ꢀ14) gemini surfactant concentration
Gemini surfactant
12ꢀE2ꢀ12
Concentration of gemini surfactant (mM)
α−helical content (%)
0
30.68
29.96
28.34
25.11
0.15
0.85
1.40
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1.75
0.15
0.85
1.40
1.75
20.83
31.84
23.79
21.14
20.40
14ꢀE2ꢀ14
3.8 Isothermal titration calorimetry (ITC)
Isothermal titration calorimetry (ITC) is an essential technique to probe protein−ligand
interactions. It directly measures the thermodynamic heat change of molecular interactions. The
calorimetric profiles of binding of mꢀE2ꢀm with the lysozyme are shown in Fig. 12. It can be
seen that the titration curves lie mainly in the endothermic domain and show maximum
endothermicity at very low mꢀE2ꢀm concentration, followed by decrease in endothermicity. The
endothermic contributions may be ascribed to hydrophobic forces governing complex formation
with mꢀE2ꢀm. Breaking of hydrogen bonding interactions due to cross linking of dimeric tails of
mꢀE2ꢀm geminis with amino acid residues of HEWL may also be responsible for positive
enthalpy values. Interestingly, the behavior of 14ꢀE2ꢀ14 is different (Fig. 12b ) from 12ꢀE2ꢀ12 .
Its reaction profile first shows high endothermicity, which decreases to zero and crosses into the
exothermic domain. The reasons for endothermicity are explained above, however,
exothermicity can be ascribed to dominance of Van der Waals/ electrostatic interactions between
the polar amino acid residues (like His, Arg, and Lys) of HEWL and the carbonyl group of the
ester gemini surfactant.63 Moreover, as the hydrophobic patches are located deeper,64 it is easier
for the shorter tail surfactant molecules to approach the residues and in order to generate the
influence. A longer tail surfactant can generate more powerful hydrophobic interactions even by
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residing at surface sites. In both cases, the hydrophobic forces remain predominant. However,
when chain length is intermediate (like in 14ꢀE2ꢀ14), chances of hydrophobic as well as
electrostatic interactions are equally probable. This could be the other reason that at higher
loadings of 14ꢀE2ꢀ14, the endothermic event changes into exothermicity. Moreover, positive ꢀH
also suggests that the interaction must have proceeded with positive entropy (ꢀS) change,
necessary to cover the criteria of spontaneity of molecular reactions.
Fig. 12 Isothermal titration calorimetric curves of 12ꢀE2ꢀ12 + HEWL (a), and 14ꢀE2ꢀ14
+ HEWL (b) systems at 298 K and pH 7.4.
Further, it can be interestingly noted (Table 3) that magnitude of binding parameters (Kb,
°
ꢀGb ), obtained in ITC, are higher than the fluorescence binding parameters; the reason being
ITC measures the global change ( i.e. enthalpy change of the binding reaction as well as the
enthalpy of all possible concomitant reactions which may accompany the binding reactions but
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not directly influencing the Kb ), while as fluorescence is specific to aromatic residues65.
Moreover, n=2, which suggests the possibility of two binding sites, which is different from one
binding site approximation of fluorescence. It may be due to absence of tryptophan residues in
the close vicinity of binding event captured by fluorescence. Higher values of binding parameters
obtained in ITC may also be ascribed to relatively longer twin tails of mꢀE2ꢀm gemini
surfactants, as it is known that longer chains (with 16ꢀ22 carbon atoms) can alter Kb values
strongly66.
Table 3 ITC derived biophysical parameters for the binding of mꢀE2ꢀm surfactants with
HEWL at at 298K and pH.7.4
°
Kb (M−1)
ꢀH°( cal mol−1) ꢀS° (cal mol−1 K−1) ꢀGb (kcal mol−1)
System
n
12ꢀE2ꢀ12ꢀHEWL 5.63× 1018 181.7
14ꢀE2ꢀ14ꢀHEWL 1.19× 1019 902.4
86.4
90.3
2
2
−25.55
−26.00
3.9 Docking analysis of HEWL-m-E2-m complex
Molecular docking is considered an essential technique to unveil interactions between ligands
and proteins67. It provides information about the binding site of ligand on protein, which has
relevance in pharmaceutical science. In this context, we have performed a molecular docking of
mꢀE2ꢀm gemini surfactants with HEWL. The bestꢀdocked structures (with lower energy
conformation) are shown in Fig. 13. Interestingly, we note that 12ꢀE2ꢀ12 binds in the close
proximity of Argꢀ128, Cysꢀ6, Alaꢀ122, Cysꢀ33, Cysꢀ30, Cysꢀ116, Argꢀ112, Alaꢀ107, Trpꢀ108,
Trpꢀ63, Trpꢀ62, Cysꢀ64, Tyrꢀ53, Aspꢀ52, Trpꢀ123, Valꢀ123, Valꢀ29, Pheꢀ34 and Valꢀ29, amino
acid residues (Fig. 13a and Fig. 13b) while 14ꢀE2ꢀ14 positions itself in the cavity lined by Valꢀ
29