Clustering of aqueous amino acids and similar molecules in the presence of phospholipid ...

50 Pages·2016·3.85 MB·English

by  Alexandra Kukharchuk

MASTER THESIS
Alexandra Kukharchuk
Clustering of aqueous amino acids and
similar molecules in the presence of
phospholipid monolayers
Institute of Physics of Charles University in Prague
Supervisor of the master thesis: Dr hab. Lukasz Cwiklik
Study programme: Physics
Study branch: Biophysics and chemical physics
Prague 2016
I declare that I carried out this master thesis independently, and only with the
cited sources, literature and other professional sources.
I understand that my work relates to the rights and obligations under the Act
No. 121/2000 Sb., the Copyright Act, as amended, in particular the fact that the
Charles University has the right to conclude a license agreement on the use of
this work as a school work pursuant to Section 60 subsection 1 of the Copyright
Act.
In ........ date ............ signature of the author
i
Title: Clustering of aqueous amino acids and similar molecules in the presence of
phospholipid monolayers
Author: Alexandra Kukharchuk
Institute: Institute of Physics of Charles University in Prague
Supervisor: Dr hab. Lukasz Cwiklik, J. Heyrovsky´ Institute of Physical Chem-
istry of the CAS; Institute of Organic Chemistry and Biochemistry AS CR
Abstract: Amino acid phenylalanine plays a key role in numerous biological pro-
cesses and is also involved in amyloid fibril diseases. The aim of the thesis is to
deepenourunderstandingofitsbehaviorandpartitioningatinterfaces, andtoin-
vestigate its clustering. Classical atomistic molecular dynamics simulations were
performed for phenylalanine and three other aromatic molecules which chemi-
cal structure is derived from it - phenylglycine, phenylacetic acid and tyrosin.
Molecules are simulated at both water-air and at water-DPPC-air interfaces.
Phenylalanine, phenylglycine and phenylacetic acid demonstrate surface activity
at the water-air interface, whereas tyrosine is not surface active. All molecules
interactwiththelipidmonolayeratthewater-DPPC-airinterfacebutonlypheny-
lalanine penetrates deep into the monolayer. Formation of transient clusters is
observed in the interfacial regions, mostly for phenylalanine.
Keywords: Langmuir monolayer, aggregates, amino acid, DPPC
Na´zev pr´ace: Agregace aminokyselin a podobny´ch molekul v pˇr´ıtomnosti fos-
folipidov´e monovrstvy
Autor: Alexandra Kukharchuk
´
Ustav: Fyzika´ln´ı u´stav UK
´
Vedouc´ı diplomov´e pra´ce: Dr hab. Lukasz Cwiklik, Ustav organick´e chemie a
ˇ ´ ˇ
biochemie AV CR, Ustav fyzika´ln´ı chemie J. Heyrovsk´eho AV CR
Abstrakt: C´ılem t´eto pr´ace bylo prohloubit naˇse znalosti o chova´n´ı fenylalaninu
a strukturnˇe chemicky podobny´ch aromaticky´ch molekul (tyrosin, fenylglycin,
kyselina fenyloctov´a) pomoc´ı metod molekulov´e dynamiky. Poˇc´ıtaˇcov´e simulace
byly provedeny na rozhran´ı voda-vzduch a na rozhran´ı voda-DPPC-vzduch. Na
rozhran´ı voda-vzduch fenylalanin, tyrosin a kyselina fenyloctova´ projevuj´ı povr-
chovou aktivitu, tyrosin se jev´ı jako povrchovˇe neaktivn´ı molekula. Na rozhran´ı
voda-DPPC-vzduch byla pozorov´ana interakce mezi vˇsemi molekulami a DPPC.
Kl´ıˇcov´a slova: Langmuirova monovrstva, klastr, aminokyseliny, aromaticky´ kruh
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Many thanks belong to Dr hab. Lukasz Cwiklik for his valuable advices and
comments to my thesis. I greatly appreciate his time, patience and attitude.
I would also like to express my sincere gratitude to prof. RNDr. Pavel
Jungwirth. Being a part of his team at the Institute of Organic Chemistry and
Biochemstry CAS meant for me both interesting investigations and inspiration.
