Master Literature study
Coiled Coils
Studying the latest research performed on coiled coils
by
Ilja Kroon
20 December 2019
Student number
11207418
Research institute
Van 't Hoff Institute for Molecular Sciences
Research group
Supervisor
2
1. Abstract
Coiled coils are two or more α-helices twisted around one other (figure 1). They consist of an almost complete systematical orientation, due to a heptad repeat and a C2-symmetry axis. The heptad repeat
is indicated by (abcdefg)n and formed by 3.5 amino acids per helical turn. Coiled coils have different
functions based on specificity and their stability. In this thesis the natural processes of coiled coils as transcription factors and their use in membrane fusion will be discussed. As transcription factor coiled coils regulate DNA transcription, so a specific binding to the RNA polymerase II and DNA is needed. In membrane fusion a heterodimeric interaction is needed for the fusion of two membranes. From these natural processes, artificial adjustments will be derived. First structural changes and systematic orientations will be discussed. Already much research has been performed on coiled coils and new insights have been created. Especially structural changes of the coiled coils obtained a broader overview. For example preferences in hetero- or homodimerization or their topology. Further, artificial membrane fusion is already successfully applied, however artificial transcription factors need more research. Not much effective changes in the artificial transcription factors have been obtained. By performing more research an even broader scope of coiled coil systems can be derived, thereafter coiled coil interactions can be applicable in the medicinal area, for drug delivery or bioimaging in combination with nanotubes.
Figure 1. Schematic coiled coil. On the left a dimer is presented and on the right trimer coiled coil is presented.1
3 2. Index 1 Abstract p.2 2 Index p.3 3 Introduction p.4 3.1 Aim p.5
4 Functionalities of coiled coils in nature p.6
4.1 Coiled coils as transcription factor p.6
4.2 Membrane fusion induced by coiled coils p.7
4.2.1 Vesicle tethering p.7
4.2.2 Membrane fusion p.8
4.2.3 Initiation of HIV into host cells p.9
4.3 Summary p.9
5 Artificially adjusted coiled coils p.10
5.1 Mutation in the amino acid sequence p.10
5.1.1 Mutations in the a positions p.10
5.1.2 Mutations in the e-and g- positions p.11
5.2 Coiled coils in nanomaterials p.12
5.3 Artificial transcription factor p.13
5.4 Artificial membrane fusion p.13
6 Discussion and prospects p.14
7 Acknowledgements p.15
8 References p.15
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3 Introduction
Coiled coils are two or more α-helix structures twisted around one other. An α-helix is the secondary structure of a protein formed by interacting amino acids.2 These amino acids have an interaction with
one other over a distance of four amino acids(i-i+4) via hydrogen bonds of the C=O groups (i) and the N-H groups (i+4). α-Helices and coiled coils are present in all types of organism, for example yeast, viruses and mammals.
Coiled coils have a systematic organization in their amino acid sequence; a heptad repeat.3 This is due
to the helical orientation with two turns per helix. The heptad repeat indicates that a pattern is repeated after seven residues, these amino acids
are often denoted as (abcdefg)n in which positions
a and d are occupied by hydrophobic amino acids and positions e and g are occupied by charged amino acids.4, 5 The coiled coil structures can vary on
parallel and antiparallel orientations. A parallel configuration means that the N-terminus of the compounds are oriented at the same side. The antiparallel conformation consist of a C- terminus side connected to the N-terminus side of the other α-helix. The electrostatic interactions between the flanking amino acids e and g provide stability of the coiled coil, in antiparallel g-g’ and e-e’ interactions are present as shown in the top picture of figure 2 and in the parallel conformer g-e’ and e-g’ interactions are present as shown in the bottom picture of figure 2.
