Accepted Manuscript

Title: In silico investigation of propofol binding sites in human

serum albumin using explicit and implicit solvation models

Authors: Sergey Shityakov, Norbert Roewer, Carola Förster,

Jens-Albert Broscheit

PII: S1476-9271(17)30350-X
DOI: http://dx.doi.org/doi:10.1016/j.compbiolchem.2017.06.004
Reference: CBAC 6702

To appear in: Computational Biology and Chemistry

Received date: 23-5-2017

Revised date: 24-6-2017
Accepted date: 27-6-2017

Please cite this article as: Shityakov, Sergey, Roewer, Norbert, Förster, Carola,
Broscheit, Jens-Albert, In silico investigation of propofol binding sites in human
serum albumin using explicit and implicit solvation models.Computational Biology
and Chemistry http://dx.doi.org/10.1016/j.compbiolchem.2017.06.004

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
In silico investigation of propofol binding sites in human serum albumin using explicit
and implicit solvation models
Sergey Shityakov1,*, Norbert Roewer1,2, Carola Förster1, and Jens-Albert Broscheit1,2
1
Department of Anesthesia and Critical Care, University of Würzburg, 97080 Würzburg,
Germany
2
Sapiotec Ltd., 97078 Würzburg, Germany

* Author to whom correspondence should be addressed:

Sergey Shityakov
Department of Anesthesia and Critical Care
University of Würzburg
97080 Würzburg, Germany
E-Mail: Shityakov_S@ukw.de
Tel: + 49 (931) 201 - 30016
Fax: + 49 (931) 201 - 30019

Graphical Abstract:

1
Highlights

 Different propofol binding sites (PR1 and PR2) for human serum albumin are
proposed.
 Propofol exhibits different binding affinity to PR1 and PR2 binding sites.
 Implicit solvation models correctly predict propofol binding affinity.

Abstract
All-atom molecular dynamics (MD) simulations are presented on general anesthetic propofol
bound to human serum albumin (HSA) due to the drug pharmacokinetics and
pharmacodynamics in the circulatory system. We implemented the explicit and implicit
methods to compare the binding affinity of propofol at the different binding sites (PR1 and
PR2) in the HSA protein. Only the implicit model provided the evidence in accordance with
the experimental data indicating that the HSA-ligand interactions are dominant by hydrophobic
forces due to the higher drug affinity at the PR1 position with a ΔGMM-PB/SA value of −28.84
kcal*mol-1. Overall, this study provides important information on the accuracy of explicit and
implicit solvation models to characterize the propofol interaction with different HSA binding
sites.

Keywords: propofol, human serum albumin, binding sites, ligand promiscuity, molecular
docking, molecular dynamics, explicit and implicit solvation.

Introduction
Human serum albumin (HSA) is one of the most abundant proteins in blood plasma (40-50
mg/mL) and it plays a crucial role in the transport of different drugs, metabolites, and fatty acids
around the body. Therefore, a drug binding to human serum albumin is an important parameter
to determine the pharmacokinetic and pharmacodynamic properties for chemical substances in
the human body (Carter and Ho, 1994; Peters, 1996). However, it has been shown that some
monomeric proteins, such as HSA, have different binding sites, which are involved in the
“ligand promiscuity” (Torres and Aburto, 2005; Deeb et al., 2010) for various drugs, including
different anesthetics (Liu et al., 2005).
Propofol (2,6-di-isopropylphenol) is a short-acting general anesthetic agent for controlled
anesthesia when administered by injection (Langley and Heel, 1988; Trapani et al., 2000).
Approximately 50-80% of general anesthetics, including propofol will be bound to HSA after

