Master Literature study
Coiled Coils
Studying the latest research performed on coiled coils
by
Ilja Kroon
1 April 2020
Student number
11207418
Research institute
Van 't Hoff Institute for Molecular Sciences
Research group
Supervisor
Computational Chemistry
Dr. Jocelyne Vreede
2
1. Abstract
Coiled coils are two or more α-helices twisted around one other (figure 1). Their orientation is almost entirely systematical, due to a heptad repeat and a C2-symmetry axis. The amino acids in a heptad
repeat are 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 role of coiled coils in biological processes, such as transcription factors and their use in membrane fusion will be discussed. 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 naturally occurring processes, artificial adjustments will be derived. First structural changes and systematic orientations will be discussed. Currently, many research has been performed towards coiled coils to understand their structure and activity. This research is helpful for artificial adjustments to coiled coils. Analysis of the effect of certain structural changes, created an overview in preferences for hetero- or homodimerization and their topology. Further, artificial membrane fusion and drug delivery systems in different topology are already successfully applied. However, more research towards artificial transcription factors need to be performed, as few 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. Overall, coiled coil interactions can be applicable and promising in the medicinal area, for drug delivery or bioimaging.
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 viruses into host cells p.9
4.2.3.1 HIV p.9
4.2.3.2 CoV-2 virus p.10
4.2.3.2.1 CoV-2 vaccines p.11
4.3 Summary p.12
5 Artificially adjusted coiled coils p.13
5.1 Mutation in the amino acid sequence p.13
5.1.1 Mutations in the a- position p.13
5.1.2 Mutations in the e-g position p.14
5.2 Encapsulation of nanomaterials with coiled coils p.15
5.2.1 Unbound nanostructures p.16
5.2.1.1 Hydrogels p.16
5.2.1.2 fibres p.17
5.2.1.3 Tubes p.18
5.2.2 Directly bound nanostructures p.19
5.2.2.1 SAGEs p.19
5.2.2.2 Vesicles p.20
5.2.2.3 Polyhedrons p.21
5.2.3 Origami nanostructures p.22
5.3 Artificial transcription factor p.23
5.4 Artificial membrane fusion p.23
5.5 Labelling with use of coiled coils p.24
6 Discussion and prospects p.26
7 Acknowledgements p.27 8 References p.28 9 Appendices p.35 9.1 Appendix 1 p.35 9.2 Appendix 2 p.36 9.3 Appendix 3 p.37
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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 another 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) in the backbone. α-Helices and coiled coils are present in all types of organism, for example yeast, viruses and mammals.
Coiled coils have a systematic organisation in their amino acid sequence; a heptad repeat.3 This is
caused by the helical orientation with two turns per repeat. The heptad repeat indicates that a pattern is repeated after seven residues, these amino acids are 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 connected to the N-terminus of the other α-helix. The electrostatic interactions between the flanking amino acids e and g provide stability of the coiled coil, in parallel orientation e-g’ interactions and hydrophobic a-a’ and d-d’ interactions are present as shown in the top picture of figure 2. In the antiparallel conformer, g-g’ , e-e’ and a-d interactions are present as shown in the bottom picture of figure 2.6
The heptad repeats in the coiled coils results in a systematic orientation of an alpha-helical bundle. The symmetrical C2- axis also provide a more systematical
structure. This symmetry indicates that after a rotation of 1800 around the C
2-axis the exact same structure is
obtained. In the case of a parallel structure this axis is parallel with the helices as shown in the top picture of figure 2. In case of an antiparallel conformation the axis is perpendicular to the helixes as shown 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 tighter packing creates a
regularity in the packing interactions, which provides a more stable compound than separate α-helices.5
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.9,10 Additionally, discontinuities in the repeat can occur via insertion and deletion of residues.
When four residues are deleted (or three are inserted) a stammer is formed.11 This leads to an even
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
helixesand in the bottom picture the antiparallel conformation is shown with a perpendicular C2-axis.6
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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 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, which contain eleven and fifteen residues respectively. These differences also change the handedness of the coiled coil. Periodicities of 3.5 (heptads) cause of a left-handed turn as shown in figure 3A. In contrast to hendecads and pentadecads, which contain 3.67 and 3.75 amino acids per turn respectively, result in 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.11 The further away from the 3.6 periodicity the
more strain in the helix is created, that is the reason that 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 synthesised 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 and possible future 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 transcription and membrane fusion.