Due to the heptad repeats in the coiled coils there is a more systematic orientation. The symmetrical C2
-axis also provide a more systematical structure. This symmetry indicates that after a rotation of 1800
around the C2-axis the exact same structure is obtained. In the case of a parallel structure this axis is
parallel with the helices as seen in the top picture of figure 2. In case of an antiparallel conformation the axis is perpendicular to the helixes as seen in the bottom picture of figure 2.7
The side chains on the a- and d-position of two or more α -helices are packed in such a way that a side chain (knob) from one helix is placed in the space surrounded by sidechains of the opposite α-helices (holes).8 This interlocking provides a tighter packing in the coiled coils, where 3.5 amino acids per turn
are preferred, instead of 3.6 amino acids per turn as in a single α-helix.2,9 This tight packing creates van
der Waals interactions and a regularity in the packing interactions, which provides a more stable compound than separate α-helices.5 Especially short coiled coils with a three to five heptad repeat are
stable.
Coiled coils can differ in the amount of repeats, such as short coiled coils as the leucine zipper with four heptad repeats or vesicle tether coiled coils, which consist of more than 100 amino acids per complex.10,11 Additionally, discontinuities can be formed by insertion and deletion of residues. When
four residues are deleted (or three are inserted) a stammer is formed.9 This leads to an even tighter
packing of the coiled coil, due to a decrease in periodicity of amino acids per turn (<3.5) as shown in figure 3C. In the case of an insertion of four (or deletion of three) the coiled coil is more unwound and
Figure 2. Interaction of the coiled coils; the orange dotted lines are hydrophobic interactions (a-d) and the red dotted lines are electrostatic interactions (e-g).On top the parallel conformation with its C2-axis parallel to the
helixes and in the bottom picture the antiparallel conformation is shown with a perpendicular C2-axis. 6
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a stutter is formed. If a stutter occurs every 18 residues, the periodicity changes to 3.6, which is the ideal conformation for an α-helix.12 With regular repeats of the stutters in the heptads a difference in
repeats can occur, providing hendecad and pentadecad repeats, each eleven and fifteen repeats respectively. These differences also change the handedness of the coiled coil. Periodicities of 3.5 (heptads) consist of a left-handed turn as shown in figure 3A. In contrast to hendecads and pentadecads, 3.67 and 3.75 amino acids per turn respectively, which are right handed (figure 3D). These structures consist of 3 turns per 11-residues and 4 turns per 15-residues, due to the exceeded 3.6 residues per turn.9 The further away from the 3.6 periodicity the more strain in the helix is created,
and that is why stammers, with a periodicity of 3.4 are rare.
Figure 3. Changes in conformation by insertion or deletion of amino acids. A) undistorted structure B) insertion of 1: "skip" C) Stammer D) Stutter
3.1 Aim
Coiled coils occur in great variety with multiple functions, such as selective binding and creating specificity and stability. More insight in the differences and the systematical orientation of the coiled coils leads to a greater insight for artificial synthesized coiled coils and their applications, for example in the medicinal area as drug delivery systems. In this thesis the important biological activities of coiled coils will be discussed, focusing on membrane fusion and coiled coils as transcription factors. Additionally, the computational design of the coiled coils will be discussed and some artificial applications will be proposed to generate an overview in the current coiled coil research.
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4 Functionalities of coiled coils in nature
Coiled coils can have different functions due to their great versatility in structure. In this chapter a few natural processes will be discussed, focusing on membrane fusion and transcription.
4.1 Coiled coils as transcription factor
Coiled coils can be active as transcription factors. Transcription factors are proteins, which possess certain binding areas for the interaction with DNA. Due to this interaction RNA polymerase II can bind and transcribe the DNA to RNA. The binding of a transcription factor controls the gene activity.13 A
transcription factor can be a processor and induce transcription or be a repressor and repress transcription. An example for a well-known repressor is the Lac repressor, which is present in bacteria and plays a role in the lactose metabolism. When lactose is not present the transcription factor is bound and the DNA is not transcribed.14 An example of a processor transcription factor is GCN4, which
is present in yeast and regulate almost all genes involved with the amino acid synthesis. GCN4 is activated by amino acid starvation.15
Structural examples of transcription factors are leucine zippers (LZ) and helix-loop-helix (HLH) domains. Both consist of dimerized coiled coils, so interaction with the DNA can take place. Due to the dimerization step in advance the cell can regulate the activity of the transcription factors.16 The structure of the leucine
zipper is a Y-shape, in which the stem is the leucine zipper, that creates stability.13
The arms of the Y are the amino acid terminal basic regions, which interact with the major groove of the DNA (figure 4 left panel).