2
intravenous administration (Altmayer et al., 1995), which could cause significant changes in
the drug’s pharmacokinetics and pharmacodynamics. Propofol binding to HSA is considered
an exothermic and enthalpy-driven process, established by a combination of hydrogen bonding,
van der Waals forces and hydrophobic interactions (Liu et al., 2004). Franks and co-authors
(Bhattacharya et al., 2000) subsequently published a high-resolution crystal structure of HSA
that identified two principal propofol binding sites in the domain III (PR1 and PR2), that has
encouraged others to extend and further this work. However, these sites are also opioid binding
sites, as demonstrated by Liu and co-authors, indicating that general anesthetics may share these
sites with opioids in the central nervous system, a potential mechanism for the minimal alveolar
concentration decrease (Zhou et al., 2012).
Although the HSA molecule transports a significant amount of propofol in the blood, the PR1
or PR2 binding preferences of this drug have not been investigated as of yet in detail. Moreover,
it was proposed that one of the propofol binding sites most likely had a higher affinity due to
the reduction in drug concentration, where the crystals were partially back-soaked, causing the
PR2 electron density disappearance (Bhattacharya et al., 2000).
Various computational methods, including X-ray crystallography, nuclear magnetic resonance
(NMR) spectroscopy, scanning electron microscopy, molecular docking, and molecular
dynamics (MD) simulations can be used for the visualization of ligand binding with HSA and
for the measurement of ligand affinity, in comparison to the experimental results. However,
despite numerous experimental and theoretical trials to characterize the propofol binding to the
different HSA binding sites associated with “ligand promiscuity” (Torres and Aburto, 2005;
Schywalsky et al., 2005; Deeb et al., 2010), none of these attempts has employed an explicit
and implicit solvation methods to predict binding affinities for different PR binding sites in
HSA.
The LIE (linear interaction energy), GB/SA (Generalized Born Surface Area), and MM-PB/SA
(Molecular Mechanics Poisson-Boltzmann Surface Area) approaches have been proven to be
particularly attractive for lead optimization and rely on a proper representation of protein-ligand
interactions, considering both solvation and entropic effects. In this current study, the authors
perform classical all-atom MD simulations on a general anesthetic propofol bound to HSA, to
describe the drug binding affinity to different protein binding sites using the LIE, GB/SA, and
MM-PB/SA methodology.

Computational methods

3
The protein crystal structure of propofol-HSA complex (ID: 1E7A) was obtained from the
Protein Data Bank (PDB) with a resolution of 2.2 Å as the only x-ray model available in the
PDB database (Bhattacharya et al., 2000). The PROCHECK software (Laskowski et al., 1996)
was implemented for stereochemical validation of the HSA molecule. The AutoDock (Morris
et al., 2009) and DOCK6 (Kuntz et al., 1982) programs were used to dock propofol into the
binding sites of HSA. These algorithms consider ligand flexibility by changing the
conformations of the ligand in the active site while keeping protein rigid. The final scores for
all DOCK6 solutions were calculated by a grid score function (GS) and used for docked pose
rankings. Propofol was further analyzed inside the HSA active site using the GB/SA approach
devised by Hawkins and Zou for DOCK6 (Zou et al., 1999; Hawkins at al., 1995) to calculate
energy scores (ESHawkins and ESZou). Molecular mechanics potential energy minimization and
MD simulations were carried out using the program package GROMACS (GROningen
MAchine for Chemical Simulations) v.4.5.5 (Pronk et al., 2013). The Amber99SB force field
(Hornak et al., 2006) was used in all MD simulations. The GROMACS software has the
limitation to parameterize the ligand heteroatom group in the PDB file. To overcome this, the
ligand topology file was prepared using the ACPYPE server (Sousa da Silva et al., 2012),
utilizing the general amber force field (GAFF) and AM1-BCC charges. Energy minimized
structures of propofol-HSA complexes were used as a starting point for MD simulations. The
complexes was solvated in a cubic of simple point charge (SPC) water molecules (Starr et al.,
2000) with 7.5 (complex) and 30 (ligand) Å distances between the solute and the box. Further,
NaCl was added to a concentration of 0.15 M in the periodic box to make an overall neutral
system in terms of formal charges. The position restrained run was performed for 1 ns of NVT
(constant volume and temperature) ensemble dynamics to relax the water while applying
restraints to the protein and equilibrating the system. The production run was then performed
at constant pressure and temperature (NPT) for analyzed systems at 300 K for 50 ns. The
particle mesh Ewald method (Perera et al., 1997; de Souza et al., 1997) was used to treat long-
range Coulomb interactions. The LINCS (LINear Constraint Solver) algorithm was used to
constrain bond lengths involving hydrogens, permitting a time step of 2 fs. vdW forces and
Coulomb interactions were maintained at 10 Å according to the MD protocol established by
Prof. John E. Kerrigan (The Cancer Institute of New Jersey, New Brunswick, NJ, USA
The predicted interaction energy (Eint) and dissociation constant (Kd) for the PR1 and PR2
binding sites were calculated using the following equations namely:
𝐸𝑖𝑛𝑡 = 𝐸𝐿𝐽 + 𝐸𝐶𝑜𝑢𝑙 (1)

