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1222 Mol. BioSyst., 2012, 8, 1222–1229 This journal is cThe Royal Society of Chemistry 2012
Cite this:
Mol. BioSyst
., 2012, 8, 1222–1229
Probing the binding of two fluoroquinolones to lysozyme: a combined
spectroscopic and docking study
Pengfei Qin,
a
Baoling Su
b
and Rutao Liu*
a
Received 12th October 2011, Accepted 20th December 2011
DOI: 10.1039/c2mb05423j
Ciprofloxacin (CPFX) and enrofloxacin (ENFX) are two of the most widely used
fluoroquinolones (FQs) in human and veterinary medicines. Their occurrence in the environment
has received much attention because of the potential adverse effects on humans and ecosystem
functions. In this paper, we investigated the interaction mechanism between the two FQs and
lysozyme by the spectroscopic and molecular docking methods. As shown by the fluorescence
spectroscopy, additions of CPFX or ENFX effectively quenched the intrinsic fluorescence of
lysozyme, which was attributed to the formation of a moderately strong complex. The enthalpy
change (DH) and entropy change (DS) indicated that van der Waals forces and hydrogen bonds
were the dominant intermolecular forces in the binding of two FQs to lysozyme. Furthermore,
data obtained by UV-vis absorption, synchronous fluorescence and circular dichroism (CD)
suggested that both CPFX and ENFX could lead to the conformational and some
microenvironmental changes of lysozyme. Finally, the molecular docking illustrated that the two
FQs had specific interactions with the residues of Trp62 and Trp63.
1. Introduction
The introduction of fluoroquinolones (FQs) more than 20 years
ago provided clinicians with a range of antibacterial agents that
have a broad spectrum of activity to act against both Gram-
negative and Gram-positive bacteria.
1
Ciprofloxacin (CPFX)
and enrofloxacin (ENFX) (structures shown in Scheme 1),
which belong to the group of FQs, are frequently used in many
human and veterinary applications.
2,3
However, due to the
incomplete metabolism and the relative ineffectiveness of
conventional water treatment technologies in removing them,
4
they have recently been detected in wastewaters, surface and
ground water and in drinking water as well.
5–7
There is a
growing concern in their presence, persistence and fate in the
environment because low levels of antibiotics could favor the
proliferation of antibiotic resistant bacteria, which could pose
serious threats to human health and ecosystem functions.
8–11
Besides, treatment of FQs in animals could also induce some
adverse effects like fatal liver failure, renal dysfunction and
embryo toxicity to humans.
12–14
Nonetheless, there are few
detailed studies on the enzyme binding properties and potential
toxicological relationship of FQs in the same class, which are
potentially important for understanding and predicting their
toxicity and distribution in the body.
Lysozyme is an antimicrobial protein widely distributed in
various biological fluids and tissues, including tears, saliva,
skin, blood, liver and lymphatic tissues of humans and other
animals.
15
It has been extensively used in the pharmaceutical and
food fields because of its physiological and pharmaceutical
functions, such as anti-inflammatory, anti-viral, immune
modulatory, anti-histaminic and anti-tumor activities.
16–18
Lysozyme is a small monomeric globular protein formed by
129 tactic amino acid residues containing 6 tryptophan (Trp),
3 tyrosine (Tyr) and 4 disulfide bonds (a schematic of lysozyme
secondary structure is presented in Scheme 2). Its high-resolution
crystal structure shows that the two dominant fluorophores,
Trp62 and Trp108, are arranged close to the substrate binding
site.
