Beruflich Dokumente
Kultur Dokumente
Biomedical Research Foundation, Academy of Athens, 4 Soranou Ephessiou, 11527, Athens, Greece
ABSTRACT: (Word Style BD_Abstract). All manuscripts must be accompanied by an abstract. The abstract should briefly state the problem or purpose of the research, indicate the theoretical or experimental plan used, summarize the principal findings, and point out the major conclusions. Abstract length is one paragraph.
Introduction
The octapeptide angiotensin-II (H-AspArgValTyr
IleHisProPhe-OH, AII) is one of the oldest peptide
hormones, known for its multiplicity of biological actions
related to endocrine or connected to the central and peripheral nervous system1. It is produced by the conversion
of angiotensin-I (H-AspArgValTyrIleHisProPhe
HisLeu-OH, AI) to AII by the action of the angiotensin-I
converting enzyme (ACE) of the vascular endothelium.
AII is a potent pressor agent, which has a vital role in the
regulation of blood pressure, in the conservation of total
blood volume and salt homeostasis. AII binds to the AII
receptor AT1, which is a transmembrane protein. As the
binding site of AT1 receptor is exposed to the lipid membrane, AII is directly exposed to the lipid bilayer. An X-ray
structure of Angiotensin II-Fab complex shows that the
hormone peptide adopts a compact U-shaped structure
when it is bound to the receptor2. However, photolabeling
studies of [Bpa3]AngII with the AT1 receptor indicate a
model where the peptide in its bound form adopts a rather extended -strand structure3. Furthermore, several
NMR studies of AII in aqueous and organic solvents indicate the existence of a mixture of unfolded and folded
conformations ranging from extended -structures to the
more compact U-shaped ones4-8. Organic solvents such as
TFE and heating have been reported to favor the fold of
the angiotensin amide in a rather compact structure determined through circular dichroism measurements9.
However, recent NMR and Molecular dynamics studies of
[Val5] AngII analogue in a water/ethanol (35%v/v) binary
solvent at low temperatures, suggest that the peptide is
primarily in an extended -structure and that most regions of the peptide are preferentially solvated by ethanol
molecules10-11. Despite the inherent flexibility of the octapeptide, another possible reason for the divergent structural models proposed over the years could be the use of
different solvents and experimental conditions.
Mixtures of ethanol and water are solvents that can influence stability and conformational properties of biological molecules. Use of such solvents in experiments with
biomolecules that usually operate in environments that
are only partly aqueous may produce results that help in
understanding the role(s) of water in maintaining macromolecule structure and activity. In other arenas, mixtures of water and ethanol provide reaction media that
enable enzymes to act on substrates that are not soluble
in water, although substrate specificity, reaction rates and
protein stability may be altered by the solvent mixture. In
all of these systems, the effects observed may be the result
of direct interactions of solvent alcohol molecules with a
protein. In general, preferential solvation of proteins by
organic co-solvents and clustering of organic molecules in
the binary solution are considered possible mechanisms
that could either allow or block proteins from jumping
between different conformational states12-14.
Moreover, ethanol/water mixtures may be considered
as a bilayer mimic considering the amphiphilic nature of
ethanol.
In this paper, we provide an atomic-level picture of the
intermolecular interactions governing AngII-water and
AngII-water-ethanol mixtures using MD simulations.
Structural and dynamical properties of these systems are
outlined in the context of identifying the solvent microenvironment around the AngII peptide and in describing
the representative conformations of AngII in these two
media. Our results are in excellent agreement with relevant experimental data and provide insights into the bioactive conformation of this hormone.
Methods
System Preparation
Human Angiotensin II (AngII) was chosen -Asp-ArgVal-Tyr-Ile-His-Pro-Phe-for the conformational study of
the peptide in water and ethanol/water-35% (v/v) solvent
at different temperatures. The initial structure of the octapeptide was taken from the Protein Data Bank,
1N9V.pdb7. The apparent pH was chosen to be 4 in order
to match most of the experiments. So, in the model peptide the terminals are ionized. Asp is not protonated and
charged (-1), Arg is protonated and charged (+1) and His is
protonated and charged (+1). The system was solvated in
pure water or 35% ethanol-1,1-d2-water (v/v) and neutralized with 1 Cl- ion.
ically, the distance between the center of mass of the Cand N-terminal residues (ASP-PHE) reaches the lowest
values ( ~1.4-1.1nm ) in water and (~1.4-1.0nm) in water/ethanol the same time periods that the radius of gyration adopts also its minimum values (Fig.1 iii, iv). NMR
experiments have revealed that the C- and N- terminal
residues distance of AngII in water is 0.72nm8 while in
water/trifluoroethanol and in dimethylsulfoxide (Spyroulias, 2003) is around 1.78 nm. In our simulations we observe that the folding/unfolding events of AngII at elevated temperatures (310K, 323K) seem to be significantly
more frequent in the binary solvent (water/ethanol) than
in water.
Arg2 hydrogen bond of about 1-4% in pure water and 312% in water/ethanol (see Table S2).
In Water
In Water/Ethanol
Gerigs sim.
*Exp.
278K
0,1846( 0,0458)
0,0579 ( 0,0025)
0,053 ( 0,007)
0,041
298K
0,6045 ( 0,0245)
0,3497 ( 0,0899)
0,11 ( 0.02)
0,14
310K
0,9412 ( 0,3155)
0,1563( 0,0141)
323K
2,7192 ( 0,6804)
0,4776 ( 0,1569)
*Simulations and experiments for AngII in ethanol/water-35% (v/v), reported in Gerig, 2013.
In Water/Ethanol
Gerigs sim.
*Exp.
278K
0,7344( 0,0034)
0,342( 0,013)
0,22
298K
1,2809 ( 0,0105)
0,839 ( 0.022)
0,68
310K
1,7711 ( 0,0645)
323K
2,3184 ( 0,0908)
*Simulations and experiments for AngII in ethanol/water-35% (v/v), reported in Gerig, 2013.
Table 3. Coordination numbers of each peptide residue with O ethanol and Owater in the first solvation shell for the system of AngII
in water/ethanol.
NOwater
NOethanol
Res. 1
16
2
Res. 2
11
2
Res. 3
2
1
AUTHOR INFORMATION
Corresponding Author
* (Word Style FA_Corresponding_Author_Footnote). Give
contact information for the author(s) to whom correspondence should be addressed.
Present Addresses
If an authors address is different than the one given in the
affiliation line, this information may be included here.
Author Contributions
The manuscript was written through contributions of all
authors. / All authors have given approval to the final version
of the manuscript. / These authors contributed equally.
(match statement to author names with a symbol)
Res. 4
14
8
Res. 5
2
1
Res. 6
14
2
Res. 7
15
3
Res. 8
12
2
ACKNOWLEDGMENT
ABBREVIATIONS
CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand
2; CCR5, CC chemokine receptor 5; TLC, thin layer chromatography.
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