4
 𝛥𝐺𝑏𝑖𝑛𝑑 (2)
𝐾𝑑𝑝𝑟𝑒𝑑 = 𝑒𝑥𝑝
𝑅𝑇

, where 𝐸𝐿𝐽 and 𝐸𝐿𝐽 are the short-range Lennard-Jones and Coulomb energy components; R
(gas constant) is 8.31 J(mol*K)−1, and T (room temperature) is 298.15 Kelvin, respectively.
The trajectory files were analyzed through the GROMACS utilities in order to compute the
appropriate functions. The g_lie program used the LIE equation (Hansson et al., 1998) to
calculate the free energy of binding constant (ΔGLIE) as:
𝑝−𝑙 𝑤−𝑙 𝑝−𝑙 𝑤−𝑙
𝛥𝐺𝐿𝐼𝐸 = 𝛼(𝑉𝐿𝐽 − 𝑉𝐿𝐽 ) + 𝛽(𝑉𝐶𝑜𝑢𝑙 − 𝑉𝐶𝑜𝑢𝑙 ) (3)
𝑝−𝑙 𝑤−𝑙 𝑝−𝑙 𝑤−𝑙
, where the 𝑉𝐿𝐽 , 𝑉𝐿𝐽 , 𝑉𝐶𝑜𝑢𝑙 , and 𝑉𝐶𝑜𝑢𝑙 parameters are vdW (Lennard-Jones potential) and
electrostatic (Coulomb potential) terms for protein-ligand (p-l) or water-ligand (w-l)
interactions with scaling factors (𝛼, 𝛽). The g_mmpbsa program used the Poisson–Boltzmann
equation to calculate the binding affinity from the free energetic terms to account for the change
in the free energy (ΔGMM-PB/SA) of binding (Kumari et al., 2014). Molecular graphics and
visualization were performed with the Chimera v1.10 (Goddard et al., 2005) and LigPlot
programs (Wallace et al., 1995) in order to visualize the binding sites and build two-dimensional
(2D) interaction diagrams or three-dimensional (3D) Ramachandran plots. Non-ligand residues
and atoms involved in hydrophobic contacts and SAburied visualizations were performed with
the Naccess v2.1.1 program (Hubbard and Thornton, 1993). Statistical analyses were performed
using the MATLAB and GraphPad Prism v.6 software for Windows (GraphPad Software, Inc.,
San Diego, CA, USA) followed by graphic representations.

Results and Discussion

Propofol is an intravenous general anesthetic classed with the alkyl phenol group of compounds
(Sebel et al., 1989). This drug is a preferred agent for day-patient surgeries due to its rapid
metabolism and reduced post-anesthetic nausea (Duke, 1995; Wallentine et al., 2011). This
general anesthetic is thought to act by enhancing the action of pentameric ligand-gated ion
channels (Trapani et al., 2000). Its overall distribution, metabolism, and efficacy can be changed
based on the affinity to plasma proteins, especially HSA (Servin et al., 1998; Lysakowski et al.,
2001, Xu et al., 2015). Therefore, the interactions of general anesthetic propofol with HSA were
analyzed to describe the drug binding affinity to different receptor binding sites using molecular
docking and MD simulations using explicit and implicit solvation models.
Since the success of all computational methods highly depends on the quality of the protein–
ligand models, it is mandatory that even crystallographic data undergo the stringent validation

5
due to the misinterpretation of the electron density of the crystal (Dauter et al., 2014; Shityakov
and Dandekar, 2010). Therefore, HSA was subjected to stereochemical validation in order to
investigate the φ-ψ dihedral angles in 2D and 3D plots (Figure 1 [A, B]).