19
They play important roles in binding with a substrate or an
inhibitor and in stabilizing the structure. Hence, it offers the
possibility of correlating its dynamics with enzymatic activity
Scheme 1 Chemical structures of CPFX and ENFX.
a
Shandong Key Laboratory of Water Pollution Control and Resource
Reuse, School of Environmental Science and Engineering,
Shandong University, China–America CRC for Environment &
Health, Shandong Province, 27# Shanda South Road, Jinan 250100,
P.R. China. E-mail: rutaoliu@sdu.edu.cn; Fax: +86-531-88364868;
Tel: +86-531-88364868
b
Department of Logistic Management, Shandong University,
27# Shanda South Road, Jinan, Shandong 250100, P.R. China
Molecular
BioSystems
Dynamic Article Links
www.rsc.org/molecularbiosystems PAPER
This journal is cThe Royal Society of Chemistry 2012 Mol. BioSyst., 2012, 8, 1222–1229 1223
using the fluorescence analyses of these tryptophan residues,
which could provide information on the lysozyme–ligand inter-
action and ligand-induced conformational change around the
binding site.
20
In the present work, we investigated the mechanism of two
FQs, CPFX and ENFX, binding to lysozyme by means of
spectroscopic and molecular modeling methods under simulative
physiological conditions. Information about the binding para-
meters, thermodynamic parameters, binding modes and affinity,
conformational investigation and binding location is discussed.
This report is helpful to clarify the biological mechanism of FQ
toxicity in vivo. In addition, it could also provide basic information
for understanding the transportation of FQs in the body.
2. Results and discussion
2.1 Effects of CPFX and ENFX on lysozyme fluorescence
Fluorescence measurements have been widely used to study
the binding mechanism of small molecules to proteins.
21,22
Changes in the intrinsic fluorescence of lysozyme could provide
considerable information about its structure and dynamics. The
fluorescence spectra of lysozyme in the presence of CPFX and
ENFX at 300 K are illustrated in Fig. 1. The fluorescence
intensity of lysozyme at 340 nm decreases regularly upon
increasing the concentration of CPFX or ENFX with a red shift
of the emission peak. The results suggest that both FQs could
interact with lysozyme and the fluorescence chromophores of
lysozyme (Trp and Tyr) are placed in a more hydrophilic
environment after additions of CPFX and ENFX.
23
The peak
located at 415 nm in Fig. 1 is the intrinsic fluorescence of FQs.
The effects of lysozyme on the fluorescence intensity of two FQs
were also evaluated (Fig. 2). The fluorescence intensity of either
CPFX or ENFX around 415 nm quenches with a blue shift as
the concentration of lysozyme increases, which provides evidence
for formation of a FQ–lysozyme complex.
24
2.2 Fluorescence quenching mechanism
The different mechanisms of fluorescence quenching are
usually classified as either dynamic quenching, resulting
from collisional encounters, or static, due to the formation
of a ground-state complex between the fluorophore and
quencher.
25
In order to confirm the quenching mechanism induced by
CPFX and ENFX, the graphs plotted for F
0
/Fagainst [Q]
according to the Stern–Volmer equation are shown in Fig. 3.
The curves are linear with high Rvalues, and the calculated
quenching constants at the corresponding temperatures are
listed in Table 1.
Generally speaking, during the dynamic process, the
quenching rate constants of the fluorescent complexes will
increase with temperature, because the higher temperatures
will result in faster diffusion and hence lead to larger amounts
of collision quenching. In contrast, increased temperature is
likely to result in decreased stability of complexes, and thus the
static quenching constants are expected to decrease with
increasing temperature. Besides, the maximum dynamic
quenching constant k
q
of the various quenchers is 2.0
10
10
L mol
1
s
1
under normal circumstances.
26
As shown
in Table 1, the K
sv
values decrease with increasing temperature
and the k
q
is greater than 2.0 10
10
L mol
1
s
1
.
This indicates that the quenching of both systems is not
initiated from dynamic collision but from the formation of a
complex.
Therefore, the data should follow the modified Stern–Vol-
mer equation. Fig. 4 displays the modified Stern–Volmer plots
and the corresponding results of K
a
values at different tem-
peratures are shown in Table 2. The quenching constants are
found to decrease as temperature rises. And the data again
confirm that the quenching process in both systems belongs to
static quenching.