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 can be a repressor and repress transcription. 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.14 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.15
Structural examples of transcription factors are leucine zippers (LZ) and helix-loop-helix (HLH) domains. Both consist of dimerised coiled coils, so interaction with the DNA can take place. Due to the dimerization, 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).17
Figure 4. Left leucine zipper structure, right the helix-loop-helix structure. (LZ=leucine zipper, HLH=Helix-loop-helix, BR= Basic region).17
Leucine zippers are relatively short coiled coils.The leucine zipper consists of a four heptad repeat with leucine at the d-position, which is needed for the dimerization. Yet, a single replacement of leucine does not necessarily eliminate this dimerization property. Another important amino acid is asparagine, which is present in 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
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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 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, whereafter 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
(ADPribosylation 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, which brings 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 5.25 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
Additional to 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 signalling cascade.27
Membrane fusion occurs in a three step process. First, the coiled coils present on different membranes connect, creating 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) which 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 to fuse with one other. In that case the mechanism would be disturbed and neurotransmitters will not be released, and no signal will be transferred.
Figure 6. Mechanism of membrane fusion by SNARE.28
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4.2.3 Initiation of viruses into host cells 4.2.3.1 HIV
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
Figure 7. Mechanism of viral membrane fusion.34
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 of vaccines is the use of a trimeric coiled coil complex that fits on the trimeric gp41 complex.36 The end terminus of gp41 is the carboxy terminus, 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 disulphide bonds at the C-terminus were placed to increase stability.37 This coiled coil based drug is not optimal, because the side-effects of this binding
are yet unknown. Additionally, this complex has a high molecular weight and would need to be injected, which is not preferable. 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
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4.2.3.2 CoV-2 virus
In December 2019 the virus SARS-CoV-2 was identified in Wuhan, China, whereafter it has spread globally.38, 39 Over one year the world has been impacted by this virus, which led to several societal
measurements as social distancing and lockdowns. The virus can directly spread via aerosols and other indirect ways, such as shaking hands. The virus is highly contagious and its clinical features include severe acute respiratory distress syndrome and multiple organ failure.40 According to the WHO (world
health organisation), the impact of the virus differs per person, as elderly, diabetes and asthma patients are more vulnerable to the virus.
SARS-CoV-2 is a single stranded RNA envelop virus in the trimeric class and is thus similar to the previously described HIV-virus.41 This class I fusion proteins have different conformations during the
membrane fusion: native state, metastable (pre-hairpin) and a stable post-fusion conformation. The CoV-2 virus consist of two subunits, the first subunit consist of a receptor binding domain that recognizes and binds to the receptor angiotensin-converting enzyme 2 (ACE2).42 The second subunit
consist of two trimeric coiled coils (HR1 and 2) of 100 and 50 Å respectively.43
After the receptor binding domain (RBD) of subunit one has bound to the angiotensin converting enzyme 2 (ACE2) receptor, the transmembrane protease serine 2 (TMPRRS2) on the host cell membrane activates the spike protein and promotes the entrance of the virus.41 After the first step of receptor
recognition the dissociation of subunit two is induced, whereafter the trimeric fusion peptide (FP) is fused into the host cell membrane. Thereafter, subunit two mediates the cell membrane fusion by folding the heptad repeat core in an antiparallel manner to form a six helical coiled coil bundle. During this conformation a large amount of energy is released which drives the membrane fusion (figure 8).44
For the membrane fusion there are several interactions between the proteins of the host and viral cells. These interactions among others consist of aromatic-aromatic and ionic interactions.41 One of
the aromatic-aromatic interaction is the phenylalanine of SARS-CoV-2, which is inserted in the hydrophilic pocket of ACE2 and connects to tyrosine.45 This interaction is stronger than SARS-CoV as
this virus consist of a leucine- tyrosine binding which is weaker, due to the lack of aromatic-aromatic interactions of the coiled coil and thus consist of a lower binding affinity. For the structural changes of the virus to enhance invasion, it is important to neutralize the two hotspots of the ACE2 receptor both consisting of lysine amino acids. The virus neutralizes these amino acids with an interaction with tyrosine.45
As mentioned the corona virus impacts the respiratory system, this is because the ACE2 receptor is often found in the lungs and heart.41 The SARS-CoV-2 virus is also has a 10 to 20 times higher binding
activity than the SARS virus, which makes it more difficult to treat as vaccines with compatible binding affinity has to be discovered.42 Additionally, viruses such as SARS-CoV-2 can mutate, which is the case
in among others the UK and brazil.46 Due to the many infections, the virus is pressured to evolve. Also,
these mutations can occur when people are partly vaccinated against the virus, which provides an opportunity for the virus to adjust to the vaccines. Overall, viruses are mutating constantly due to RNA
Figure 8. Mechanism of viral membrane fusion of CoV-2.Top is the viral membrane and bottom is the host cell membrane.44
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copy errors, yet the SARS-CoV-2 mutates relatively slow, this is due to better RNA proofreading capability of SARSCoV-2. The mutation that often occurs in SARS-CoV-2 is a single amino acid mutation in the spike receptor domain. The aspartate is changed to a glycine and is called the D614G mutation.46
