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2 Repeat Proteins as Scaffolds for Nanofabrication

2 Repeat Proteins as Scaffolds for Nanofabrication

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4



Designed Repeat Proteins as Building Blocks for Nanofabrication



bottom-up design of complex protein nanostructures [12, 18, 20].

Repeat proteins are non-globular structures

that are involved in essential cellular processes

acting typically as scaffolds for the mediation of

protein–protein interactions. Repeat proteins are

composed by a variable number of tandem

repeats of a basic structural motif of 18–47 amino

acids, and are dominated by short-range and regularized interactions [21, 22]. There are a variety

of repeat protein families composed of units with

different structures, being alpha helical, betastrand or a mixture of the two secondary structure

elements. Some of the most abundant and wellstudied classes of repeat proteins are formed by

the repetition of simple building blocks: tetratricopeptide repeats (TPR) which consists of 34

amino acid sequence that folds in helix-turn-helix

motif [23], ankyrin repeats (ANK) which consist

of 33 amino acid sequence that folds in helixloop-helix motif [24], leucine rich repeats (LRR)

which consist of 20–30 amino acids that fold in a

beta-turn-helix motif [25], armadillo repeats

(ARM) [26], and transcription activator-like

(TALE) [27] (Fig. 4.1). As shown in Fig. 4.1 the

different repeated units form elongated structures

with defined twists due to the different packing

between the units leading to structures with distinct shapes. These building blocks are widely

used in protein engineering, and consensus

designed proteins have been constructed for

many of these repeat families [24–26, 28].



4.3



Repeat Protein-Based

Assemblies



Considering the main features of repeat proteins

previously described, it is evident that they are

ideally suited for nanobioengineering. Their

structures are modular which simplifies the

design problems to the level of simple units and

the interactions between the neighboring units

are local and predictable. Thus, each repeat unit

can be used as a building block with individually

engineered properties (stability, function, and

interactions between modules) in order to generate designed proteins and higher order assem-



63



blies [29–31]. Because repeat proteins are

simplified systems, it is possible to control how

protein sequence-structure-function relate in

these type of proteins. Indeed, some recent works

confirm the level of understanding of those

repeated systems, and showed that it is possible

by rational computational design to engineer proteins with different properties that expand the

sequence and structure space observed in Nature

[32–34].

The tetratricopeptide repeat (TPR) is an example of the wide range of possibilities that repeat

proteins give to the field of protein assemblies

[23]. To create new TPR proteins that capture the

sequence-structure relationship of the TPR fold,

a consensus TPR (CTPR) sequence was designed

by the Regan Laboratory from the statistical

analysis of natural TPRs (Fig. 4.2a) [28]. CTPR

sequence presents only a few highly conserved

small and large hydrophobic amino acids, being

involved in intra- and inter-repeat packing

interactions that encode the TPR fold [23, 35,

36]. The amino acids at the other positions admit

variations, giving the flexibility to introduce

novel functionalities such us different chemical

reactivities and ligand-binding specificities [37,

38]. Additionally, CTPRs are thermodynamically

more stable than their natural counterparts, which

make them more tolerant to the destabilizing

effects of mutations. If necessary, their stability

can be modulated in a predictable manner by

changing either the sequence of the repeats or the

number of repeated units [39, 40]. In Nature,

TPRs occur in arrays of tandem repeats, from 3 to

20 and their cellular role is mostly to mediate

protein-protein interactions and the assembly of

multi-protein complexes. Similarly, CTPR

repeats can be combined in tandem to form CTPR

proteins that present a continuous right-handed

superhelical structure with eight repeats per one

full turn of the superhelix (Fig. 4.1 and 4.2) [28].

The aforementioned properties of these repeat

proteins allow a good control at the molecular

level. In order to use these proteins as building

blocks for supramolecular assemblies is also

important to control the structure at different

length scales. This control can be achieved

through the specific protein interactions that will



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S.H. Mejias et al.



Fig. 4.1 Representation of repeat protein scaffolds. (a)

For each repeat protein family, the structure of an individual repeated unit is shown together with an schematic

representation of each building block: ANKyrin repeat in

blue (ANK), Tetratrico Peptide Repeat (TPR) in orange,

and Leucin Rich Repeat (LRR) in green. (b) The crystal

structures of repeat proteins composed by 4 repeats of each

building block are represented using the same color code

as in the panel (a) (front view on the left side and top view

on the right side). The structures illustrate the different



packing arrangements between the building blocks as

displayed in the schematic representations of the packing

from N-terminal to C-terminal of the proteins below the

crystal structures. (c) Crystal structures of long repeat

arrays. A repeat protein form by 12 ANK repeats (PDB ID:

2XEE); a repeat protein form by 20 TPR repeat (PDB ID:

2AVP); and a repeat protein form by 16 LRR repeat (PDB

ID: 1A4Y). Depending on the packing of the building

blocks the different repeat proteins show different twist

and therefore different shapes



drive the assembly and the environment that will

affect the assembly process. Given the above, a

variety of modified CTPR modules are designed

with desired amino acid compositions for selected

applications (Fig. 4.3a). The combination of

these building blocks into long arrays leads to

proteins with modules that encode different

structural and functional properties (Fig. 4.3b).