Scheme 2 Schematic presentation of the secondary structure of
lysozyme (the Trp residues are marked).
Fig. 1 Fluorescence quenching of lysozyme at 340 nm in the absence
and presence of various concentrations of CPFX (a) and ENFX (b);
conditions: CPFX (10
6
mol L
1
) 1–9: 0, 1, 2, 3, 4, 5, 6, 7, 8; ENFX
(10
6
mol L
1
) 1–9: 0, 1, 2, 3, 4, 5, 6, 7, 8; lysozyme: 2 10
6
mol L
1
;
buffer: NaH
2
PO
4
/Na
2
HPO
4
,pH=7.40;T=300K.
1224 Mol. BioSyst., 2012, 8, 1222–1229 This journal is cThe Royal Society of Chemistry 2012
2.3 Thermodynamic parameters and binding modes
Generally, the acting force between a small molecule and a
macromolecule mainly includes hydrogen bonding, van der
Waals force, electrostatic and hydrophobic interactions. The signs
and magnitudes of the thermodynamic parameters (DHand DS)
can account for the main forces involved in the binding reaction.
Ross and Subramanian
27
have summed up the thermodynamic
laws to determine the types of binding associated with various
interactions. That is, if DHo0andDSo0, van der Waals’
interactions and hydrogen bonds play main roles in the binding
reaction. If DH40andDS40, hydrophobic interactions are
dominant. Electrostatic forces are more important when DHo0
and DS40. The thermodynamic parameters and K
a
values of the
binding systems are listed in Table 2. The negative sign for free
energy (DG) indicates that the interaction process is spontaneous
in the FQs binding to lysozyme. And the negative values of DH
and DSin both systems suggest that hydrogen bonding and van
der Waals forces are predominant in the acting forces. The K
a
in
the ENFX/lysozyme system is much greater than that in CPFX/
lysozyme, indicating that ENFX binds more strongly than CPFX.
2.4 Identification of the binding parameters
According to a plot of log[(F
0
F)/F]versus log[Q] in eqn (5),
the binding constant for the CPFX/lysozyme system at 308 K
is 1.87 10
5
L mol
1
and the number of binding sites is 1.13.
As for ENFX/lysozyme, the binding constant at 308 K is
2.26 10
5
L mol
1
and nis 1.12. The value of nin the two
systems is approximately equal to 1, indicating that there is
only one site in lysozyme for either CPFX or ENFX during
their interaction. Besides, the value of K
b
suggests that there is
a strong interaction in the binding of both molecules to
lysozyme, and thus even low concentrations of CPFX and
ENFX in the body can interact with lysozyme easily.
2.5 Conformational investigations
In order to determine the binding effects of two FQs on the
structure and the microenvironment of lysozyme, we utilized
the methods of UV-vis absorption, synchronous fluorescence
and CD spectroscopy to further investigate the conforma-
tional changes of lysozyme.
2.5.1 UV-vis absorption spectra. UV-vis absorption
spectroscopy has been widely used to explore the structural
changes of protein and to investigate the ligand–protein
Fig. 2 Effects of lysozyme on the fluorescence intensity of CPFX (a)
and ENFX (b) at 415 nm; conditions: lysozyme (10
6
mol L
1
) 1–7:
0, 1, 2, 3, 4, 5, 6; CPFX, ENFX: 8 10
6
mol L
1
; buffer: NaH
2
PO
4
/
Na
2
HPO
4
, pH = 7.40; T= 300 K.
Fig. 3 Stern–Volmer plots of CPFX (a) and ENFX (b) quenching
with lysozyme at three different temperatures. Conditions: lysozyme:
210
6
mol L
1
; buffer: NaH
2
PO
4
/Na
2
HPO
4
, pH = 7.40.