A B
180

135

90

45
Psi (Degrees)

-45

-90

-135

-180
-180 -135 -90 -45 0 45 90 135 180
Phi (Degrees)

Figure 1: 2D (A) and 3D (B) Ramachandran plots of the HSA-propofol structure. In 2D plot
the most favored regions are colored in red, whereas the allowed, generously allowed and
disallowed regions are indicated in yellow, light yellow, and white, respectively. The Gly
residues are highlighted with circles.

Altogether, summary statistics showed that 87.6% (474 residues) and 12.4% (67 residues) of
all observed non-Gly and non-Pro residues were in the most favored and allowed regions. The
boundaries of these default regions are based on the calculations by Morris et al., 1992. The
expected values for the comparison were about 90% for the most-favored regions based on the
analysis of 118 structures with a resolution of at least 2.0 Å (Laskowski et al., 1996).

6
Since the number of propofol binding sites in HSA has been reported to be more than 20 (Mazoit
and Samii, 1999), we investigated the PR1 and PR2 binding sites associated with the crystal
structure that were additionally confirmed by continuous elution chromatography (Liu et al.,
2004). These sites are formed amphiphilic clefts or pockets on the protein receptor for
anesthetic binding, causing only very minor changes in the local protein structure (Bhattacharya
et al., 2000).
The reference PR1 and PR2 binding cavities of HSA with propofol are displayed in Figure 2;
where propofol is buried in both the binding variants. From Figure 2 [A], it can be observed
that the PR1 binding locus contains conserved water molecules connected to each other by an
H-bond at a distance of 2.81 Å. By contrast, the PR2 cavity was characterized by the absence
of co-crystallized water molecules (Figure 2 [B]).

A B

2.81

Figure 2: PR1 (A) and PR2 (B) binding sites of the HSA crystal structure as a reference model
are shown to demonstrate the binding site complex geometry. In both cases the aromatic ring
lies within an apolar pocket of HSA. Propofol is presented in its active conformation. H-bond
between water molecules colored in red is depicted with dashed yellow line and measured in
Å. Hydrogen atoms have been omitted for clarity.

As previously determined, the majority of the conserved water molecules were either attached
to deep grooves at the ligand-protein interface or completely buried in binding cavities

7
(Poornima and Dean, 1995). In our case, these water molecules are not directly linked to the
ligand, but they might mediate its binding to HSA via hydrogen bond formations and changing
the shape and complementarity of binding sites (Poornima and Dean, 1995). Probably for these
reasons, propofol located in subdomain IIIA (PR1) was proposed to bind more strongly than the
molecule in PR2 (subdomain IIIB), judging by the disappearance of electron density after the
substance concentration reduction (Bhattacharya et al., 2000). However, these data were never
confirmed by any additional experiments.
Beyond establishing the HSA-propofol hydrogen-bonding network, the main mechanism
through which HSA may trigger drug affinity, is by the formation of the hydrophobic packing
of the drug into the protein binding pocket. Through the inspection of non-ligand residues
involved in hydrophobic contacts with propofol in the static HSA crystal structure, it has been
previously shown that the PR1 and PR2 binding sites have a different number of hydrophobic
contacts associated with “ligand promiscuity” (Bhattacharya et al., 2000). To investigate this
mechanism, subsequent analysis of intermolecular interactions, including hydrophobic and
hydrogen-bonding contacts was carried on the HSA-propofol crystal structure as a reference
model. The binding modes and key amino acids shown in Figure 3 are quite similar to those
detected in the reference X-ray structure indicating the consistency of experimental and
theoretical results. In addition, this binding study conducted through the molecular docking
might provide the implicit information on the approximate duration of propofol half-life in the
blood revealing the efficacy of this medication.
The diisopropylphenol fragment of propofol binds to the active site formed by the PR1 (Leu387,
Ile388, Asn391, Ala449, Val433, Phe403, and Leu407) and PR2 (Leu532, Lys536, and Phe502)
region residues involved in the hydrophobic interactions. In particular, the single hydroxyl
group of the propofol phenolic moiety forms the hydrogen bond with oxygen atoms of the
Leu430 side chain in PR1 at the distance of 3.15 Å (Figure 3 [A]) and Ser579 in PR2 at the
distance of 2.9 Å (Figure 3 [B]).