This bottom-up approach mimics the routes to

complex structures in living systems.



The structural characterization of CTPRs

revealed some interesting inherent self-assembly

properties of these building blocks. In the crystal

form, CTPR proteins showed “head-to-tail” and

“side-to-side” intermolecular interactions that

resulted in different crystallography packing

geometries of the same molecules [41, 42]. These

interactions can serve as models to develop synthetic systems in which the assembly of the units

is controlled by design. In this sense, the modular



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Designed Repeat Proteins as Building Blocks for Nanofabrication



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Fig. 4.2 CTPR repeat as building block. (a) CTPR

repeat unit structure is represented showing helix A in

green and helix B in orange. On the right, a schematic

representation of the structure of the CTPR building block

using the same color code. Below, the CTPR consensus



sequence is shown highlighting in red the conserved

amino acids. (b) Crystal structure of a repeat protein composed of 4 CTPR repeats (A helices in green and B helices

in organe). Below, it is shown the schematic representation of the CTPR packing from N-terminal to C-terminal



Fig. 4.3 Tailored CTPR repeat proteins formed by modified CTPR-based building blocks. (a) CTPR-based repeat

units with variable sequences and properties are represented in different colors. Below: schematic representation of the blocks as cylinders. (b) Examples of the

formation of CTPR protein variants by combination of

different CTPR repeat units. On the top, schematic representation of the proteins and below the three dimensional



models based on the structure of CTPR8 and CTPR20

(PDB ID: 2AVP). From the top to the bottom: CTPR20

protein formed by 20 repeats of two different CTPR units

colored in blue and orange alternatively; a CTPR16

formed by 16 identical CTPR repeats; a CTPR12 protein

formed by 12 repeats of three different CTPR variants

combined in groups of three repeats



structure of the CTPR repeat proteins and the

basic understanding of their sequence-structure

relationships opens the possibility to modulate

the interaction between the units. Thus, it is possible the formation of different protein assemblies in a controlled manner through a hierarchical



self-assembly including nanofibers, monolayers,

nanotubes, and nano-structured thin films

(Fig. 4.4). These nanostructures and materials

will constitute the basis for future functional

materials and structures with many potential

applications in nanotechnology.



S.H. Mejias et al.



66



Fig. 4.4 Different types of CTPR-based assemblies

designed by the modulation of interactions between

CTPR blocks. The left column shows the structure of the

designed CTPR proteins highlighting the modifications

introduced into the blocks. The right column shows the

different assemblies constructed using the modified

blocks. From top to bottom: Protein nanofibers generated

using the intrinsic “head-to-tail” interactions and a simple

disulfide bond staple to fix those interactions between

molecules [43]; CTPR protein monolayer formed by the



oriented immobilization of the CTPR units on a gold surface and the “side-to-side” lateral packing interactions

between long CTPR units [45]; CTPR protein nanotubes

formed by the introduction of a second interacting interface that provides an extra dimension to the final structure

by allowing two superhelical CTPR molecules to assemble; Nano-structured protein thin films are generated

using the intrinsic “head-to-tail” and “side-to-side”

assembly properties of CTPRs through specific noncovalent interaction [45]



4.3.1



the intrinsic “head-to-tail” interaction long linear

polymers based on CTPR blocks have been

obtained [43, 44] (Fig. 4.5b). These thin protein

nanofibers represent the simplest higher order

structures derived from CTPR proteins [43].