Moreover, I would like to thank my beloved boyfriend Otto for his enormous
support during my studies.
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Contents
Introduction 2
1 Essential concepts of the cell membrane 4
1.1 Cell membrane structure, lipid bilayers and monolayers . . . . . . 4
1.2 Interactions of membranes with aminoacids . . . . . . . . . . . . . 5
2 Classical molecular dynamics 8
2.1 Introduction to Molecular dynamics . . . . . . . . . . . . . . . . . 8
2.2 Classical MD fundamentals . . . . . . . . . . . . . . . . . . . . . 9
2.3 Empirical force fields . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.4 Methods of solving Newton’s equations of motion . . . . . . . . . 12
2.5 Statistical ensembles . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.6 Selected methodological issues . . . . . . . . . . . . . . . . . . . . 13
2.6.1 Periodic boundary conditions . . . . . . . . . . . . . . . . 13
2.6.2 Interactions cut-offs . . . . . . . . . . . . . . . . . . . . . . 14
2.6.3 Constraints . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.6.4 Temperature scaling . . . . . . . . . . . . . . . . . . . . . 16
2.6.5 Pressure scaling . . . . . . . . . . . . . . . . . . . . . . . . 16
2.7 Types of analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
3 Simulation parameters 20
3.1 Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.2 Software for simulating . . . . . . . . . . . . . . . . . . . . . . . . 20
3.3 System’s preparation . . . . . . . . . . . . . . . . . . . . . . . . . 21
4 Simulations and simulation results 25
4.1 Equillibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.2 Localization of molecules at the interfaces . . . . . . . . . . . . . 26
4.2.1 Density profiles in Water-Air system . . . . . . . . . . . . 26
4.2.2 Density profiles with DPPC . . . . . . . . . . . . . . . . . 28
4.3 Surface excess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.4 Order parameter . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.5 Radial distribution function . . . . . . . . . . . . . . . . . . . . . 34
4.6 Clustering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5 Comparing simulations results with experiment 37
5.1 Surface activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
5.2 Clustering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Conclusion 39
Bibliography 41
List of Figures 44
List of Abbreviations 46
1
Introduction
Majority of all biologically essential processes takes place in a cell. The plasma
membrane, a key cell element, borders all organelles in the cell’s cytoplasm and
encloses the cell itself. Hereby, the plasma membrane plays an important role in
many processes such as cell transport, signaling, apoptosis and many others. The
plasma membrane is a complex dynamic system with a huge variety of its types.
Nevertheless, the basic chemical architecture stands on the combination of lipids,
proteins and sterols. Due to its amphiphilic nature, lipids create a bilayer, with
hydrophobic chains inside the cell membrane and hydrophillic headgroups on its
surface. Such a bilayer, for instance, enables simple diffusion of small uncharged
molecules inside the cell and protects the cytosol from the influx of unfavorable
ions. Together with the incorporated proteins and sterols, that contribute to
the membrane’s properties, the cell membrane represents a fluid semipermeable
barrier [1].
It is obvious, that cell membrane’s interactions with different molecules are
very important. In terms of our work we concentrated on investigation of inter-
actions between the cell membrane and small aromatic molecules. What is so
special on aromatic molecules? Aromatic molecules play key roles in such phe-
nomenaasmembranechannelgating, proteinfolding, DNAstacking, andamyloid
fibril formation. In particular, among amino acids, there are three aromatic ones
- phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr). Phenylalanine is a
unique amino acid due to its ability of self-assembly. Because of both clustering
and previously suggested involvement of membranes in this phenomenon [2], we
focused on Phe in our investigations. On practical side, clustering of Phe is a key
factor in development of phenylketonuria because long fibrils of Phe monomers
tend to be cytotoxic. The disease is characterized by a disrupted mechanism of
Phe metabolism, particularly the synthesis of Tyr from Phe. In order to deepen
our understanding of the Phe behavior in the presence of lipid assemblies, we
employed atomistic-level computer simulations of Phe, Tyr and two other aro-
matic molecules derived from Phe - phenylacetic acid (PhAA) and phenylglycine
(PhGly). By introducing small chemical modifications of Phe molecule, we aimed
at explaining the role played by individual chemical moieties of Phe.