8
 A B

Figure 3: Protein-ligand 2D interaction diagrams for the PR1 (A) and PR2 (B) binding sites of
the HSA-propofol crystal structure as a reference model are shown to demonstrate the key non-
ligand residues involved in hydrophobic contacts and H-bonds that are essential for the binding.
All H-bonds and distances are depicted with dashed lines and measured in Å. Hydrogen atoms
have been omitted for clarity.

In the AutoDock experiment, propofol as a neutral compound binds to HSA with a ΔGbind value
of −6.13 kcal*mol-1 (Kdpred = 30.93 μM) for PR1 and −5.98 kcal*mol-1 (Kdpred = 39.87 μM) for
PR2 in the vacuum surrounding with no contribution to the overall energy function. In
comparison, the experimental binding affinity of propofol to HSA was determined as having a
Kd of 65 µM with a stoichiometry of approx. 2 using isothermal titration calorimetry (Liu et
al., 2004). These results also agree with those determined by the DOCK6 algorithm of propofol
within the PR1 (GS = −29.91 kcal*mol-1, ESHawkins = −23.22 kcal*mol-1, and ESZou = −3.64
kcal*mol-1) and PR2 (GS = −26.73 kcal*mol-1, ESHawkins = −19.02 kcal*mol-1, and ESZou =

9
−1.85 kcal*mol-1) binding sites for the reference HSA structure using the GB/SA implicit
solvation model (Table 1).

Table 1: Summary of protein-ligand energy terms for different propofol binding sites of the
HSA crystal structure calculated in the gas phase (E int) or by using the Hawkins(*) and Zou
(**) GB/SA approaches.
Energy Binding site
terms PR1 PR2
(kcal/mol)
Eint 9.14 7.32
EvdW* −22.21 −17.33
EGB* 3.18 2.64
ESA* −4.18 −4.34
EvdW** −12.13 −9.01
EGB** 6.25 6.14
ESA** 7.41 8.04
ΔESAS_hp**1 −5.18 −7.02
1
: change in the hydrophobic and total solvent accessible surface area upon ligand binding

In general, HSA displays several structural movements during MD simulations (Supplementary

material 1 and 2), but maintained its initial 3D structure under certain physiological or
pathological conditions that alter pH, protein charge, and osmotic blood properties due to the
disulfide Cys-Cys and hydrogen bonds (Deeb et al., 2010). The interaction energies between
the propofol molecule and HSA based on the short-range energy terms of Lennard-Jones (ELJ)
and Coulomb (ECoul) interactions were analyzed in MD simulations as a crude estimate of the
stability of propofol-HSA. The average Eint value was found to be −30.79 kcal*mol-1 for PR1
and −32.03 kcal*mol-1 for PR2 (Figure 4 [A, B]).

10
 A B
20 20

10 10

0 0
Energy (kcal/mol)

Energy (kcal/mol)
-10 -10

-20 -20

-30 -30
E E
-40 Coul -40 Coul
E E
LJ LJ
-50 -50
Eint E
int
-60 -60
0 10 20 30 40 50 0 10 20 30 40 50
Time (ns) Time (ns)

Figure 4: Lennard-Jones (ELJ), Coulomb (ECoul), and interaction (Eint) energy terms measured
for the PR1 (A) and PR2 (B) binding sites of HSA during the 50 ns MD simulation using the
explicit solvation model.

