The Open Nutraceuticals Journal, 2010, 3, 213-219

213

Open Access

Molecular Dynamics Simulation Studies on Effects of Erythritol on

Alcohol Dehydrogenase Folding
Surya Prakash Dwivedi1, Neeraja Dwivedi1, Abhishek Tyagi1,3, R.B. Singh2 and Sanjay Mishra1,4,*
1

Department of Biotechnology, College of Engineering & Technology, IFTM Campus, Lodhipur Rajput, Delhi Road,
Moradabad 244 001, U.P., India
2

Halberg Hospital and Research Institute, Civil Lines Moradabad 244 001, U.P., India

Indian Institute of Technology, Kharagpur 721304, West Bengal, India

Department of Biotechnology & Microbiology, Institute of Foreign Trade & Management, Lodhipur Rajput, Delhi
Road, Moradabad 244001, U.P., India
Abstract: Liver Alcohol dehydrogenase (LADH) catalyzes reversible oxidation of primary and secondary alcohols to
aldehyde using NADH+ as a coenzyme. The aim of the present study was to validate preexisting biophysical characterization of erythritol using simulation studies of protein-erythritol-water interaction in solution at the molecular level. Two
molecular dynamics simulations of the protein ADH in solution at room temperature were carried out, one in the presence
(about 0.77 M) and another in the absence of erythritol. The erythritol molecules were observed to cluster and move
toward the protein, and expel water from the protein surface and ultimately form hydrogen bonds with protein. Besides,
the coating by erythritol was noticed to reduce the conformational fluctuations of the protein compared to the erythritolfree system. Conclusively, at a moderate concentration of erythritol solution, erythritol molecules cluster in the protein
region and interact with ADH via many H-bonds that prevent the protein folding, although the data shown in the present
study are contrary probably due to providing a minimum solution from water with an osmolyte like erythritol with 10 ns
simulation and subsequently studying the desired interaction with protein using above solution using 15 ns of simulation.
Over findings provide new insights into further exploration of the studies pertaining to ADH activity in alcoholic liver
diseases leading to lever Cirrhosis.

Keywords: ADH, erythritol, molecular dynamics simulation, H-bonds, protein folding.

INTRODUCTION
Erythritol (2R, 3S)-butane-1, 2, 3, 4-tetraol) is a natural
sugar alcohol, a sugar substitute [1]. It occurs naturally in
fruits and fermented foods. Industrially, it is produced from
glucose by fermentation with yeast, Moniliella pollinis [2].
Horse liver alcohol dehydrogenase (HLADH, EC 1.1.1.1)
catalyzes reversible oxidation of primary and secondary
alcohols to aldehyde using NADH+ as a coenzyme. The
oxidation involves alcohol hydroxyl proton ionization and
passage of the H+ through a hydrogen bond network to water
via His51 imidazole and hydride transfer from alkoxide to
NAD+. This enzyme has a molecular weight of 80 kDa and is
a dimer of two identical subunits as reported in the X-ray
structure. The enzyme has a twelve-stranded
- sheet, which
makes up the central core of the dimer. Each subunit of this
dimeric enzyme binds one molecule of NAD+ and two Zn
(II) ions. One of the zinc ions is in the active site, while
another is structural. Upon enzyme substrate complex formation a rigid body rotation closes the cleft between the coenzyme and catalytic domains. This conformational change is
*Address correspondence to this author at the Department of Biotechnology
& Dean (Research) College of Engineering & Technology, IFTM Campus,
Lodhipur Rajpoot, Delhi Road, Moradabad (U.P.) India; Tel: +91 0591
2360817; Fax: +91 0591 2360818; E-mail: [email protected]
1876-3960/10

described by a rotation of 9 to 10 in the monoclinic form.