By combining the “head-to-tail” interactions

observed in the CTPR crystals with the introduction of specific reactivities into the CTPR units,

linear higher order structures have been assembled. A designed CTPR variant was constructed



Protein Nanofibers



As mentioned above, when CTPR proteins are

crystallized, it is observed the formation of continuous superhelices along the crystal through

“head-to-tail” interactions between molecules [41,

42]. Since all the CTPR units are identical, the

crystallographic inter-molecular packing interface

is the same as the intra-molecular interface

between repeats (Fig. 4.4). Taking advantage of



4



Designed Repeat Proteins as Building Blocks for Nanofabrication



67



to encode directional “head-to-tail” packing

according to the schematic in Fig. 4.5. Two

unique cysteine residues were introduced at the

N- and C-terminal ends of a long CTPR20 protein to act as a staple of the “head-to-tail”

interaction (Fig. 4.5a). The CTPR polymerization

in solution is facilitated by the association of two

CTPR20 molecules through their packing

interfaces and the formation of a disulfide bond

between the cysteines. If the “head-to-tail”

interactions are not fixed by the disulfide bonds,

there was no significant polymer formation. In

this case, even if the “head-to-tail” interaction

between CTPR proteins should occur, the dissociation of the complex is faster than the association and the equilibrium is therefore shifted

towards the monomeric form when the proteins

are in solution.



The described approach is simple and

provides some advantages for the fabrication

and patterning based on protein scaffolds [43].

The nanofibers can be reversible disassembled

to monomeric units by breaking the disulfide

bonds between the CTPRs under reducing

conditions, while keeping the structure of the

individual building blocks. Their modularity

permits the combination of repeated blocks with

different characteristics, including stability and

functionality. Additionally, the nanofiber

formation can be described by a predictive

simple polymerization model, that can be used

to rationally control the polymerization tuning

the experimental conditions such as protein

concentration, temperature and polymerization

time to achieve the desired size distributions of

the polymeric chains [43].



Fig. 4.5 CTPR proteins as building blocks for controlled polymerization and nanofiber formation. (a)

Schematic representation modified CTPR protein unit

where the repeats at the N and C-terminal ends of the

protein have been modified by adding a cysteine.

Modified units are colored in black and the other repeats

in blue. (b) On the left, schematic representation of the

bottom-up strategy to generate protein-based polymeric

nanofibers. The modified CTPR proteins interact through

“head-to-tail” inter-molecular interactions and the cysteine mediated disulfide bonds act as staples of the interaction. The inter-molecular packing interfaces in the



polymers are the same as the intra-molecular interfaces

between two repeats in the same molecule. On the right,

the structural arrangement of CTPR fibers based on the

crystal structure of the CTPR20 protein. (c) CTPR20 protein polymerization growth monitored by the increase in

the size of the Cys-CTPR20-Cys polymers as a function

of time by dynamic light scattering (DLS) [43]. (d)

Negative stained TEM images of CTPR polymerization

process. Panel 1 shows a micrograph of CTPR20 monomers circled in black. Panel 2 shows CTPR20 samples

after polymerization in which linear nanofibers are

observed [43]



S.H. Mejias et al.



68



4.3.2



Protein Monolayers



CTPR protein can also be assembled into ordered

monolayers by a combination of oriented immobilization and the potential of “side-to-side”

interactions between CTPR proteins. In the CTPR

crystal forms, it was observed that not only “headto-tail” but also “side-to-side” interactions are

essential for the crystal packing [41]. In order to

generate protein monolayers, a long CTPR protein composed of 20 CTPR repeats was modified

with a single cysteine at the C-terminal to permit

the oriented immobilization of the protein onto a

gold surface through gold-sulfur bond (Fig. 4.6)

[45]. Several factors, including the oriented

immobilization, the large aspect ratio of CTPR20

molecules, and the propensity to form tight sideto-side interactions between the molecules are

expected to drive the assembly of the CTPR

molecules into highly packed and oriented protein

self-assembled monolayers (SAMs) (Fig. 4.6).

In order to characterize these types of assemblies, it is needed to apply surface characterization techniques including quartz crystal

microbalance (QCM), surface plasmon resonance

(SPR) and high-resolution surface imaging such

as atomic force microscopy (AFM), or scanning

electron microscopy (SEM). By QCM and SPR it

is possible calculate the surface coverage, which



Fig. 4.6 CTPR proteins as building blocks for the controlled formation of protein monolayers. (a) Schematic

representation of the modified CTPR protein unit. The

building blocks at the C-terminal end of the protein, modified with a single cysteine, are colored in black and the

rest of the repeated units in green. (b) On the left, schematic representation of “side-to-side” interactions



showed that the CTPR protein units were indeed

assembled in a compact manner onto the gold surface by thiol chemisorption. In addition, QCM

provides information about the viscoelastic properties of the deposited protein layer, showing that

protein concentration is the parameter that mostly

determines the state of the deposited material. At

low concentrations there are not enough neighboring CTPR molecules immobilized to give rise

to the assembly. However, when the protein concentration was increased, denser packing is promoted by lateral interactions, leading to a more

rigid monoloayer. Finally, high compact protein

monolayers were imaged by AFM [45].