Similarly, the binding free energy values were estimated for PR1 and PR2 based on the
difference in the energetic profiles of the interactions between propofol and HSA in the “bound”
versus “unbound” endpoints. The PR2 site located in subdomain IIIB of HSA was found to
establish the highest affinity for propofol with ΔGLIE = −9.18 kcal*mol-1 and Kdpred = 0.17 µM,
while PR1 only provided ΔGLIE= −7.77 kcal*mol-1 and Kdpred = 1.92 µM (Figure 5 [A]).

A B
0 0
PR1
-5 PR2
GMM-PB/SA (kcal/mol)

-5
-10
GLIE (kcal/mol)

-15
-10
-20

-15 -25

PR1 -30
11
PR2
-20 -35
0 10 20 30 40 50 0 10 20 30 40 50
Time (ns) Time (ns)
Figure 5: Gibbs free energy of binding measured for the PR1 and PR2 binding sites of HSA
during the 50 ns MD simulation using the explicit LIE (A) and implicit MM-PB/SA (B)
solvation models.

On the contrary, the MM-PB/SA calculation confirmed by the molecular docking studies and
experimental results providing the evidence that, at clinically relevant concentrations of
propofol in the blood, the PR1 binding site would probably be occupied as one with higher
affinity to this drug (Figure 5 [B] and Table 2).

Table 2: Summary of protein-ligand energy terms for different propofol binding sites of the
HSA crystal structure calculated by using the MM-PB/SA approach.
Energy Binding site
terms PR1 PR2
(kcal/mol)
EvdW −32.95 −31.73
Eelec −2.49 −2.77
Epolar_solv 9.69 13.34
ESASA −3.07 −3.08
ΔGMM-PB/SA −28.84 −24.25

To understand protein-ligand affinity at the molecular level, it is useful to determine the protein
residues contributing in this binding. To test this, we calculated the contribution energy (Econt)
per HSA residue showing higher summarized affinity (Esum) for propofol (Econt = −14.23
kcal*mol-1) at PR1 (Esum = −21.31 kcal*mol-1) than at PR2 (Esum = −11.51 kcal*mol-1) due to
the greater amount of residues with negative interaction energy for the former site (Figure 6 [A,
B]).

12
 A B

Figure 6: Energetic contribution (Econt) of HSA residues at PR1 (A) and PR2 (B) in the propofol
binding during the 50 ns MD simulation using the MM-PB/SA solvation model. The residues
with minimal contribution energy are labeled.

The data derived from this study has both clinical and pharmacological relevance: the relatively
strong binding of propofol (PPBprop > 95%) to HSA should competitively release other drug-
like molecules bound to this site (PR2) with less affinity, transiently increasing their free
concentration and physiological effects. The analysis and comparison of propofol binding
mechanisms have also provided valuable information about how this medication might interact
with target proteins in the brain to produce general anesthesia.

Conclusion
All-atom molecular dynamics (MD) simulations are presented on general anesthetic propofol
bound to human serum albumin (HSA) due to the drug PK/PD in the circulatory system. We
implemented the explicit and implicit methods to compare the binding affinity of propofol at
the PR1 and PR2 binding sites in the HSA protein. The MM-PB/SA(GB/SA) implicit model
provided the evidence in accordance with the experimental data (Bhattacharya et al., 2000)
indicating that the HSA-ligand interactions are governed by the vdW forces due to the higher
drug affinity at the PR1 with ΔGMM-PB/SA = −28.84 kcal*mol-1. Finally, our data provides
important information on the accuracy of explicit and implicit solvation models to characterize
the propofol interaction with different HSA binding sites.

13
Conflict of interests
The authors do not have conflicts of interests regarding the publication of this paper.

Acknowledgments
Special thanks are extended to Dr. Biswapriya B. Misra from the Department of Biology,
Genetics Institute, University of Florida for his assistance in the paper's writing. The authors
are also grateful to the BMBF (Bundesministerium für Bildung und Forschung) for their support
of this work by providing the BMBF01 grant to Jens Broscheit.