Leucine zippers are relative short coiled coils.The leucine zipper consist of a four heptad repeat with leucine at the d-position, which is needed for the dimerization. A single replacement of leucine does not necessarily eliminate this dimerization property. Another important amino acid is asparagine present at the a- position. This forms a hydrogen bond with the asparagine from the opposite helix, inducing the parallel orientation. Distortion in this area would cause an interaction of the asparagine with the apolar site of the opposite helix and so destabilize the structure.16
The helix-loop-helix proteins are slightly different than the leucine zippers. They also consist of the leucine zipper end but with a connection to a helix. This first helix is linked via a loop to the second helix and this second helix is attached to the basic area that connect to the DNA. In this structure the first helices interact with one other and with their hydrophobic side it binds to the second helix (figure 4 right panel). The helix-loop-helix proteins work in a cooperative way, this is because they occur as tetramers, but bind to the DNA as dimers. Their tetrameric structures are bound by a head to tail fashion, which forms an antiparallel four-helix bundle.18 When one dimer has bound the second prefers
also binding to DNA over existing free in solution.16
Figure 4. Left leucine zipper structure, right the helix-loop-helix structure. (LZ=leucine zipper, HLH=Helix-loop-helix, BR= Basic region).17
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4.2 Membrane fusion induced by coiled coils
Membrane fusion can occur in different types of cells. In this section three types of membrane fusion will be discussed. Vesicle tethering in the Golgi apparatus, membrane fusion in for example synaptic cells and membrane fusion induced by viruses.
4.2.1 Vesicle tethering
The Golgi apparatus is part of the endomembrane system of eukaryotic cells. Its function is to store products from the endoplasmic reticulum for future transport.19 The Golgi apparatus consists of a cis-
and trans-side. The cis-side receives the vesicles released from the endoplasmic reticulum, where they will be fused with the Golgi-membrane, and thereafter the content is released in the Golgi apparatus. For further transportation new vesicles from the Golgi apparatus are released at the trans-side, which is faced to the cell membrane.20
In the Golgi complex more than 15 different coiled coils are bound, called Golgins. Golgins can extend to capture transport vesicles.21 Golgins can be bound to the membrane in different ways. For example
direct to the membrane (transmembrane Golgins) or via an adaptor protein, such as GRASP family proteins (a stacking protein).22 Other adaptor proteins include Rab (G-protein), ARL or ARF (
ADP-ribosylation factor) families. The interaction between Rab and the Golgin is unknown. However, the interaction of ARL and the Golgin occurs via a RIP (receptor interacting protein) domain and for the ARF a GRAB (GRIP-related ARF binding) domain is bound, both being able to bind as dimers.23
One residue in a coiled coil can extend the complex with 0.148 nm.24 This elongated feature makes
them suitable for connection between membranes.21 This increase in length indicates the distance the
coiled coil is able to reach into the cytoplasm. Yet, there is no exact mechanism of the vesicle tethering. The current opinion in biology will be discussed here. For the capturing of vesicles, Rab-GTP is loaded on the vesicles. This connects to the tethering factor; an unbound coiled coil, presented in yellow in figure 5. The C-terminus of this tethering factor binds to the basic N-terminus of the Golgin.23 After connection the
whole tether complex will collapse or bend, depending on rigidity, bringing the vesicle to the membrane. Instead of collapsing the vesicle can also “hop” along the tether to the membrane.21,25 The process of tether collapsing
is presented on the bottom left of figure 5, and the vesicle hopping is presented on the bottom right of figure 5. After this transfer the vesicle is fused with the cis-membrane of the Golgi apparatus, where it will be stored for later release on the trans-side.