The hydroxyl oxygen of the alcohol substrate is ligated to the
active site zinc.
One of the common methods in industry for stabilizing and
keeping of enzymes is use of osmolytes. Osmolytes are
compounds that support proteins and cells in unfavorable
condition. These materials are usually small and non-charge
organic compounds that dont change enzymatic activity.
Besides, osmolytes can act as molecular chaperon and can
enumerate suitable compound for trapping molten globule of
some proteins. Nevertheless, pattern of melting of helixes
can be changed accompanied with amplification of hydrophobic interaction, and hydrogen bonds. Osmolytes are small
organic compound that are ubiquitous in living systems.
Osmolyte molecules in aqueous solution can have profound
effects on protein stability, structure, and function [3], and it
has long been known that osmolytes as solvent additives
have significant effects on protein folding or unfolding [4].
Various osmolytes such as trimethylamine N-oxide [5], glycerol [6], betaine [7], and erythritol [8] were used to study
protein folding experimentally. An osmolyte can enhance
and inhibit protein folding, and the favoring and inhibiting
effects of an osmolyte on protein folding depend on its concentration. Despite its widespread use, the molecular basis
for osmolytes ability to assist and inhibit protein folding
2010 Bentham Open

214 The Open Nutraceuticals Journal, 2010, Volume 3

remains unknown. It is reported that an osmolyte stabilizes a

folded state of protein, while it binds to the protein by hydrogen bonds. The stabilizing effect shifts the equilibrium
toward the native state, thereby favors the protein folding
[9]. It has been mentioned that an osmolyte directly influences protein folding by being preferentially excluded from
the backbone of the protein, which raises the free energy of
the unfolded state and favors the folded population [10]. In
fact, protein stability is the result of a balance between the
intramolecular interactions of protein functional groups and
their interactions with the solvent environment. Thus, as
mentioned above, presence of small organic molecules like
osmolyte in aqueous solution can also have basic effects on
protein stability, structure, and function.
The influence of osmolytes on biomolecules has been extensively investigated. Currently available experimental techniques have often led to contradictory conclusions. Three
main hypotheses have been put forward involving (1) the
direct interaction between osmolyte molecules and the protected biostructure through hydrogen bonds (water-replacing
hypotheses) [11-14]; (2) the trapping of water molecules
close to the biomolecular surface (water-layer hypotheses)[15]; and (3) the entrapment of a particular biomolecu;ar
conformation in a high-viscosity osmolyte glass (mechanical-entrapment hypotheses) [16, 17].
Theoretically, it revealed that trehalose could enhance and
inhibit protein folding and the favoring and inhibiting effect
of trehalose on protein folding depends on its concentration
[18]. At higher trehalose concentrations, trehalose prevents
the peptide from folding to its native structure. In another
theoretical study, it is reported that at moderately concentrated trehalose solution, trehalose does not reduce the conformational fluctuation of the protein [19]. It is apparent
from mentioned study [20] that in lower concentration of an
osmolyte such as trehalose, the peptide and protein folds
faster than that in water. Molecular-level insights into how
osmolyte molecules effect protein folding would be invaluable for the rational design of small molecular additives for
enhancing or hindering the folding of protein. However, it
seems unlikely that experimental approaches can provide the
molecular details. Molecular simulation is expected to contribute greatly to this end. The aim of the present study was
to: (a) validate preexisting biophysical characterization of
erythritol using simulation studies of protein-erythritol-water
interaction in solution at the molecular level; and (b) provide
new insights into further exploration of the studies pertaining
to ADH activity in alcoholic liver diseases leading to lever
Cirrhosis.
MATERIALS AND METHODS
All molecular dynamics (MD) simulations were performed
using the gromacs 3.3 program together with the G43a1
force field [20-23] for each simulation. The MD simulations
were carried out by particle mesh Ewald method [24] for the
electrostatic interactions. The Vander Waals cutoff was 14
. The integration time step was 1 femtosecond (fs), with the
neighbor list being updated every fifth step by using the grid
option and a cutoff distance of 12 . Periodic boundary condition was used with constant number of particles in the systems, constant pressure, and constant temperature simulation
criteria (NPT). In this simulation the systems were coupled

Dwivedi et al.