This change ensures a more open conformation, due to fewer interactions between amino acids as visualised in figure 9. The conformation is more open as aspartate has a carboxylate ion and glycine does not consist of any functional groups, meaning that there is a decrease in ionic interactions. When there is an even further decrease in intermolecular interactions, the virus can be more easily bound to the receptors on the host cell membrane. The consequence is a more easy infection of the host cells.46
SARSCoV-2 has an improved binding between the heptad repeats one and two in the six helix bundle. With this stronger binding between the coiled coils the membrane fusion is enhanced.39
4.2.3.2.1 CoV-2 vaccines
To conquer the global pandemic it is necessary to create immunity. As the immunity via infection is not clearly proven to be effective, it is necessary to vaccinate the population. Vaccines are divers as different areas can be targeted. In the Netherlands there are currently eight accepted vaccines, from which the four most common are Pfizer and Moderna, which are mRNA vaccines, AstraZeneca and Janssen, which are antigen based vaccines.
mRNA vaccines can consist of non-replicating RNA or viral RNA. mRNA based vaccines encode the antigen of interest. As RNA is easily degraded, it is encapsulated for the transportation to the correct cells. The encapsulation should therefore consist of some target, such as a transmembrane protein, that brings the vaccine to the correct cells, in case of COVID cells in the respiratory area. This transportation can also be provided by coiled coils as they easily denature and gradually release the vaccine. This is discussed more in detail in section 5.2. When the mRNA vaccine is taken up in the cell, the cell can produce antibodies against the virus.47
The other type was antigen based vaccines. These can target different cells such as B-type or T-cells. Targeting B-cells are based on forming antibodies, as antibodies block the interaction of the virus with the host cell. As mentioned in the previous sector, CoV-2 enters the cell via the RBD, which would make this a good candidate to inhibit. Yet, RBD is glycosylated and methylated, which makes it difficult for vaccine development to create the correct fit and activity. Therefore, the receptor interaction site (RIS), which is directly bound to ACE2 is an easier target as it is not glycosylated. In addition to inhibiting the Figure 9. Open conformation of CoV-2 relative to degree of contagiousness.46
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receptor binding for the virus, vaccines can also bind to receptors of cells that produce antibodies. Receptors as TLR7-9 are suitable as these enhance the production of IgG and IgA antibodies, which are active in the respiratory tract.48
The infection with CoV-2 can be measured via these antibodies. After infection the body produces IgM followed by IgG and IgA antibodies. The amount of IgM antibodies peaks in day 7 to 10. When these IgM antibodies are measured with use of serology, it indicates that there is still an ongoing infection. When only IgG antibodies are found, it indicates that the virus is absent. The IgA antibodies are an easy indication as they are found in saliva, yet they can provide of a false negativity as they are present in low levels.48
4.3 Summary
Analysing the natural processes results in a greater insight in the structures of the coiled coil complexes and their mechanisms. For the structure in general the a- and d-position are occupied by hydrophobic amino acids. These positions 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 into the b, c and f position should be performed to investigate the specific importance of these positions. The mechanism of membrane fusion in particular relies on the presence of a heterodimeric coiled coil, to ensure fusion of the vesicles to the cell membrane instead of with each other. The part of the transcription factor shows 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. The analysis of the viral entrance of HIV and SARSCoV-2 is important for the research to vaccines.
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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 (dimers). 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 synthesised, which can be applicable in different areas. In this chapter, differences in the structure will be analysed 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- or 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.49 The leucine zipper is often used as model for coiled coils, due
to its short monomeric structure and the ability for hetero- and homodimerization. The thermal stability of these dimers were analysed by using circular dichroism spectroscopy, which uses left and right polarised 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.50
With a double mutated alanine thermodynamic cycle as reference the coupling energy between the different amino acids was 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). With 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 (kcal/mol). 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.49 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. 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.49
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 and form electrostatic interactions. By analysing the Lac repressor, it was seen that it can self-associate into an antiparallel tetramer.51 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 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.52 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 10A). 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 10B). 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, a parallel heptamer will form.51 A parallel
heptamer has a 3-1-2-1 interaction as shown in figure 10C. 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 10. A) canonical coiled coil 3-4 interaction (a-d). B) antiparallel tetramer, 3-3-1 interaction (a,d,g) C). parallel
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5.2 Encapsulation of nanomaterials with coiled coils
Nanomaterials are compounds with features of size in the order of 1-100 nm.53 In this thesis, the focus
is on nanomaterials derived from natural processes as inspiration. Coiled coils can be used in nanomaterials to achieve different goals, for example assist in drug delivery systems or in bioimaging. In drug delivery systems, the transported drug is surrounded by transport molecules. Bioimaging is the linking of fluorescent compounds to certain receptors of cells, which can be irradiated and give an illuminated signal. This indicates the presence or overexpression of the receptors present on specific cells.