Table 1 Stern–Volmer quenching constants of the CPFX/lysozyme
and ENFX/lysozyme systems at different temperatures
T/K
K
sv
(10
4
L mol
1
)
K
q
(10
12
L
mol
1
s
1
)R
a
S.D.
b
CPFX 300 5.445 5.445 0.99137 0.02108
308 4.545 4.545 0.98569 0.02276
314 4.382 4.382 0.9906 0.01771
ENFX 300 5.844 5.844 0.99625 0.01486
308 5.598 5.598 0.99569 0.01527
314 5.406 5.406 0.99509 0.01574
a
Ris the correlation coefficient.
b
S.D. is the standard deviation of the
K
sv
values.
This journal is cThe Royal Society of Chemistry 2012 Mol. BioSyst., 2012, 8, 1222–1229 1225
complex formation.
28
The absorption spectra of lysozyme as a
function of FQs concentration are presented in Fig. 5. There
are two major absorption peaks in the lysozyme spectra. The
peak located at 200 nm to 230 nm designates the secondary
structure due to the protein skeleton, while the band from 260 nm
to 300 nm indicates the chromophore microenvironment.
29
Fig. 5 shows that after the addition of CPFX, the absorption
peak of lysozyme around 210 nm decreases with a red shift, while
the peak intensity around 280 nm is slightly changed. Also,
lysozyme in the presence of ENFX exhibits a similar trend with a
decrease of absorbance and a red shift around 210 nm. All these
results indicate that the interaction between FQs and lysozyme
leads to the loosening and unfolding of the enzyme skeleton.
2.5.2 Synchronous fluorescence spectra. Synchronous
fluorescence spectra can provide characteristic information
on the molecular environment in the vicinity of chromosphere
molecules. When the wavelength intervals (Dl) are stabilized
at 15 or 60 nm, synchronous fluorescence gives the character-
istic information of Tyr residues or Trp residues, respec-
tively.
23
Therefore, synchronous fluorescence spectroscopy
focusing on the Tyr and Trp residues of lysozyme was adopted
to explore the structural changes of lysozyme in the presence
of various concentrations of CPFX and ENFX.
It is apparent from Fig. 6A(a) and B(a) that the emission
peaks do not shift over the investigated concentration range,
which indicates that both CPFX and ENFX have little effect
on the microenvironment of the Tyr residues in lysozyme. But
the emission peaks of the Trp residues (Fig. 6A(b) and B(b))
exhibit a slight red shift upon the addition of CPFX or ENFX.
This phenomenon expresses the microenvironmental alteration
of the Trp residues, around which the polarity is increased and
the hydrophobicity is decreased.
30
These results further confirm
that the conformational and microenvironmental changes occur
in lysozyme upon the additions of CPFX and ENFX.
2.5.3 Circular dichroism (CD) analysis. To ascertain the
possible influence of FQs binding on the secondary structure
of lysozyme, CD measurements were performed in the
presence of different FQs concentrations (Fig. 7). There are
two negative peaks in the UV region at 208 and 222 nm, which
are characteristic of the a-helical structure of a protein.
31,32
As shown in Fig. 7, both CPFX and ENFX could cause a
gradual increase in the a-helix content of lysozyme, suggesting
Fig. 4 Modified Stern–Volmer plots for the quenching of lysozyme
by CPFX (a) and ENFX (b) at different temperatures. Conditions:
lysozyme: 2 10
6
mol L
1
; buffer: NaH
2
PO
4
/Na
2
HPO
4
, pH = 7.40.
Table 2 Modified Stern–Volmer association constants K
a
and relative
thermodynamic parameters of the CPFX/lysozyme and ENFX/lysozyme
systems
Reagents T/K
K
a
(10
4
L mol
1
)R
a
DH/
kJ mol
1
DG/
kJ mol
1
DS/
J mol
1
K
1
CPFX 300 2.25 0.99035 54.46 24.99 98.23
308 1.04 0.99817 23.69
314 0.85 0.99784 23.62
ENFX 300 3.18 0.99097 47.52 25.86 72.20
308 2.40 0.99581 25.81
314 1.36 0.9992 24.85
a
Ris the correlation coefficient for the K
a
values.