to external constant temperature (100, 200, 300 K,
= 0.1

ps) in three steps and external constant pressure bath (1 atm,

= 0.5 ps). For all simulations, the water molecules were
added as a simple point charge (SPC) model.
The force field parameters for the erythritol were extracted
from the website pertaining to Dundee PRODRG Server
[25]. Besides, force field parameters of glucose were positively considered for the simulation studies. Protonated state
of various residues were determined from the pKa calculation
by PROPKA at PH=7 [26]; and the point charges for erythritol was obtained from a HF/6-31G* single point calculation
in Gaussian 98 using the CHELPG fitting procedure [27-28].
Note that the limitation of this approach is that the polarization effect associated with the condensed phase environment
is not explicitly included, although the tendency for the
HF/6-31G* QM level of theory to overestimate dipole moments has been suggested to account for this deficiency [29].
RESULTS AND DISCUSSION
In the present study the attempts have been made to determine effect of erythritol on folding of ADH, and therefore
ADH had to be studied in a box including water molecule
and ions in absence and presence of erythritol with a moderate concentration. Initially, a mixture of water and erythritol
molecules was prepared considering it in a minimum location, followed by filling latter solution in a box involving
ADH. Ultimately, equilibrium and minima geometries of
ADH could be obtained in a box with water molecule and
ions both in absence and presence of erythritol. The data
extracted from the present studies are summarized as follows:
For the Preparation of a minimum solution of water molecules with erythritol molecules, an erythritol molecule was
first placed in a cubic box with periodic boundary conditions. The size of the cubic box was 50. Then, 65 molecules of erythritol were added to the box. Ultimately, the box
was filled with 3491 SPC water molecules. Since this system
is neutral, addition of ions is not required. Referring to
erythritol density, its molecular weight and ratio between the
number of erythritol molecules and that of water molecules
which is equal to ~ 1/ 54, erythritol concentration would be
about 0.95M in the box. Thus, erythritol has a moderate concentration. Note that erythritol molecules were randomly
added to the aforementioned box [18]. Initially, the water
molecules alone were subjected to energy minimization with
the erythritol molecules kept fixed in their initial configuration. The water molecules were then allowed to evolve using
a molecular dynamics simulation for 20 ps with a step time
of 1 fs, again keeping the structure of the erythritol molecules fixed. Next, the entire system was minimized using the
steepest descent of 1000 steps followed by conjugate gradients of 9000 steps. In order to obtain equilibrium geometry
at 300 K and 1 atm, the system was heated at a weak temperature (
= 0.1 ps) and pressure (
= 0.5 ps) coupling by
taking advantage of the Berendesen algorithms [23]. Heating
time for molecular dynamics simulations at 100 K, 200 K
and 300 K was 100 ps. All above simulations were performed at constant temperature and pressure with a nonbonded cutoff of 14 . Molecular dynamics simulation was
further carried out for 3 ns at 300 K, followed by structural
minimization calculation. The latter minimization was per-

Erythritol Versus Alcohol Dehydrogenase Folding

The Open Nutraceuticals Journal, 2010, Volume 3

formed at the steepest descent of 1000 steps followed by

conjugate gradients of 9000 steps. This way, a minimum
geometry was obtained for solution of erythritol with water
molecules. Variation in kinetics and potential energy components versus time in MD simulation has been well illustrated (Figs. 1a & b). By having a look at this figure, it reveals that the kinetics and potential energy components
would be expected to fluctuate in equal and opposite direction. This point is explicitly specified by referring to Fig.
(1b). Data of afore mentioned Figure belongs to last 300 ps
of simulation. Latter facts show that energy conservation is
satisfied in MD simulation performed [30]. Thus, the simulation has been done accurately and the extracted equilibrium
structure was obtained by employing accurate algorithms
and parameters in solving motion equations and under energy stability conditions.

215

strong chance to pass probable barrier energies in phase

space and reach to more deep minima of a solution studied.
Simulated annealing is often a convenient way to ensure the
conformational space is explored effectively [31], and that in
which the conformational space is sampled at sufficiently
high temperatures. A simulated annealing algorithm starts
from a given geometry and has an ability to cross barrier to
other conformers.
In simulated annealing, starting point has been the final system obtained after 3 ns MD simulation. First, mentioned
system has been heated to 600K by employing of an MD
simulation of 200ps. Strategy being decrease in temperature
from 600 K to 300 K in time duration of 200 ps, followed by
keeping temperature in 300K for 200ps. Then, temperature
was decreased from 300K to 50K followed by increase from
50 K back to 300 K. Time duration for each increase or decrease of temperatures was 50 ps. This way carrying out a
simulated annealing of 7 ns, 10 near local minima structures
(10 geometries in 50K) is resulted form starting system. Inspection of the annealed systems showed no indication of
any bond breakage or tension in structures as expected. Ultimately, the annealed near local geometries were optimized