References
1. Carter, D. C. and J. X. Ho (1994). "Structure of serum albumin." Adv Protein Chem 45:
153-203.
2. Torres, E. and J. Aburto (2005). "Chloroperoxidase-catalyzed oxidation of 4,6-
dimethyldibenzothiophene as dimer complexes: evidence for kinetic cooperativity."
Arch Biochem Biophys 437(2): 224-232.
3. Deeb, O., M. C. Rosales-Hernandez, et al. (2010). "Exploration of human serum
albumin binding sites by docking and molecular dynamics flexible ligand-protein
interactions." Biopolymers 93(2): 161-170.
4. Liu, R., J. Yang, et al. (2005). "Truncated human serum albumin retains general
anaesthetic binding activity." Biochem J 388(Pt 1): 39-45.
5. Langley, M. S. and R. C. Heel (1988). "Propofol. A review of its pharmacodynamic and
pharmacokinetic properties and use as an intravenous anaesthetic." Drugs 35(4): 334-
372.
6. Trapani, G., C. Altomare, et al. (2000). "Propofol in anesthesia. Mechanism of action,
structure-activity relationships, and drug delivery." Curr Med Chem 7(2): 249-271.
7. Altmayer, P., U. Buch, et al. (1995). "Propofol binding to human blood proteins."
Arzneimittelforschung 45(10): 1053-1056.
8. Liu, R., Q. Meng, et al. (2004). "Comparative binding character of two general
anaesthetics for sites on human serum albumin." Biochem J 380(Pt 1): 147-152.
9. Bhattacharya, A. A., S. Curry, et al. (2000). "Binding of the general anesthetics propofol
and halothane to human serum albumin. High resolution crystal structures." J Biol Chem
275(49): 38731-38738.
10. Zhou, R., J. M. Perez-Aguilar, et al. (2012). "Opioid binding sites in human serum
albumin." Anesthesia and Analgesia 114(1): 122-128.

14
11. Schywalsky, M., H. Ihmsen, et al. (2005). "Binding of propofol to human serum
albumin." Arzneimittelforschung 55(6): 303-306.
12. Laskowski, R. A., J. A. Rullmannn, et al. (1996). "AQUA and PROCHECK-NMR:
programs for checking the quality of protein structures solved by NMR." J Biomol NMR
8(4): 477-486.
13. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.;
Olson, A. J., AutoDock4 and AutoDockTools4: Automated Docking with Selective
Receptor Flexibility. J Comput Chem 2009, 30 (16), 2785-2791.
14. Kuntz, I. D.; Blaney, J. M.; Oatley, S. J.; Langridge, R.; Ferrin, T. E., A Geometric
Approach to Macromolecule-Ligand Interactions. J Mol Biol 1982, 161 (2), 269-288.
15. Zou, X. Q.; Sun, Y. X.; Kuntz, I. D., Inclusion of solvation in ligand binding free energy
calculations using the generalized-born model. J Am Chem Soc 1999, 121 (35), 8033-
8043.
16. Hawkins, G. D.; Cramer, C. J.; Truhlar, D. G., Pairwise Solute Descreening of Solute
Charges from a Dielectric Medium. Chem Phys Lett 1995, 246 (1-2), 122-129.
17. Pronk, S., S. Pall, et al. (2013). "GROMACS 4.5: a high-throughput and highly parallel
open source molecular simulation toolkit." Bioinformatics 29(7): 845-854.
18. Hornak, V., R. Abel, et al. (2006). "Comparison of multiple Amber force fields and
development of improved protein backbone parameters." Proteins 65(3): 712-725.
19. Sousa da Silva, A. W. and W. F. Vranken (2012). "ACPYPE - AnteChamber PYthon
Parser interfacE." BMC Res Notes 5: 367.
20. Starr, F. W., J. K. Nielsen, et al. (2000). "Hydrogen-bond dynamics for the extended
simple point-charge model of water." Phys Rev E Stat Phys Plasmas Fluids Relat
Interdiscip Topics 62(1 Pt A): 579-587.
21. Perera, L., L. Li, et al. (1997). "Prediction of solution structures of the Ca2+-bound
gamma-carboxyglutamic acid domains of protein S and homolog growth arrest specific
protein 6: use of the particle mesh Ewald method." Biophysical Journal 73(4): 1847-
1856.
22. de Souza, O. N. and R. L. Ornstein (1997). "Effect of periodic box size on aqueous
molecular dynamics simulation of a DNA dodecamer with particle-mesh Ewald
method." Biophysical Journal 72(6): 2395-2397.
23. Hansson, T., J. Marelius, et al. (1998). "Ligand binding affinity prediction by linear
interaction energy methods." J Comput Aided Mol Des 12(1): 27-35.