In nanomaterials, molecular self-assembly is an efficient method to organise molecules in a precise and predetermined structure.54 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 functionalised at the C- or N-terminus.55
Well known structures of coiled coils in artificial applications are the unbound nanostructure, where the coiled coils are oriented like fibres, tubed and networks for the use in for example hydrogels. Another orientation is the discrete nanostructure, such as self-assembled cage-like particles (SAGEs), vesicles and polyhedron structures. The final oligomerising state are the origami nanostructures.56
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5.2.1 Unbound nanostructures 5.2.1.1 Hydrogels
Hydrogels adsorb and retain large amounts of water relative to their mass. Characteristics of hydrogels are that they are elastic and soft. The hydrogels can be used for drug delivery systems or wound treatment. Previous to the use of coiled coils in hydrogels beta-sheet orientations were applied. The benefit of the switch to the use of coiled coils is that coiled coils are less sensitive to folding mistakes, as they consist of more stable and specific interactions.57 Additionally, the structural and mechanical
properties of coiled coils are well known, which makes it easier to work with. In general the alteration of the coiled coil properties can easily be adjusted by substitution of amino acids.
Hydrogels are often di-block (AB) or tri-block (ABA) oriented, where A is the coiled coil sequence and B is the hydrophilic linker (figure 11A). After addition of water the coiled coils collapse due to hydrophobic forces and form interactions with another coiled coil, whereafter a grid structure is formed which encloses water molecules by the polar interactions of the hydrophilic linker (figure 11B).
57,58 The water molecules can again be released, due to the reversible structural properties of the coiled
coils units, as with denaturation the coiled coils unfold. This denaturation could occur due to pH or temperature changes. Also, the addition of salt could release water as the water molecules are substituted by ions, which have a stronger interaction with the linkers and therefore releases water.59,60 The change of temperature has an impact on the binding of the coiled coils as they have a
relative small net free energies of stabilisation. After temperature increase the coiled coils will unfold and the grid structure is opened and water release could occur.61
Figure 11. A) Tri-block hydrogel (ABA) A=coiled coil and B= flexible linker. B) Abstract orientation of hydrogels and the coiled coil interaction. Blue and red are coiled coils, grey is the flexible linker. 57
For their application in wound treatment, it is based on the water release of the hydrogels as this moisturizes, cools and protect the wounds. Hydrogels are often used on dry wounds such as burns. Also, additions such as collagenase to the hydrogels helps to improve wound healing, as collagenase enzymatically removes necrotic tissue form wounds.62, 63
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In the application of drug delivery systems the coiled coils can have a different function than just the build-up of the grid. In this case the drug or nanoparticle and the grid are functionalised with coiled coils. In research of Roth et al. they analysed the release of gold
nanoparticles via hydrogels (figure 12). Gold nanoparticles are interesting for biomedical applications in cancer. This research group synthesised gold nanoparticles functionalised with polyethylene glycol for stability and coiled coils via thiol gold chemistry (appendix 2). The hydrogel itself consisted of a commonly used complex: Alginate. These chains were derived with glycidyl propargyl ethers (GPE), which could couple to an azidohomoalanine coiled coil via azide alkyne click chemistry (appendix 3). The coiled coils on the nanoparticle and on the hydrogel chains interacted via E/K amino acids. This hydrogel could be injected and due to coupling of the nanoparticle to EGF, it could specifically couple to EGF receptors and inhibit the phosphorylation. The insertion of the nanoparticle via coiled coils interactions creates a gradually release of the drug.64
5.2.1.2 fibres
Coiled coils can also orient themselves in a lateral manner. During the synthesis of coiled coil nanofibers both elongated, but also stacked structures were observed (figure 13)65. The elongation of