Molecular dynamics (MD) is a computer simulation method that enables in-
vestigating system structure and dynamics at the atomistic level. On one hand,
MD gives the opportunity to perform a virtual ’experiment’ involving individual
atoms and molecules. It also allows for preparation of the system in the desired
way and elimination of the unfavorable influence of the environment. Hence, due
to MD, we can explore interactions between specific molecules that is not directly
possible in experimental methods. On the other hand, MD is an in silico exper-
iment dealing with simplified molecular models in the computer reality. It has,
therefore, several drawbacks among which the most important are relatively lim-
ited length- and time scales as well as the use of approximate force-fields. Typical
time scale of simulations is within hundreds of nano seconds up to few microsec-
onds whereas numerous molecular biological processes take place in micro- and
even milliseconds. The force fields, being an approximation of the interaction
potentials, differ by their nature from the real interactions.
2
In this thesis, I employed atomistic MD simulations to obtain molecular level
information about interactions between specific small aromatic residues (Phe,
Tyr, PhGly, PhAA) and a plasma membrane. One-component phospholipid
monolayer was used as a plasma membrane model; this particular choice was
made to directly mimic the system used in experiments performed in the collab-
orating group. Note that the structure of a real cell membrane is much more
complex, and undoubtedly influenced by proteins and sterols incorporated in it.
Nevertheless, the simplified model enables to investigate both lipid-water inter-
face interactions and penetration of molecules into the lipidic phase.
Phe and several small aromatic molecules, derived from it, were studied in
order to observe the influence of the presence or absence the specific chemical
moieties in the Phe structure. MD simulations were performed both in pres-
ence and absence of phospholipid monolayer (pure water-air interface) with the
main aim to explore the surface behavior of the molecules and the way how they
influence/interact with lipids.
This work contains five chapters. The first one introduces molecules under
investigation. In the second one, I discuss theoretical fundamentals of classical
moleculardynamicswiththeemphasesonmethodsthatwereusedinthiswork. In
thethirdchapter,Ispeakdirectlyaboutthemethodologicalissuesandtheprocess
of preparation of the simulated systems. The next, forth, chapter presents the
results of simulations together with their interpretation. In the last, fifth, chapter
I discuss the obtained results in terms of comparing them with the experiments
performed by the collaborating laboratory [3]. In the last part of the thesis, I
make final conclusions.
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1. Essential concepts of the cell
membrane
1.1 Cell membrane structure, lipid bilayers and
monolayers
Cells are fundamental units of life as majority of all biological processes takes
place inside cells. Each cell is enclosed by a plasma membrane that separates
cytoplasm from the outside environment and controls molecular transport across
theboundary[1]. Itsroleiscrucialnotonlyfortransportofnutrientsandmetabo-
lites into and out of the cell, but also for such complex processes as signaling,
secretion, and prevention of infections. The architecture of the cellular membrane
is essentially based on the lipid bilayer structure with incorporated proteins. The
lipidbilayer-twomonolayersthatformatwo-dimensionalsheet-isformeddueto
amphiphilic nature of the lipids. These are species having both hydrophillic and
hydrophobic parts which enables formation of specific aggregates and assemblies
[4]. Main stabilizing factors of such aggregates are hydrophobic effects, van der
Waals forces between acyl chains of lipids, and electrostatic interactions between
polar heads. The schematic structure of a lipid molecule is shown in Fig. 1.1 a).
Fig. 1.1 b) and c) schematically depict models of lipid monolayer and bilayer.
The complexity of a cell membrane is partially related to asymmetry between
the constituting monolayers. However, due to the fact that interactions between
molecules in two monolayers are typically weaker than interactions within the
same monolayer, lipid monolayers are able to capture many physical characteris-
tics of bilayers and hence are often used as models of lipid bilayers. It gives rise
to the wide usage of Langmuir lipid monolayers as simplified biophysical models
of cell membranes. Important advantages of monolayers are the possibility to
strictly control their composition (which is not an easy task, for instance, during
formation of bilayer vesicles), control of fluidity (by controlling lateral compres-
sion), and ability to study interactions at the water-lipid interface (for instance,
between proteins and lipid headgroups). Monolayers are also relatively easily ex-
plorablebyimagingtechniques(e.g.,fluorescencemicroscopy)andhigh-resolution
spectroscopic methods (e.g., vibrational sum frequency generation).