Figure 5. Proposed mechanism of vesicle tethering (top). Vesicle transportation to Golgi membrane (bottom).25
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4.2.2 Membrane fusion
Besides Golgins, several other coiled coils are able to fuse membrane. This membrane fusion occurs in other areas than the Golgi apparatus, for example in synaptic cells.26 Here vesicles filled with
neurotransmitters will be released in the synaptic cleft by exocytosis. In the synaptic cleft, the neurotransmitters can bind to receptors and create a downstream signaling cascade.27
Membrane fusion occurs in a three step process. First, connection of the coiled coils present on different membranes, creating a heterodimeric interactions. This interaction is followed by fusion of the outer layer (hemifusion). After the hemifusion the inner layer of the bilipid membrane also fuses as shown in figure 6, resulting in complete membrane fusion.28 The exact process is yet unknown.
The coiled coil complex is created by SNARE proteins (soluble NSF attachment protein receptor; NSF=N‐ethylmaleimide‐sensitive factor) a protein that consist of an eight heptad repeat.29 The SNARE
protein is connected to the membranes via a linker. This linker consist of positively charged amino acids.30 For membrane fusion it is important that both helices prefer heterodimerization. In the case
of homodimerization, there is a possibility of two vesicles fusion with one other. In that case the mechanism would be disturbed and neurotransmitters will not be released, and no signal will be transferred.
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4.2.3 Initiation of viruses into host cells
Envelop viruses infect host cells by a temporary fusion of the membranes, an example for this is HIV (human immunodeficiency virus). HIV is a disease which spreads via sexual or blood contact and infects the immune system. The host cell does not kill the virus due to the envelope consisting of viral proteins covered by the same membrane of the host cell. This way the immune system does not recognize the virus.31 The viral proteins of HIV consist of 2 subunits, gp120 and gp41, which form a noncovalent
trimer complex on the surface.32 For the infection of the host cell (often CD4 T-cells, which play an
important role in the regulation of the immune system) binds gp120 to the CD4 receptor and the CCR5 or CXCR4 coreceptor on the host cell and creates a cascade of changes in the envelope complex.32,33 In
this way the gp41 fusion protein is able to insert in the membrane. This insertion creates a six helix bundle, which causes complete membrane fusion (figure 7).34 In this way the virus can transfer its RNA
into the host cell, where via reverse transcription the host cell is taken over by the viral DNA.33
To target HIV different ways of inhibition are used. The ideal HIV inhibitor should have a small molecular weight, is inexpensive, stable and orally active.35 An example of a coiled coil based
mechanism is the use of a trimeric coiled coil complex that fits on the trimeric gp41 complex.36 The end
terminus of gp41 is carbon so the connecting site of the inhibitor is an amine group. Research have been performed on this tetrameric structure. For this leucine zipper sequences were used to decrease aggregation and disulfide bonds at the C-terminus were placed to increase stability.37 This coiled coil
based drug is not optimal, because the effects of this binding are yet unknown. Additionally, this complex has a high molecular weight and would need to be injected, which is not optimal. Current administered peptide based drugs do not meet the optimal criteria, but many clinical trials are performed in this area, so in the future coiled coil derived drugs could be applicable.37
4.3 Summary
Analyzing the natural processes a greater insight in the structures of the coiled coil complexes and their mechanisms is obtained. For the structure there can be seen that in general the a-position is occupied by hydrophobic amino acids. In combination with the d-position it plays an important role in the dimerization of the compound. The e- and g-positions are occupied by charged amino acids and are especially important for the specificity in the dimerization. Research upon the b, c and f position should be performed to investigate the specific importance of these sequences. In the mechanism of membrane fusion it is especially important that a heterodimeric coiled coil consist. This is for the fusion of the vesicles to the cell membrane and not with vesicles among each other. In the part of the transcription factor, there could be seen that the coiled coil leucine zipper created stability in the complexes. Also certain interaction patterns became visible. For example the preference for an arginine-arginine interaction in the Leucine zipper for the inducing of a parallel configuration. These properties can be taken into account in artificial derivatives.