the fibres was due to so called “sticky ends”, which means that the C- and N-terminus of the coiled coils are charged and can therefore form a strong interaction. The stacking of the multiple heterodimers occurred in a hexagonal manner. The stacking was due to complementary charge at the surface of the coiled coils, which created higher orders. This could be induced by flanking ion pairs of the staggered orientation. Due to free energy association and low affinity interactions the higher orders are created. These bonds do not need to be strong as due to the avidity effect the coiled coils prefer to bond.65, 66
For the fibre formation a parallel heterodimer was synthesised based on the Leucine zipper. This complex had isoleucine at the a-position and leucine at the d-position. The first coiled coil strand had glutamate at the e- and g-position and the complementary strand had lysine at these positions, to induce strong heterodimerization. These electrostatic interactions are the driving force of the fibre formation. Also, asparagine was added to enhance the parallel orientation as it preferably forms interactions with other asparagine residues.66
The exact reasoning behind the stacking of the coiled coils is yet unknown and with more research more information can be retrieved so larger complexes can be formed. These larger complexes would have an increase in thermal stability.65 The nanofibers can also
be used in drug delivery systems by ionic interactions or adsorption of the drug to the fiber.67
Figure 12. Abstract visualization of the nanoparticle encapsulation in a hydrogel. Red is the nanoparticle, green is PEG, yellow is GPE, blue is coiled coil and grey is hydrogel of alginate.64
Figure 13. Abstract visualization of coiled coil nanofiber.65
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5.2.1.3 Tubes
Tube conformations are similar to the fibres, as fibres often consist of dimers, trimers and tetramers, the tube consist of pentamers, hexamers and heptamers. This is because higher ordered structures consist of a hollow area in the centre, as shown in in figure 10. Just as the fibres, tubes can stack to higher orders and also be elongated via electrostatic interactions of charged residues at the C-and N-terminus, such as lysine and glutamic acid.68
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. 68, 69 In
research of Mann et al. lysine is placed on all f-positions, which made the compound more soluble in water and created higher stability and yields charge repulsion, thus decreasing self-aggregation. In this pore a near infrared fluorescent carbon nanotube is added (figure 14).70 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.54 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.68
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.71 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 a pH change.72 The characteristic 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.72
.
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5.2.2 Directly bound nanostructures 5.2.2.1 SAGEs
A self-assembled peptide cage (SAGE) is the mimicking of a capsid from for example viruses. This capsid is a hollow structure which is formed by self-assembled molecules in high symmetry with noncovalent bonding. Coiled coils are suitable for self-assemble and are often used for the synthesis of SAGEs. In research of Woolfson et al. they developed a SAGE consisting of a homotrimer and a heterodimer. The homotrimer is connected to the heterodimer via disulphide bonds of the cysteine on the f-positions. For the synthesis of the hexagonal SAGE, the homotrimer is first formed, whereafter a single coiled coil strand is added, which couples to the trimer. This reaction is also performed with a different coiled coil strand. Whereafter both solutions are mixed and the single coiled coil strands that were connected to the trimer can for a heterodimeric structure by acid base interactions. The heterodimer formation creates the hexagonal order of the assembly (figure 15).73
The orientation was expected to be a flat hexagonal structure, yet a hexagonal globe is formed. This was not expected, yet preferred, as a hexagonal ball needs twelve pentagons to have the perfect orientation. Due to the flexibility of coiled coils and thermodynamic and geometric constrains the globe orientation occurred. According to the rules of enthalpy it showed that coiled coils preferred to have satisfying interactions (the match with the heterodimer), however this contradicts the entropic preferences. Yet, the number of coiled coil interactions is maximised. Computational studies showed that the coiled coils preferred a curve of 1200 or less, which creates the curves. Also, molecular
dynamics simulations showed that the positive charges at the end repel each other and therefore adopt a more wedge like shape.