There are three major classes of lipids molecules in cell membranes: phos-
pholipids, glycolipids and sterols. The most abundant class are phospholipids,
consequently we investigated the phospholipid monolayer [4]. The DPPC (1,2-
dipalmitoyl-sn-glycero-3-phosphatidylcholine)waschosenasaninvestigatedphos-
pholipid, as it is considered the benchmark lipid in the study of model bilayers,
both experimentally and by simulations [5]. Moreover, DPPC is often used in
phospholipid monolayer studies and hence behavior of DPPC monolayers is well
understood, inparticular, inLangmuirbalanceexperiments. Whatisparticularly
important, by varying lateral compression of DPPC films, one can very precisely
explore various phases and phase co-existence regions, and that is crucial for a
better understanding of physical basics of phases and heterogeneities present in
cell membranes. There is also an important practical reason for using saturated
4
lipid films in experiments, as their acyl chains are not prone to oxidation, the
latter being a serious problem in the case of their unsaturated counterparts.
The chemical structure of DPPC molecule is shown in Fig. 1.3. The molecule
consists of a polar hydrophilic head and a hydrophobic tail both linked to the
glycerol moiety. The head group choline is linked to glycerol via a phosphate
group. The hydrophobic tails are formed by two hydrocarbon chains of 16 carbon
atoms,correspondingtopalmiticacid. Theheadgroupisofzwitterioniccharacter,
with partial positive charge localized at the choline group and partial negative
charge at the phosphate.
Figure 1.1: a) Schematic structure of a lipid molecule with a hydrophobic tail
and a hydrophilic head, b) Lipid monolayer, c) Lipid bilayer
1.2 Interactions of membranes with aminoacids
Living cells contain a diverse set of proteins. Proteins serve an enormous variety
offunctions, dependingonwhichonecandistinguishtransport, signal, structural,
and storage proteins, as well as receptors, enzymes etc. Biological activity of a
protein is defined by its structure, that is a function of a unique sequence of
amino acids controlled by the genetic code [1]. Hence, to understand interactions
between proteins and cell membranes in detail, interactions between membranes
and individual amino acids are vital. There are twenty common amino acids
(α amino acids) found in proteins. Their general structure is the following: an
alfa carbon atom, a chiral center of a molecule, has three groups linked to it:
a carboxyl group, an amino group and R-group. The R-group is the side chain
that differs for all amino acids in its size, structure, or electrical charge. Due to
the presence of acid and basic groups, an amino acid is an amphoteric organic
compound and can occur in four (neutral, cationic, anionic and zwitterionic)
different forms depending on pH values of the environment.
In terms of our work we concentrated on investigating the surface behaviour
of certain aromatic molecules in the presence of DPPC phospholipid monolayer.
The molecules under investigation were: phenylalanine (Phe), phenylacetic acid
(PhAA), phenylglycine (PhGly) and tyrosine (Tyr), see Fig. 1.2. Aromatic
molecules play important roles in different essential processes in general. More-
over, Phe is an interesting molecule due to its unique ability of self-assembly [6].
Long fibrils of Phe monomers were observed experimentally. Interacting with the
cell membrane they change three-dimensional structure [2]. It is probable, that
this behavior is closely connected to the cause of phenylketonuria (PKU).
5
To better understand behavior of Phe at the lipid-water interface, we in-
vestigated Phe and three other small aromatic molecules with similar chemical
structure to Phe, though with differences in some particular groups. Tyr, a
proteinogennic amino acid, can be seen as Phe with the OH group added to the
aromatic ring. PhGly can be derived from Phe by removing the CH group in the
2
aliphatic chain. PhAA is similar to PhGly, however, without the NH moiety.
3
Considering the physiological environment with regular pH value, zwitterionic
forms of all molecules were assumed.
Figure 1.2: Aromatic molecules under investigation
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