Fig. 5 Absorption spectra of lysozyme as a function of CPFX (a) and
ENFX (b). Conditions: CPFX (10
5
mol L
1
) 1–6: 0, 3, 4, 5, 7, 8.
ENFX (10
5
mol L
1
) 1–6: 0, 3, 4, 5, 7, 8. lysozyme, 1 10
5
mol L
1
;
buffer: NaH
2
PO
4
/Na
2
HPO
4
,pH=7.40.
1226 Mol. BioSyst., 2012, 8, 1222–1229 This journal is cThe Royal Society of Chemistry 2012
that they could interact with the helical regions of lysozyme,
which results in an alteration of its secondary structure.
2.6 Computational modeling studies
The interaction between lysozyme and FQs was further
confirmed by molecular docking studies to determine the
preferred binding site on the protein. The best energy ranked
results are shown in Fig. 8. The oxygen atom in CPFX lies
within the hydrogen bonding distance with the hydrogen atom
on Lys 97 (1.872 A
˚, Fig. 8A(b)). In ENFX, the oxygen could
form a hydrogen bond with the Asn 103 (1.886 A
˚, Fig. 8B(d)).
These results further confirm the conclusions drawn from
the thermodynamic studies. As illustrated in Fig. 8, both
CPFX and ENFX could interact with the Trp62 and Trp63
residues, and the atoms involved in the interactions are shown
in Table 3. Their interactions could explain the observed
quenching of fluorescence and the red shift in synchronous
fluorescence of the Trp microregion. Additionally, both
Trp62 and Trp63 residues are located in the substrate binding
region of the lysozyme, and they play important roles in the
enzymatic activity of lysozyme.
33
Therefore, the binding of
FQs may result in the changes of lysozyme activity. Fig. 8 also
demonstrates that the two FQs could interact with the helical
regions of lysozyme (CPFX with Lys 97 and Asp 101, and
ENFX with Asp 101 and Ala 107), which are in accordance
with the changes of secondary structure of lysozyme in the CD
experiment.
Fig. 6 Synchronous fluorescence spectra of (A) CPFX and (B) ENFX
with lysozyme: (a) observing Tyr residues at Dl= 15 nm and (b) observing
Trp residues at Dl= 60 nm; conditions: CPFX (10
6
mol L
1
) 1–9: 0, 1,
2, 3, 4, 5, 6, 7, 8; ENFX (10
6
mol L
1
)1–9:0,1,2,3,4,5,6,7,8;
lysozyme: 2 10
6
mol L
1
;buffer:NaH
2
PO
4
/Na
2
HPO
4
, pH = 7.40.
Fig. 7 CD spectra of lysozyme in the presence of CPFX (a) and
ENFX (b). Conditions: CPFX (10
5
mol L
1
): 1–3: 0, 2, 6; ENFX
(10
5
mol L
1
): 1–3: 0, 2, 6; lysozyme: 1 10
5
mol L
1
; buffer:
NaH
2
PO
4
/Na
2
HPO
4
, pH = 7.40.
This journal is cThe Royal Society of Chemistry 2012 Mol. BioSyst., 2012, 8, 1222–1229 1227
3. Conclusions
In this paper, the binding interaction between two FQs and
the model protein lysozyme was investigated by multiple spectro-
scopic methods and the experimental results were also supported
by the molecular modeling studies. Both CPFX and ENFX could
bind to lysozyme to form a complex with only one binding site,
and the binding process was spontaneous, in which van der Waals
and hydrogen bond interactions played major roles. The UV-vis
absorption, synchronous fluorescence and CD spectra reflected
the changes in the conformation and microenvironment of
lysozyme. The binding locations for both FQs at lysozyme were
finally assessed based on the docking studies. This work provides
valuable information for clarifying the molecular binding mecha-
nism of CPFX and ENFX to lysozyme in vivo. In addition, the
data could supply some quantitative information for studies on
the molecular toxicology of this class of compounds. Further
study is also needed to provide a sound basis for health risk
assessment of these chemicals.