SAGEs can be used among others for drug delivery systems, as the coiled coils can easily be adjusted to react to certain temperatures or pH. Research of SAGEs towards endocytic uptake (via cell membrane) has been performed. In this research was visible that positive charged peptide increased the endocytic uptake. An increase in the number of positive charged residues and an increase in the length of the tetrameric expansions (multiple dimeric structures) tuned this process, as with an increase in length the charge was more easily distributed and facilitated the uptake.75 SAGEs were also
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analysed for antigen transport. The SAGEs were nontoxic in high doses and were more effective than antigens free in solution. This was seen by an increase in T-cell responses and antibody formation. SAGEs influences the immune response and with further research it may be applied in future vaccines.76
Self-assembling protein nanoparticles (SAPN) are similar to the SAGEs, only these complexes consist of triangular and pentagonal clusters. This orientation is created by two monomeric strings linked via the C- and N-terminus via two glycine residues (figure 16A).77 One monomer prefers a homopentameric
orientation and the other monomeric strand prefers a homotrimeric orientations.78 The coiled coils
stabilizes via the cysteine residues in the f-position, that forms disulphide bonds. Due to symmetry constrains the triangular/ pentagonal orientations are formed (figure 16B and 16C). SAPN are already used for immunization against variety of diseases such as malaria, HIV and influenza.76
5.2.2.2 Vesicles
For the formation of vesicle structures the hydrophilicity of the environment is the driving force. As vesicles are amphiphilic and consist of one hydrophilic end and one hydrophobic end. Vesicles could also be used for drug delivery as with use of coiled coils the drug is safely captured and protected from degradation.79 Additionally, on
the outside of the drug, cell targeting ligands can be added, such as VEGF (vascular endothelial growth factor), which targets tumour cells (figure 17). In research of Assal et al. this has been performed, and selective cell apoptosis was visible in vitro, as the drug was released via receptor binding.80
In other research of Park and Champion the vesicle formation was analysed in more detail. In this research, the hydrophilic end of the vesicle was a Leucine Zipper coiled coil combined with a hydrophobic elastin like polypeptide (ELP) as hydrophobic end. ELPs are often used in research to vesicle as drug carrier due to their biocompatibility and drug loading capacities.80 As vesicles are
solvent selective and applications for nature, such as drug delivery will be applied in water like media, the coiled coil forms the outer layer and the ELP the inner layer.81 The coiled coil of this complex, with
arginine at the a-position can form a heterodimer with another coiled coil which is linked to a fluorophore to track the vesicle formation. In this study mCherry (red) and EGFP (green) were used connected to a coiled coil with glutamic acid on the a-position.82
Figure 16. A) axial orientation of the pentamer and trimer. B) Spatial orientation of SAPNs.77 C)2D orientation of SAPNs.
Figure 17. Vesicle formation via coiled coils. White globe is VEGF or fluorophore. Yellow and blue are coiled coils. Red is ELP and green is encapsulated area. 78
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The compounds have a diameter of 1.26 µm and 1.82 µm, from mCherry and EGFP respectively. The salt concentration plays a critical role in the formation of the vesicles as concentrations of 0.3M and 0.91M (mCherry and EGFP respectively) need to be present for the vesicle formation. If the salt concentration is lower than these values, coacervation occurs. Here dispersed particles separate from solution to form a second liquid.83
Both fluorophores could be added at the same time, then a mixture of both occurred and the colour yellow was visible, as red and green were mixed. If only the conditions for mCherry were optimal, then it separated into different microphases, where the interieur is filled with the EGFP. The inner surface of the vesicle stabilizes the encapsulated protein in its coacervation state. This experiment showed that encapsulation of small hydrophobic molecules can easily be performed by the mixing of the protein amphiphiles. With use of a bi-layered vesicle it should also be possible to encapsulate hydrophilic compounds, as in a bilayer the inner and outer layer are hydrophilic. Yet, research must be performed in this area.
5.2.2.3 Polyhedrons
Polyhedron orientations are enzyme-based cages. These cages do not only consist of coiled coils, but in these structure the coiled coil symmetry provides correct orientations of the encapsulating enzyme. For the formation of capsid like molecules a C3-symmetry protein is used, such as a trimeric esterase.