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5 Artificially adjusted coiled coils
The heptad repeats of the coiled coil structure are partly systematic, which simplifies the design of these compounds. Another property, which induces specificity, is the high symmetry in the coiled coils, a C2-symmetry. However, there is not yet a complete systematic prediction of the coiled coils. A wealth
of structural information is available in the form of X-ray structures. The structures provide a greater insight in the stability and will increase knowledge to the systematical structure. With this information artificial coiled coils can be synthesized, which can be applicable in different areas. In this chapter, differences in the structure will be analyzed with mutations of the amino acids sequences. Thereafter recent research in artificial processes derived from natural processes will be discussed.
5.1 Mutations in amino acid sequence 5.1.1 Mutations in the a positions
In the research of Acharya et al. alterations of the amino acids in all the a-a’ position of coiled coil dimers were performed. This is one of the important positions, just as the d-position, which determines the preference in homo- and heterodimerization. In this research 10 different amino acids were substituted on the a-a’ positions ((I, V, L, N, A, K, S, T, E, R). These amino acids were used, because they are often found in leucine zippers.38 The leucine zipper is often used as model for coiled coils, due
to their short monomeric structure and the ability for hetero- and homodimerization. The thermal stability of these dimers were analyzed by using circular dichroism spectroscopy, which uses left and right polarized light. The compound will absorb this light and change the emission coefficient, speed and wavelength of the light. This change provides an indication of the secondary structure and the folding of the compound.39
With a double mutated alanine thermodynamic cycle as reference the coupling energy between the different amino acids were determined. This cycle was derived from an alanine-alanine complex, in which these amino acids will be altered to one of the nine other amino acids. From the circular dichroism the melting temperature (Tm) and the enthalpy (ΔH) were derived, which were converted to
the heat capacity (Cp). From this information the difference in free energy (ΔG) was derived. The final
difference in difference of the Gibbs free energy (ΔΔG) of both altered helices can be calculated and this difference (ΔΔG - ΔΔG ) indicates the coupling energy. Appendix 1 shows a schematic overview for the calculation of the coupling energy. A large difference in coupling energy implies a preference for homodimerization, such as isoleucine (I) with asparagine (N). Table 1 shows all the coupling energies of the different complexes. The positive coupling energy of isoleucine and asparagine of +10.6 kcal/mol is due to their repulsive interactions and thus prefers homodimerization. This is in contrast to charged amino acids (K, R, E) with a small difference in coupling energies, which encourage the heterodimerization.38 For example glutamic acid (E) and lysine (K), with a coupling energy of -0.9
kcal/mol. In the case of charged amino acids, heterodimerization is created by the opposite charges of the molecules. Models just as this one provide a greater overview of the coiled coil structures and so can be used in other models for stability or dimerization. From this research it can be concluded that the preference is often for homodimers due to many repulsive interactions between amino acids. Hydrophobic (I, V, L) and polar (N, T, S) amino acids form preferable homodimers by interaction with other hydrophobic or polar amino acids. As seen in table 1, with combinations as isoleucine (I) and serine (S), with a coupling energy of +2.7 kcal/mol. Charged amino acids (K, R, E) can form heterodimers if a positively charged amino acid is interacted with a negatively charged amino acid. Interactions with hydrophobic and polar amino acids with charged amino acids also prefer heterodimerization, with the exception of isoleucine (I) and glutamic acid (E); +1.3 kcal/mol and asparagine (N) and glutamic acid (E) with a coupling energy of +0.3 kcal/mol.