15
24. Kumari, R.; Kumar, R.; Lynn, A.; Consort, O. S. D. D., g_mmpbsa-A GROMACS Tool
for High-Throughput MM-PBSA Calculations. J Chem Inf Model 2014, 54 (7), 1951-
1962.
25. Goddard, T. D., C. C. Huang, et al. (2005). "Software extensions to UCSF chimera for
interactive visualization of large molecular assemblies." Structure 13(3): 473-482.
26. Wallace, A. C., R. A. Laskowski, et al. (1995). "LIGPLOT: a program to generate
schematic diagrams of protein-ligand interactions." Protein Eng 8(2): 127-134.
27. Hubbard S.J, Thornton, J.M (1993) NACCESS version 2.1.1, Computer Program,
Department of Biochemistry and Molecular Biology, University College London.
28. Sebel, P. S. and J. D. Lowdon (1989). "Propofol: a new intravenous anesthetic."
Anesthesiology 71(2): 260-277.
29. Duke, T. (1995). "A new intravenous anesthetic agent: propofol." Canadian Veterinary
Journal-Revue Veterinaire Canadienne 36(3): 181-183.
30. Wallentine, C. B., N. Shimode, et al. (2011). "Propofol in a modified cyclodextrin
formulation: first human study of dose-response with emphasis on injection pain."
Anesthesia and Analgesia 113(4): 738-741.
31. Servin, F., J. M. Desmonts, et al. (1988). "Pharmacokinetics and protein binding of
propofol in patients with cirrhosis." Anesthesiology 69(6): 887-891.
32. Lysakowski, C., L. Dumont, et al. (2001). "Effects of fentanyl, alfentanil, remifentanil
and sufentanil on loss of consciousness and bispectral index during propofol induction
of anaesthesia." British Journal of Anaesthesia 86(4): 523-527.
33. Xu, X. W., X. J. Li, et al. (2015). "Synthesis and structure of dicopper(II) complexes
bridged by N-(5-chloro-2-hydroxyphenyl)-N '[3-(methy lamino)propyl]oxamide:
Evaluation of DNA/protein binding, DNA cleavage, and in vitro anticancer activity."
Journal of Photochemistry and Photobiology B-Biology 147: 9-23.
34. Dauter, Z.; Wlodawer, A.; Minor, W.; Jaskolski, M.; Rupp, B., Avoidable errors in
deposited macromolecular structures: an impediment to efficient data mining. IUCrJ
2014, 1 (Pt 3), 179-93.
35. Shityakov, S.; Dandekar, T., Lead expansion and virtual screening of Indinavir derivate
HIV-1 protease inhibitors using pharmacophoric - shape similarity scoring function.
Bioinformation 2010, 4 (7), 295-9.
36. Morris, A. L., M. W. MacArthur, et al. (1992). "Stereochemical quality of protein
structure coordinates." Proteins 12(4): 345-364.

16
37. Mazoit, J. X. and K. Samii (1999). "Binding of propofol to blood components:
implications for pharmacokinetics and for pharmacodynamics." Br J Clin Pharmacol
47(1): 35-42.
38. Poornima, C. S. and P. M. Dean (1995). "Hydration in drug design. 3. Conserved water
molecules at the ligand-binding sites of homologous proteins." J Comput Aided Mol
Des 9(6): 521-531.
39. Stierand, K. and M. Rarey (2009). "PoseView: 2D Visualization of protein-ligand
complexes." Abstracts of Papers of the American Chemical Society 238.

17