When these enzymes are coupled via a flexible linker to coiled coils (C3-, C4- or C5-symmetry),
tetrahedral, octahedral and icosahedral structures can be created respectively (figure 18).84,85,86 The
coiled coils act as “twist ties” and keep the enzyme in the correct orientation.85 With use of this higher
order symmetries the number of geometries is restricted.84
The linker is often glycine, the length of the linker was important as the freedom of the coiled coils create the correct geometry. The linker could easily be added as the C-terminus of the enzyme was at the surface. When the linker was relative short a decrease in activity was visible, which is probably due to hinderance or distortion of the active site.85 When
the linker was eight glycine residues long the overall chemical and thermal stability of the complex increased, and the activity remained unchanged. The coiled coils stabilizes the complex as the cooperative effect of the multiple subunits.84,86
Cages based on enzymes have other advantages than nanocages based on coiled coils only. As the catalytic activity of enzymes can be used in nanoreactors or the enzymatic selectivity can be used for substrate entrance. In this cases the high stability is advantageous, as it is not the goal to degrade as the coiled coil based nanocages.86
Figure 18. Different possible orientations of polyhedrons dependent on coiled coil symmetry.84
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5.2.3 Origami nanostructures
For the formation of polyhedron structures without the need for directing groups, a polyhedral structure based on solely coiled coils can be applied. In this orientation single coiled coils are linked to one other via flexible linkers. The coiled coil will self-assemble to different polyhedron structures dependent on the amount of parallel and antiparallel interaction (figure 19). As with four parallel and two antiparallel orientations a tetrahedron is formed. With six parallel and two antiparallel a pyramid is formed and with six parallel and three antiparallel coiled coil pairs a triangular prism is formed.56,87,88,89
For the creation of these complexes computational modelling is used. The design platform CoCoPOD is often used in many studies regarding 3D-modeling of polyhedron structures and amino acids. First the geometry is specified: tetrahedron, pyramid etc. The next step is the selection of the optimal topology, based on lowest topological contact order. Third, a selection of coiled coils as building blocks is made. In the coiled coil selection, the b-, c-, and f-position were changed to negative charged residues as this decreased the stability of the coiled coils, which is necessary as higher stability led to misfolding in the structures.56 The coiled coil dimers that will be formed only consist of heterodimeric
interactions as this also reduces misfolding.89 The connections in the heterodimers lack a compact
hydrophobic core, but are stabilised by long distance interactions. Fourth, a 3D-model will be constructed of the coiled coil folding and volume, contact order and solubility will be calculated. Lastly, the design is validated in silico.88
Previous to the coiled coil origami structures, these structures were formed with use of DNA. Yet, this had many restrictions as only antiparallel interactions could be made, which limited the structures to only rectangular pyramids.87 The use of coiled coils also provided a more tuneable structure as multiple
interactions are available and not only the Watson-Crick nucleic acid interactions.
Research showed that in mammals, similar protein folding is present. This means that this specific protein folding could be used in drug delivery systems or as vaccines. If this kind of folding was not present in mammals, the body would react to the “misfolded” proteins, which would trigger adverse responses. The origami structures could also be used in non-therapeutic areas such as molecular machines and sensors.88
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5.3 Artificial transcription factors
For a greater range of transcribing possibilities, artificial coiled coil transcription factors can be synthesised. 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.90 To achieve this a greater versatility is needed than currently
available in the artificial structures. One of the first synthesised 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.91
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 20). This alteration increased the melting temperature of the coiled
coil with 270C and did not change the binding properties.92
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.93 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 2-3 nm, as mentioned earlier in section 4.1.1.29In the artificially adjusted version the SNARE proteins are mimicked by DOPE (1,2‐dioleoyl‐sn‐glycero3‐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 21). 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.94 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 also occurs in the
artificial system. This artificial process should consist of a
specific interaction between the coiled coils and fusion of both lipid layers without leakage.
With use of Förster resonance energy transfer (FRET) differences in emission between donor; nitrobenzofuran (NBD) and acceptor; lissamine rhodamine (LR) dyes were
Figure 20. Structure of isoleucine and fluorinated isoleucine
Figure 21.Systematic scheme of the artificial SNARE complex.92
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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.
5.5 Labelling with use of coiled coils
Coiled coils can be used as carriers of fluorescent labels. Labels can be selectively placed on receptors on cell membranes, with these labels the cells conformational changes and oligomerization can be tracked. In labelling it is important that the label molecule is nontoxic, and the function of the receptor is not lost.95 One of the disadvantages of labelling is that the fluorescent label and its carrier are often
large (>60kDa), which could create aggregation. Small coiled coil sequences are therefore ideal as fluorescent carrier. Several studies focused on using lysine (K) and glutamic acid (E) interactions in heterodimer formations.95,96 This is because the electrostatic interactions of these amino acids ensures
heterodimer formation and therefore hinders homodimer formation. The formation of homodimers should be prevented, as otherwise aggregation would occur with the fluorophore linked coiled coils. When this happens, the fluorophores are not linked to a cell membrane and the cell cannot be analysed. This corresponds to the values of the research of Acharya et al. as seen in table 1.
In labelling one of the two coiled coils is connected to the cell membrane, the other coiled coil which carries the fluorescent flows freely until it has found its connection possibility. In research of Gavins et al. an E3 (KIAALKE)3 coiled coil with a cysteine at the end is linked to the receptor on epidermal growth
factor receptors. A K3 (EIAALEK)3 coiled coil is bind via a thioester to a peptide nucleic acid (PNA) at
the N-terminus. PNA is chosen instead of DNA due to its higher binding strength, higher stability against degradation by nucleases and proteases and a high affinity to DNA and RNA strands.97,98
The coiled coil formation creates proximity for the PNA and the cysteine. The coiled coil interaction consist of hetero interactions of K and E at the end and homo interactions of I, L and A in the middle. The proximity of the coiled coils creates a thiol exchange of the PNA and cysteine followed by an S to N acyl transfer. After dissociation of the K3 coiled coil a E3-Cys-PNA complex is formed (reaction 1).