As mentioned elsewhere, the ability to climb over energy

barrier is limited in MD simulation. Molecular dynamics is
used to explore the conformational space in order to find a
conformation (or conformations) that only have a low intrinsic energy. Therefore, it often attempts to search conformational space. In fact, conformational analysis gives us a

Kinetic Energy

35600

-120000

33100

-125000

30600

-130000

28100

-135000

25600
0

1000

2000

Potential energy (kj/mol)

Kinetic energy (kj/mol)

Potential Energy

-140000
3000

T ime (ps)
Kinetic energy (kj/mol)

33600

-125000

31600

-127500

29600

-130000

27600

-132500

25600
2700

2800

2900

Potential energy (kj/mol)

Kinetic energy (kj/mol)

Potential energy (kj/mol)

-135000
3000

T ime (ps)

Fig. (1). Variation in kinetic and potential energies components observed in MD simulation of a box including erythritol and water molecules
at 300K.

216 The Open Nutraceuticals Journal, 2010, Volume 3

by taking advantage of steepest descent of 1000 steps followed by conjugate gradients of 9000 steps methods. Among
these minima geometries, a geometry was chosen which had
the least MM energy value. Energy of the latter geometry
was even less than that of the one which had been obtained
from optimization of system after MD simulation of 3 ns.
Thus, in order to obtain a minimum structure from solution
of erythritol with water molecules, performing conformational analysis is necessary.
As mentioned above, a minimum geometry of solution involving erythritol and water molecules was obtained using
MD simulation as well as simulated annealing. In order to
investigate erythritols effect on ADH stability, performing
two simulations are required, namely simulation of ADH in a
box including water molecules and ions in the absence as
well as in the presence of erythritol molecules. The X-ray
structure of ADH (HLADH, EC 1.1.1.1) was extracted from
protein data bank. The latter structure was placed in a solvent box with 60683 spc216 water molecules being equal to
a 1.8 distance for ADH to the box edges. Then, same Xray structure was placed in a cubic box. The size of the cubic
box was 110, 110, 160. The latter box is filled with
erythritol and water molecules belonging to minimum solution which had been obtained from previous step. In this
way, two systems were thus prepared: one including ADH
and 60683 SPC water molecules, while another involving
ADH and 45272 SPC water molecules as well as 689 erythritol molecules. Neutralization of each system required addition of eight Cl- ions. It is worth mentioning that use of cubic
box in the study of effect of osmolytes on protein is benefited in previous works [18, 19].
Referring to erythritol density, its molecular weight and ratio
between the number of erythritol molecules and that of water
molecules which is equal ~1/66, erythritol concentration
would be about 0.77M in the box. Thus, erythritol has a
moderate concentration. Initially, former system namely the
water molecules and Cl- ions alone were subjected to energy
minimization with the ADH kept fixed. Next, latter system,
the water molecules, Cl- ions as well as erythritol molecules
were exposed to energy minimization with the ADH remained fixed. Both systems along with their corresponding
constituents were then allowed to evolve using a molecular
dynamics simulation for 20 ps with a step time of 1 fs, again
keeping the structure of the ADH molecules fixed. Subsequently, following operations were performed for two systems studied.
Each of the entire above two systems was individually
minimized using the steepest descent of 1000 steps followed
by conjugate gradients of 9000 steps without imposing any
constraint. In order to obtain equilibrium geometry at 300 K
and 1 atm, the system was heated at a weak temperature (
=
0.1 ps) and pressure (
= 0.5 ps) coupling by taking advantage of the Berendesen algorithms [16]. Heating time for
molecular dynamics simulations at 100 K, 200 K and 300 K
was 100 ps. All above simulations were performed at constant temperature and pressure with a non-bonded cutoff of
14 . A molecular dynamics simulation was performed for 5
ns at 300 K. Ultimately, another molecular dynamics simulation was further carried out for 10 ns at 300 K, followed by
structural minimization calculation. The latter minimization
was performed at the steepest descent of 1000 steps followed

Dwivedi et al.