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Table 1. Coupling energies (kcal/mol) of different amino acid interactions.38
I V L N T S K R E I - V +1.1 - L +1.5 +0.9 - N +10.6 +3.7 +3.3 - T +2.6 +0.8 +1.2 +1.35 - S +2.7 +2.3 +2.0 +0.9 +0.65 - K -0.3 0 0 -0.15 -0.7 -0.5 - R 0 -0.5 -0.3 -0.2 -0.75 -0.6 +0.2 - E +1.3 +0.2 -0.15 +0.3 -0.75 -0.75 -0.9 -1.25 -
5.1.2 Mutations in the e- and g- position
Adjustments in the e- and g-position can create alterations in the parallel and antiparallel configuration and the topology. Often the flanking e- and g-positions are charged with electrostatic interactions. By analyzing the Lac repressor there was discovered, that it can self-associate into an antiparallel tetramer.40 This occurs by the uncharged amino acids present on the e- and g-position. The Lac
repressor interhelical hydrophobic interactions of a, d and e/g are of high importance for this assembly.
The alteration of uncharged amino acids on the e- and g- position were artificially tested on the GCN4 protein. By substituting the g-position to alanine or valine a tetrameric structure was formed, instead of the dimer. With use of X-ray diffraction, visible knobs-into-knobs and knobs-into-holes interactions between a, d and g were visible in an antiparallel orientation.41 The interaction of the a, d and g
positions creates a 3-3-1 interaction, in contrast to the interaction of the a- and d-position in a canonical coiled coil (figure 8A). A 3-3-1 interaction indicates that the first amino acid (a), three amino acids further (d) and three amino acids further (g) interact (figure 8B). The alteration of an uncharged amino acid on the e-position has the same effect as when performed upon the g-position. If both e- and g-positions are altered by an uncharged amino acid such as alanine, a parallel heptamer will form.40 A parallel heptamer has a 3-1-2-1 interaction as seen in figure 8C. In these complexes a hollow
middle area is created. These coiled coil complexes ranging from tetramers to heptamers could encapsulate certain molecules and thus be applicable in combination with nanomaterials.
Figure 8. A) canonical coiled coil 3-4 interaction (a-d). B) antiparallel tetramer, 3-3-1 interaction (a,d,g) C). parallel heptamer, 3-1-2-1 interaction (a,d,e,g).40
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5.2 Coiled coils in nanomaterials
Nanomaterials are compounds with features of size in the order of 1-100nm. In the design of nanomaterials there are two main approaches: ‘synthesizing up’ and ‘engineering down’. In the synthesizing up, biology as a model for artificial reproduction is used. The engineering down approach is to miniaturize complexes to macroscale.42 In this thesis, synthesizing up is from great importance because natural processes are analyzed to create artificial derivatives.
Coiled coils can be used in nanomaterials to achieve different goals, for example assist in drug delivery systems or in bioimaging. Bioimaging is the linking of a fluorescent compounds to certain receptors of cells, which can be irradiated and give a signal. This indicates the presence or overexpression of the receptors present on specific cells.