Reaction 1. First attack of the Sulphur nucleophile to the carbon, creating a thiol exchange, followed by S to N acyl transfer and the dissociation of the coiled coils.
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To the PNA tag a fluorescent label connected on a DNA strand can be added via nucleic acid hybridization. Here the single stranded PNA can hybridize with a complementary strand on which the fluorophore is present. This can also occur via an adaptor strand, which allows for brighter and reversable labelling via toehold-mediated strand displacement, which also increases specificity.97,99 In
this way PNA can attach more than one fluorescent label, which increases its intensity of brightness (figure 22).
When the fluorophores are present they can be illuminated with a certain wavelength compatible with the fluorophore structure. In general if the fluorophore consist of a longer conjugated system the absorption of wavelengths is higher, thus the emission is a lower wavelength. Then the fluorophore is excited to the excited state, where its illumination is detectable. Overtime, it relaxes back thermally to the singlet state and further to the ground state.100 This concept can also be applied in a reversed
manner, where the fluorophore is quenched after coupling. Fluorescence quenching is an often used concept in the indication of DNA hybridization. Here, the quencher molecule is attached to the end of the single strand DNA or in this case PNA, which is coupled to a DNA strand with a fluorophore.101 As
the DNA hybridises, it pairs to another strand, which detached the fluorophore quencher complex. Another labelling indication method is via Förster resonance energy transfer (FRET). Where a dipole-dipole interaction will be formed between donor and acceptor molecules, which makes the integrating molecules resonate. There is an energy transfer between both light-sensitive molecules, where the donor dye is in its excited state and transfers it energy towards the acceptor dye, where the photons fall back to the ground state and the acceptor dye illuminate. The emission of the donor is replaced by the emission of the acceptor. This difference in wavelength emission can be observed on the detectors.
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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 analysed 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. This research 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 in nanomaterials, where they are used as a transporter. The coiled coils can be orientated in different conformations, as unbound, directly bound and origami structures. The unbound conformations were subdivided in hydrogels, fibres and tubes. Coiled coils in hydrogels is a suitable use as the coiled coil interactions can easily degrade on temperature and pH increase. This degradation can release water, which can be used in wound treatment. Hydrogels can also be used in drug delivery, as coiled coils can be bound to the grid and the drug, after degradation it gradually releases the drug in the body. Multiple drugs and the in vivo application can be tested to enlarge the scalar and specificity of drug delivery systems. Other drug delivery systems can be in fibre or tube orientation. Fibres are formed by multiple dimers, elongated via ionic interactions at the C- and N-terminus and stacked with use of charged flanking amino acids. Tubes work in a similar manner, but they are not stacked and consist of a hollow centre due to the tetrameric orientation. In the research of Mann et al. a soluble environment for the nanomaterials was created with use of the coiled coils tubes. This is advantageous, because nanomaterials are often insoluble by itself. Also, encapsulation is needed as many drugs are sensitive to the environment and can easily degrade.
Directly bound nanomaterials were subdivided in SAGES, vesicles and polyhedrons. The SAGEs provided an easy way for encapsulation. With use of homotrimers and heterodimers, which self-associated into a hexagonal globe. Similar, SAPN formed a globe structure by homopentameric and homotrimeric coiled coils, which were linked via a linker. The formation of vesicles by coiled coils, can also occur easily. Due to the hydrophilicity of the environment, the vesicle formation occurs spontaneous. If all these complexes can be formed while encapsulating drugs or nanomaterials, there is a large potential for coiled coils in drug delivery systems. Research into a bilayer vesicle can yet be performed, which would be beneficial in hydrophilic drugs, which easily degrade.
Polyhedron formation is created by enzymatic encapsulation stirred by coiled coils. C3-symmetric
enzymes could encapsulate nanomaterials, and due to the Cx-symmetry of coiled coils, different
formations could be created; pyramid, tetrahedral, octahedral etc. Polyhedrons can be used in nanoreactors, due to their catalytic activity. They can also be applicable for substrate entrance, due to their enzymatic selectivity. All directly bound nanomaterials can be used in drug delivery systems, SAPN are already used. In polyhedrons more research into the length of the linker could be performed, as the optimal length and the exact reasoning for the optimal length are yet unknown.