4. Materials and methods
4.1 Materials
Lysozyme (Sigma) was dissolved with ultrapure water to
form 2 10
5
mol L
1
solution, and then preserved at
0–4 1C. CPFX (98% purity) and ENFX (98% purity) were
purchased from Beijing Huamaike Biotechnology, Co., Ltd,
and stock solutions of CPFX (1 10
2
mol L
1
) and ENFX
(1 10
2
mol L
1
) were prepared and diluted as required. The
0.2 mol L
1
phosphate buffer (mixture of NaH
2
PO
4
2H
2
O and
Na
2
HPO
4
12H
2
O, pH 7.4) was used to control pH. All other
chemicals were of analytical grade. Ultrapure water was used
throughout the experiments.
4.2 Apparatus and measurements
4.2.1 Fluorescence measurements. All fluorescence spectra
were recorded on an F-4600 fluorophotometer (Hitachi,
Japan) equipped with a 10 mm quartz cell and a 150 W Xenon
lamp. For the fluorescence measurements, a certain amount of
FQs, 1 mL phosphate buffer at pH 7.4, and 1 mL lysozyme
were added in turn to a series of 10 mL colorimetric tubes and
made up to the mark with water. After equilibration for
20 min, the fluorescence spectra were obtained.
Fig. 8 Surface and cartoon representations of CPFX (A) and ENFX (B)
to lysozyme. (a) The structure of lysozyme is displayed in its hydrophobicity
surface mode. (b) The atoms of CPFX and ENFX are marked with blue.
And the lysozyme is shown in ribbon mode. The hydrogen bonds between
CPFX (ENFX) and lysozyme are illustrated by the cyan-blue solid line.
Table 3 Atoms involved in the interaction of CPFX and ENFX with
the Trp residues of lysozyme
Ligand Ligand atom Nearest residues Distance/A
˚
CPFX H18 Trp62 (CZ3, CH2) 2.88
C9 Trp62 (CE3, CZ3, CD2) 3.63
N2 Trp62 (CZ3) 3.55
C1 Trp63 (CZ3, CH2) 3.29
C6 Trp63 (CH2, CZ3) 3.32
C10 Trp63 (CZ2) 3.97
F1 Trp63 (CH2, CZ2, CZ3) 3.11
ENFX C1 Trp62 (CE3, CB, CD2) 3.62
C2 Trp62 (CB, CE3, CD2, CG) 3.60
F1 Trp62 (CE, CB, CD2) 2.98
C2 Trp63 (CZ2, CH2, CE2) 3.56
C3 Trp63 (CZ2, CH2) 3.45
C43 Trp63 (CZ2) 3.08
O44 Trp63 (CZ2, CE2, HE1, NE1) 3.04
1228 Mol. BioSyst., 2012, 8, 1222–1229 This journal is cThe Royal Society of Chemistry 2012
The fluorescence emission spectra were measured at 300, 308
and 314 K, maintaining the temperature of the samples in a
thermostated water bath. The widths of the excitation and
emission slit were both set at 5.0 nm. The excitation wave-
length was chosen to be 280 nm, the emission wavelength was
recorded from 290 to 500 nm. The synchronous fluorescence
spectra were recorded at Dl= 15 and 60 nm. The wavelength
range scanned from 250 to 310 nm.
4.2.2 Principles of fluorescence quenching. The fluorescence
quenching data were first analyzed according to the Stern–Volmer
equation,
29
F
0
/F=1+K
sv
[Q] = 1 + k
q
t
0
[Q] (1)
where F
0
and Fare the fluorescence intensities of protein in the
absence and presence of quencher, respectively; k
q
is the
quenching rate constant of the bimolecular reaction; K
sv
is
the dynamic quenching constant; t
0
is the average lifetime of a
molecule without quencher and is generally taken as 10
8
s;
[Q] is the concentration of a quencher.