In nanomaterials, molecular self-assembly is an efficient method to organize molecules in a precise and predetermined structure.43 Coiled coils are well suited for self-assembly for the following reasons:
Coiled coils consist of a well-defined shape, size and surface functionality, the intra- end interhelical interactions are well understood, self-assembly is possible at low concentrations and they can easily be functionalized at the C or N-terminus.44
As seen in the previous section tetrameric to heptameric compounds consist of a hollow middle area. This is due to their tube conformation. This tube formation is shown on the left in figure 9. The tube contains a certain pore size which is accessible for hydrophobic substances, due to the hydrophobic side chains inside the coiled coil tube, often consisting of isoleucine or leucine.45 Lysine is placed on
all f-positions, which made the compound more soluble in water. Additionally, lysine created higher stability and yields charge repulsion, thus decreasing self-aggregation.46 In this pore a near infrared
fluorescent carbon nanotube is added (figure 9). This is a tubular carbon grid with a honey comb structure, which is fluorescent when irradiated with infra-red light. Carbon nanotubes are used in this complex, due to their facile differentiation in diameter and great thermal stability.43 Nanotubes should
consist of a fitted diameter, to match the pore size of the coiled coil. Coiled coils can self-assemble around the nanotube, using the carbon tube as a template. The multiple coiled coil tubes interact with each other via opposite charges on the C- and N-terminus and exposed hydrophobic side chains. This end-to-end self-assembly is constructed through hydrophobic and electrostatic interactions.47
Due to the coiled coil encapsulation and its solubility, the nanotube can be transported through the human body and connect to the target cells. Peptides can recognize receptors, which are often overexpressed in pathological cells.48 After recognition the coiled coil tube will degrade and release the
drug. Another possibility is that the coiled coil complex transport through the membrane and degrade in the cell due to an pH change.49 The property of the carbon nanotube with infrared fluorescence is
advantageous, because infra-red radiation is less harmful to the human tissue and can penetrate deeper though the tissue, with less interference of other proteins and so will be suitable for bioimaging. Also with use of IR light, When the complex is linked to the cells it can use thermal destruction to kill for example cancer cells.49
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5.3 Artificial transcription factors
For a greater range of transcribing possibilities, artificial coiled coil transcription factors can be synthesized. Heterodimers are preferred for use as transcription factor. Due to the two different coiled coils a greater diversity in RNA polymerase binding can occur and so create a larger variety of sequences that can be transcribed.50 To achieve this a greater versatility is needed than currently
available in the artificial structures. One of the first synthesized heterodimer was ‘Velcro’ which had glutamate on the e- and g-position of one dimer and lysine at the e- and g-position of the other dimer. This interaction was created by electrostatic interaction.51
An example of a synthetic adjustments in coiled coils as transcription factors was made in GCN4 were on the a-position was fluorinated. Isoleucine was converted to 5,5,5-trifluorisoleucine (figure 10). This alteration increased the melting temperature of the coiled
coil with 27 0C and did not change the binding properties.52
Changes in amino acid sequence can influence the stability and binding affinity of the coiled coils. Fluorination of certain amino acids increases the stability of thermal unfolding and chemical denaturation of the coiled coils by increasing the buried hydrophobic area.53 Further influences of this
fluorination, such as toxicity, are yet unknown.
5.4 Artificial membrane fusion
The fusion of membranes can be applicable in the delivery of drugs or proteins into a cell. Examples that can occur in nature are vesicle tethering or viral infections. The main purpose of these coiled coils is inducing membrane fusion. Research performed by Marsden et al. focused on the membrane fusion with use of SNARE proteins. These proteins create a four helix structure to bring two membranes in close proximity of 2-3nm, as mentioned earlier in section 4.1.1.29
In the artificially adjusted version the SNARE proteins are mimicked. This is performed by DOPE (1,2‐dioleoyl‐sn‐glycero‐ 3‐phosphatidylethanolamine) as an anchor to the membrane. DOPE is connected via a linker; a short poly(ethyleneglycol) chain (PEG), which connects to the helix (figure 11). For the coiled coil a simplistic structure is used; a three heptad repeat coiled coil. This is one of the shortest coiled coil complexes possible.29 These coiled coils should have a strong
heterodimeric interaction and therefore glutamic acid (K) and lysine (E) are placed on the a-a’-positions.54 This dimer is
created by the electrostatic interactions of the charged amino acids. Due to the difference in charge the formation of homodimers are prevented. The three step fusion process as discussed in the section 4.1.2, consisting of coiled coil interaction creating a close proximity of both membranes and further hemi- and complete membrane fusion. This artificial
process should consist of a specific interaction between the coiled coils and fusion of both lipid layers without leakage.