4.3.3



Protein–Based Thin Films



One advantage of using CTPR proteins for the

generation of highly ordered materials and devices

is the fact that these proteins can maintain their

structure in the solid state, as previously reported

[46]. As described before, in the crystal structures

of CTPR arrays, individual molecules stack

“head-to-tail” to form virtually continuous superhelix [39, 47, 48]. In addition, “side-to-side”

interactions are also observed. Therefore, one can

hypothesize that under some experimental conditions, due to these specific interactions between



between the CTPR proteins and the formation of a protein

monolayer by oriented immobilization onto the gold surface. On the right, representation of a CTPR monolayer

using the crystal structure of the CTPR20 protein. (c)

AFM image of CTPR20-Cys protein monolayer deposited

on gold surface [45]



4



Designed Repeat Proteins as Building Blocks for Nanofabrication



69



superhelices, CTPR proteins would self-assemble

into higher order structures giving rise to ordered

solid thin films. This hypothesis was demonstrated

by depositing CTPR protein solutions on teflon

surface and evaporating the solvent. This process

resulted in solid macroscopic films in which the

proteins assembled by specific contacts between

superhelices similar to the ones present in the

crystalline forms (Fig. 4.7) [46, 49].

The macroscopic order in the final material

was verified by X-ray diffraction, showing a

characteristic pattern of macroscopic alignment

and a dependence on the angle, indicative of a

directional order [46]. Furthermore, circular

dichroism (CD) experiments verified that, in the

case of CTPR protein solid films based on the

self-assembly of CTPR superhelices, CTPR proteins retained their characteristic alpha-helical

secondary structure [46]. Therefore, the structural information from the crystal structures can

be applied to the solid films in order to generate



functional materials with functionalities specifically arranged within the material.

If individual protein molecules retain their

secondary structure in the film, it is expected that

the proteins will also retain their function. This

would be the simplest manner to obtain functional materials in which both, structure and

functionality, are encoded by the protein

molecule. The activity of CTPR proteins within

the film was confirmed by the generation of films

with CTPR proteins that recognize specifically

the C-terminal peptide of Hsp90. In the presence

of the ligand molecule the films are formed and

the CTPR units impose order to the Hsp90 peptide upon specific recognition [46].

These results provide clear evidence that

CTPR proteins are an ideal model to design

novel biomaterials and devices in which

molecular order and specific functionalities can

be modulated by design of the CTPR protein

sequence.



Fig. 4.7 CTPR protein-based solid film. (a) Schematic

representation of a long CTPR protein with each repeat

colored in orange. (b) Schematic of the generation of protein-based thin films from CTPR protein molecules. A

drop of a concentrated protein solution (1–3 %) is deposited on Teflon surface, upon water evaporation the protein

molecules arrange to form a solid thin film. Within the



films the protein molecules are organized leaded by “headto-tail” and “side-to-side” interactions [46]. (c) Structural

characterization of CTPR protein film. Circular dichroism

(CD) spectrum of CTPR protein film shows the characteristic signal for alpha helical secondary structure indicating

the of preservation of the structure within the solid material

[46]. (d) Optical image of a macroscopic CTPR solid film



S.H. Mejias et al.



70



4.4



Repeat Proteins as Scaffolds

for Biomolecular Patterning



In the last years, the development of new hybridmaterials for different nanotechnological applications, such as optoelectronics, cell signaling,

plasmonics, and catalysis has attracted many

research efforts. In this sense, the performance of

the final hybrid-materials highly relies on the

properties and organization of the molecules

within the materials. Therefore, many approaches

are being used in order to have reliable control

over the arrangement of the active components of

the materials at different length scales. The use of

different building blocks as the basic units to

engineer defined supramolecular assembly has

been acknowledged as a powerful strategy for the

fabrication of functional materials.

Many bottom-up approaches have been

reported, for example, using inorganic building

blocks and nanoparticles [50, 51]. In addition,

small organic templates have been also explored

to control the formation of supramolecular architectures based on the organization of different

molecules, at the nanometer scale, for improving

their properties [52, 53]. However, these methods

often do not achieve the selective orientation and

arrangement of the different functional components, and the control of the monodispersity at

different scales is still missing.

Bioinspired scaffolds can be used to control

the order at different scales and therefore to precisely pattern active components (Fig. 4.8). For

example, bioinspired self-assembling and nanostructure patterning based on small peptides and

nucleic acids have been recently reported [3, 53].

However, peptides and nucleic acids do not provide the functional diversity and potential number of reactivities of proteins [18].