Otherwise, the data should follow the modified Stern–Volmer
equation:
29
F0
DF¼1
faKa
1
½Qþ1
fa
ð2Þ
where DFis the difference in fluorescence intensity with and
without the quencher at concentration [Q]; K
a
is the effective
quenching constant for the accessible fluorophores; and f
a
is the
fraction of accessible fluorescence. The dependence of F
0
/DFon
the reciprocal value of the quencher concentration [Q]
1
is linear
with slope equal to the value of (f
a
K
a
)
1
.
4.2.3 Analysis of thermodynamic parameters and binding
parameters. If the enthalpy change (DH) does not vary signifi-
cantly over a certain temperature range, the temperature-
dependent thermodynamic parameters were investigated on
the basis of the van’t Hoff equation,
25
ln K2
K1
¼DH
R
1
T1
1
T2ð3Þ
DG=DHTDS=RT ln K(4)
where K
1
and K
2
are the binding constants (analogous to K
a
in
eqn (2)) at T
1
and T
2
, and Ris the universal gas constant.
For a static quenching interaction, the binding constant (K
b
)
and the number of binding sites (n) can be determined using
the following formula,
26
log F0F
F¼log Kbþnlog½Qð5Þ
where F
0
,F, and [Q] are the same as in eqn (2), K
b
is the
binding constant and nis the number of binding sites per
lysozyme molecule. According to eqn (5), values of nand K
b
at
physiological pH 7.4 were calculated.
4.2.4 UV-vis absorption analysis. The UV-vis absorption
spectra of lysozyme in the presence and absence of FQs
were recorded at room temperature on a UV-vis-2450 spectro-
photometer (Shimadzu, Japan) equipped with 10 mm quartz
cells in the range from 190 to 350 nm.
4.2.5 CD spectroscopic measurements. CD spectra were
measured from 200 to 250 nm on a J-810 Circular Dichroism
Spectrometer (Jasco, Tokyo, Japan) using a quartz cell with a path
length of 1 mm. The scanning speed was set at 200 nm min
1
.
Each spectrum was the average of two successive scans.
The a-helical contents of lysozyme in the absence and
presence of FQs were calculated from the following equations:
29
MRE ¼observed CD ðmdegÞ
Cpnl 10 ð6Þ
a-Helixð%Þ¼MRE208 4000
33000 4000 100 ð7Þ
where C
p
is the molar concentration of the protein; nis the
number of amino acid residues and lis the path length.
4.2.6 Molecular modeling study. Lamarckian Genetic
Algorithm (LGA) implemented in AutoDock 4.2 was applied
to calculate the possible conformation of the drug that binds
to the protein. The molecular structure of both CPFX and
ENFX was created by the Gaussian 03W program,
34
using the
B3LYP functional and the 6-31(d) basis set. The crystal
structure of lysozyme (PDB entry 2LYZ) was taken from
Protein Data Bank.
35
Docking calculations were first carried
out on a lysozyme protein model, by the addition of essential
hydrogen atoms and computing Gasteiger charges. Initial
positions, orientations, and torsions of the ligand molecules
were set as default. Affinity (grid) maps of 70 70 70 A
˚
grid points and 0.375 A
˚spacing were generated using the
Autogrid4 program. Each docking experiment was derived
from 10 different runs that were set to terminate after a
maximum of 250 000 energy evaluations. The population size
was set to 150. The conformer with the lowest binding free
energy was used for further analysis.
Acknowledgements
The work is supported by NSFC (81161120413), the Cultivation
Fund of the Key Scientific and Technical Innovation Project,
Ministry of Education of China (708058), and Key Science–
Technology Project in Shandong Province (2008GG10006012)
are also acknowledged.
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