Figure 11. Systematic scheme of the artificial SNARE complex.29
Figure 10. Structure of isoleucine and fluorinated isoleucine
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With use of Förster resonance energy transfer (FRET) differences in emission between donor; nitrobenzofuran (NBD) and acceptor; lissamine rhodamine (LR) dyes were measured.26 The inner and
outer lipid layer of the membranes were covered with the dyes. When the membranes are not yet fused the acceptor dye will give a strong emission. After fusion a higher emission of the donor dye would be visible due to the larger distance between the donor and acceptor dye. To test if a complete fusion occurred, the dyes on the outer layer were deactivated, so only the emission of the inner layer dyes would be visible. To check if the cells leaked, ethylenediaminetetraacetic acid (EDTA) was used. EDTA react with the Tb(DPA)33− complex present inside the vesicle and would then provide a
fluorescent signal. No signal was visible so the fusion occurred without leakage. This was substantiated by the reaction time of 1 minute, which indicates that the driving force was created by the E-K coiled coil interaction and not by curvature strain by the membranes. In this research a successful artificial membrane fusion occurred.
6. Discussion and Prospects
In this literature thesis, an outlook of the structure and artificial adjustments are provided. Also the main functionalities of the coiled coil are discussed, among which membrane fusion and as transcription factor.
Looking at the natural structure of coiled coils, there can be seen that the a- and d-position are hydrophobic. The flanking e- and g- position are often charged, creating electrostatic interactions and providing stability. In the research of Acharya et al. the preferences for homo- or heterodimerization was analyzed by using their coupling energies. From this research there could be concluded that most interactions prefer homodimerization. Heterodimerization often occurs in combination with a charged amino acid. In another research by Grigoryan et al. the e- and g-positions were altered. In this research was showed that a change from charged to uncharged amino acid of only one of the two positions create an antiparallel tetrameric structure. When both positions consist of an uncharged amino acid a parallel heptameric structure consist.
These changes in the structure provide a greater overview of the systematic structure of coiled coils. This information can be applied in different artificial applications, for example membrane fusion. As seen in the research performed by Meyenberg et al. a successful membrane fusion is created. Such artificial membrane fusions can be used in the transfer of medicine, when these have to be transported through a membrane. Which is often needed, because drugs are often large molecules which do not flow easily though the membrane and often have to connect to a receptor for transportation. Further research in this area can include analyzing the precise mechanism of the natural membrane fusion process among which vesicle tethering. With a greater insight in this mechanism, there could be more anticipated on this membrane fusion in artificial processes.
Looking at transcription factors, there is not many artificial adjusted transcription factors. The research of Son et al. fluorinated the isoleucine present on the a-position. Which provided the complex from greater stability, due to a more buried hydrophobic core. . But, no changes in binding properties were visible. Yet the influences on for example toxicity are unknown Further research should be performed on these effects. Furthermore, artificial transcription factors do not seem applicable in humans, due to the great change in gene activity it can create. These transcription factors should only be used in prokaryotic cells, due to morality issues. In prokaryotic cells transcription factors can cause transcription of certain parts of DNA and create desired proteins, which can later be used in other applications.
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Coiled coils can also be applied in nanomaterials, where they are used as a transporter. In the research of Mann et al. a soluble environment for the nanomaterials was created, because nanomaterials are often insoluble by itself. Personally, I see a great future for this application, due to the nontoxicity and easy transportation. I hope more research in coiled coils in combination with nanomaterials will be performed.
Concluding, coiled coils have different application possibilities. Nowadays, a lot of research has been performed in coiled coils. This creates many possibilities in applications such as with nanomaterials and in the medicinal area. Yet a lot more research has to be performed, because a lot is still unknown of the coiled coils and their mechanism.
7. Acknowledgements
I would like to thank dr. Jocelyne Vreede for her help during this literature thesis. She always had time for me and provided me from great feedback on my report and on my presentation. Additionally, I would like to thank dr. Francesco Mutti for being my second corrector.
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Appendix 1
Schematic explanation of the thermodynamic cycle. The differences in both ΔΔG is the coupling energy in kcal/mol.