As described above, modular approaches display advantages for the design of complex supramolecular structures. At the same time,

modularity allows for the patterning of mono and

multicomponent systems by having a set of scaffolding modules that carry different active components (Fig. 4.8). Here we will show that repeat

proteins present advantages not only for the generation of protein-based assemblies, but also for



the development of the next level of complexity

toward the generation of hybrid functional

materials.

Specifically, this section explains the potential

of CTPR proteins as scaffolds to create biohybrid functional materials. Designed CTPR can

be used as a particular type of biomolecular scaffolds which encompass the structural simplicity

of DNA and short peptides and the functional

versatility of proteins. In order to generate these

ordered functional materials is necessary to combine the control of the building block assembly

and the control over the functionalization of the

protein modules.



4.4.1



Repeat Proteins as Scaffolds

for Patterining Metallic

Nanoparticles



Proteins and in particular repeat proteins have

potential to be used as scaffolds for the fabrication of nanoelectronic devices, nanowires or

plasmonic sensors. As previously reported,

metallic nanoparticles can be adsorbed or covalently bound onto macromolecules including

DNA templates [14, 54], proteins and polypeptides [54–57], polysaccharides [58], autoassembled peptides [59], microtubules [60],

enzymes [61], and even viruses [62–66] to form

patterned hybrid nanomaterials and evolve to

nanowires or nanosheets with electrical conduction properties.

The use of repeat proteins as scaffolds for

nanoparticle patterning has advantages associated

to their modularity. Indeed, the repetition of

protein motifs allows the introduction of close

and periodical reactive sites to coordinate

metallic conductive species such as nanoparticles.

The easy and well-controlled genetic modification

and production are the key characteristics of

repeat protein in these field of applications.

Overall reactive sites can be well-designed

regarding type, number, and disposition among

the protein, for the specific functionalization with

metallic nanoparticles through different selected

interactions. Additionally, through advanced

molecular biology and biochemistry techniques



4



Designed Repeat Proteins as Building Blocks for Nanofabrication



71



Fig. 4.8 Schematic representation of bottom-up

approach for the fabrication of functional supramolecular architectures. (a) Schematic representation of

different elements with great potential for applications in

different nanotechnological fields, such as nanoparticles,

metal ions, small organic molecules, and functional peptides or proteins. (b) Schematic representation of different

scaffolding units such as small organic templates, DNA,

peptides and proteins. In this chapter we will focus on

proteins as scaffolds that can be modified with different



reactivities. The colored surfaces represent orthogonal

reactivities that will be used to conjugate the different

components. (c) Conjugates in which different components are linked to the scaffolding units by the selected

reactivity. (d) Supramolecular hybrid architectures formed

by the arrangement different functionalized scaffolds. The

scaffolding units are used to arrange active components

into defined patterns, as required to achieve optimal properties in the final structure



un-natural amino acids are incorporated into proteins, expanding the potential reactivities of proteins for bioconjugation. These strategies allow

the introduction of specific binding properties to

conjugate any metallic nanoparticle or even

explore organo-metallic strategies. Moreover, the

recent developments of in situ reduction of metallic salts into nanoclusters or nanoparticles in the

presence of protein open the field of a one-pot

fabrication of nanowires by a combination of

metal salts, scaffolding proteins and reducing

conditions, followed by thermal annealing of

metal growth process.

Hybrid materials based on chiral molecules

and metallic nanostructures are also of interest in

chiral plasmonics applications [67], non-linear

optics [68], or negative refractive index matter

[69]. One example is the use of supramolecular

patterning to create novel chiral superstructures

of gold nanorods [70]. Also the preparation of

metamaterials based on plasmonic mesophases

with switchable polarization-sensitive plasmon

resonances shows several potential applications

strate [173] or

in combination with adhesion-promoting layers

such as secondary cell wall polymers [66, 67, 138,

150], lipids [31, 151], polyelectrolytes [151] or

complete ultrafiltration membranes [203]. These

sensory-active materials can also be comprised of

commercial synthetic polymers and S-layer proteins, which in turn can be further chemically

modified. To ensure their functionality, the introduction of additional spacer molecules has proven

to work in some cases [66, 67, 96]. In many

instances, these modifications can have a positive

effect on both the stability and sensitivity of the

sensory-active layers. In turn, interactions

between the sensor and the respective target molecules are capable of generating signals that can

be converted on a sensor chip with a suitable

transducer in measurable, mostly electrical signals, which can be read out after appropriate

amplification. These optical, electrical or mechanical signals can be determined via optoelectronic,

amperometric, potentiometric or piezoelectric



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