by conjugate gradients of 9000 steps. This way, a minimum

geometry was obtained for ADH at moderately concentrated
erythritol solution. As far as variations in total energy versus
time is concerned, for 10ns of the MD simulations is about
less than 1 part in 1000 and it is quite apparent that the kinetics and potential energy components would be expected to
fluctuate in equal and opposite direction for MD simulation
of ADH in absence and presence of erythritol molecules.
Latter facts show that law of energy conservation is fulfilled
in MD simulations [29]. Thus, the simulation has been done
accurately and the extracted equilibrium structures were obtained by benefitting accurate algorithms and parameters in
solving motion equations and under energy stability conditions. In addition, temperature fluctuation for the simulation
at 300 K for each of the systems was less than 0.5K. Therefore, the final equilibrium structure at 300K belonging to
every system was obtained under temperature stability conditions.
Data obtained from the experiments carried out on conformational changes of the ADH in absence and presence of
erythritol molecules entails that the RMSD from the initial
structure for backbone atoms is used to represent the conformational changes of the ADH during the folding. The
Backbone RMSD is displayed as a function of time in Fig.
(2), during simulation of ADH in absence and presence of
erythritol molecules. Having a look at Fig. (2), it reveals the
final values of the Backbone RMSD as well as these values
on the average are decreased in presence of erythritol molecules, and they are smaller than that in pure water. This point
is obvious from values of Backbone RMSD during simulation and belonging to last 300ps of simulations which has
been decreased about 5
and 6
in presence of erythritol
molecules, respectively. Besides, fluctuations in aforementioned values are considerably weakened by ~ 16
and 37

in presence of erythritol molecules than that in pure water,
respectively. Latter fact has been clarified by referring to
standard deviation for RMSD values which are equal to 0.02
and 0.017 and RMSD values belonging to last 300ps of that
are equal to 0.0095 and 0.006 in absence and presence of
erythritol molecules in last 300ps of simulation, respectively.
Besides, the RMSF per residues show that the mobility of
residues in the presence of the erythritol molecules is decreased by ~
6, when compared with the mobility in the
absence of erythritol molecules. Standard deviation for theses RMSF values are equal to about 0.041 and 0.031 in absence and presence of erythritol molecules, respectively.
Thus, fluctuations in these RMSF are considerably decreased
by ~ 22
in presence of erythritol molecules than that in pure
water. Also, mobility of some residues is considerably in the
presence of erythritol molecules. As examples, the RMSF
values belonging to residues 1-45 of ADH have been decreased ~
20 on the average due to presence of erythritol
molecules. Also, fluctuations in these RMSF are considerably weakened by ~ 42
in presence of erythritol molecules
than that in pure water. Latter point is completely clear from
standard deviation for theses RMSD values which are equal
to 0.075 and 0.043 in absence and presence of erythritol
molecules, respectively.
Considering the RMSF per atoms, it is obvious that the mobility of atoms belonging to ADH have been reduced in the
presence of erythritol molecules ~7
as observed for RMSF

Erythritol Versus Alcohol Dehydrogenase Folding

The Open Nutraceuticals Journal, 2010, Volume 3

217

In the absence of erythritol molecules (a)

0.25

RMSD (nm)

0.2
0.15
0.1
0.05
0
0

2000

4000

6000

8000

10000

T ime (ps)

Fig. (2). Root-mean-square atomic positional deviation (RMSD) obtained from the initial structure for Backbone atoms (a) in the absence of
erythritol molecules (b) in the presence of erythritol molecules.

per residues. This reduction is completely figured out by

referring to atoms 1-431 which belong to residues 1-45.
These atoms have a mobility decrease of ~ 16 in moderately
concentrated erythritol solution relative to the atoms of ADH
in pure water solvent. Further, RMS deviation of residues
and atoms belonging to ADH is decreased in the presence of
erythritol molecules. These findings completely corroborate
to RMSD Backbone values obtained and summarized in Fig.
(3). Thus, the results depicted above implicate that erythritol
inhibits the folding of ADH at moderate concentration of
erythritol solution. Such a behavior is qualitatively consistent
with the experimental observation of firefly luciferase
refolding in presence of erythritol [32], and a speculative
theory about
-hairpin folding [11].
The ADH folding via H-bond interactions involved in the
absence and presence of erythritol molecules has been analyzed. Table 1 lists the total number of inter-molecular and
intra-molecular H-bonds for final geometries of ADH which
were obtained from MD simulations of 15ns in the absence
and presence of erythritol molecules. It has been known that
backbone H-bonds increase during the folding [33]. The
backbone H-bonds are closely connected during the peptide
desolvation, because H-bond between water and the ami-

dogen and carboxyl groups must be broken before the backbone H-bond can be formed. In fact peptide desolvation is a
deriving force in peptide folding [34]. Data of Table 1 show
that intra-molecular H-bonds belonging to ADH as well as
inter-molecular H-bonds between ADH and water molecules
are decreased in presence of erythritol molecules about 4
and 19, respectively. Also, some H-bonds are formed between ADH and erythritol molecules. Thus, it can be concluded that erythritol molecules accumulate around the protein and exclude water. Besides, forming H-bonds between
protein and erythritol has an effect on the formation of the
protein backbone H-bonds. Thus, although erythritol molecules can dissolve protein, but formation of more H-bond
between erythritol and ADH can act a factor for inhibition of
folding protein, while backbone H-bonds is also changed.
Ultimately, the protein cannot fold into its native state in
moderately concentrated erythritol solution due to the direct
interaction between osmolyte molecules and the protected
biostructure through hydrogen bonds (water-replacing
hypotheses). Nevertheless, at a moderate concentration of
erythritol, more erythritol-erythritol interaction occurs (Table
1). This is because of clustering of most of the erythritol
molecules in the simulation system. Nevertheless, the num-

218 The Open Nutraceuticals Journal, 2010, Volume 3

Dwivedi et al.

Distance (nm)

1.6

1.2

0.8

0.4

0
0

2000

4000

6000

8000

10000

Time (ps)

Distance (nm)

1.6

1.2

0.8

0.4

0
0

2000

4000

6000

8000

10000

Time (ps)
Fig. (3). The average distances of the water (a) and erythritol (b) molecules with the center of geometry of the protein as a function of time
for the simulation including erythritol molecules.
Table 1.

The Number of Inter-Molecular and Intra-Molecular Hydrogen Bonds for Conclusive Geometries of ADH Obtained from
MD Simulations of Femtosecond in the Absence and Presence of Erythritol Molecules

Number of Hydrogen Bonds

Absence of Erythritol Molecules

Presence of Erythritol Molecules

Water-ADH

1056

852

ADH-ADH

563

541

Water-erythritol

ND

4658

Erythritol-ADH

ND

214

Erythritol-Erythritol

ND

754

ND= not detected.

ber of hydrogen bonds between ADH and water molecules

as well as the number of hydrogen bonds between erythritol
and water molecules is decreased during simulation, respectively and number of hydrogen bonds between ADH and
erythritol molecules is increased during simulation (Table 1).
Consequently, more H-bonds can form between the protein
and erythritol molecules when protein folds in the presence
of moderately concentrated solution of erythritol. Also, the

average distances of the water and erythritol molecules with

the center of geometry of the protein are displayed in Fig. (3)
for simulation including erythritol molecules.
CONCLUSION
Conclusively, the major findings from the present study provide new insights into the development and projection of
new design of putative molecular additives to exaplain com-

Erythritol Versus Alcohol Dehydrogenase Folding

The Open Nutraceuticals Journal, 2010, Volume 3

prehensively the probable involvement of a novel mechanism of osmolysis of erythritol molecules, ultimately, leading to inhibiting the folding of alcohol dehydrogenase, an
enzyme catalyzing a reversible oxidation of primary and
secondary alcohols to aldehyde using NADH+ as a coenzyme. Further work along these notions and obejectives is in
progress in our laboratory.

[14]

ACKNOWLEDGEMENTS

[17]

The authors are grateful to Prof. R. M. Dubey (Managing

Director) and Prof. A. Srivastav (Director), CET, IFTM,
Moradabad, U.P, India for providing the necessary facilities
and encouragement. Besides, Dr. R.S. Sangwan and Dr.
(Mrs.) N.S. Sangwan (Senior Scientists, CIMAP, Lucknow
India) are duly acknowledged for critical reading of the
manuscript and valuable suggestions.

[18]

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Accepted: June 08, 2010

Dwivedi et al.; Licensee Bentham Open.

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