Advanced protein design opens new avenues for nanotechnology

(Nanowerk Spotlight) The manufacture of nanoparticles has reached a very high level of control of their shape, size and chemical nature. However, assembling nanoparticles in a controlled manner and with clearly defined functionalities in three-dimensional space remains quite a challenge.

Dr. Erik Dujardin, Research Director, Groupe Nanosciences, PicoLab at CEMES in France explains to Nanowerk what the challenges are:

Controlling order at the desired length scale, up to macroscopic dimensions; Controlling the relative arrangement of the building blocks when these are of different chemical nature, hence bearing different physical properties (this is a unique aspect of self-assembly: nanoparticles could be mixed in various combinations and their respective properties could therefore lead to unforeseen synergies); Controlling the spatial organization of nanoparticles in three dimensions; and Introducing dynamics, i.e. the possibility to reconfigure the assemblies at will. This would open the way to have nanomaterials with a portfolio of properties and one would be able to go from one to the other by switching from one form of the assembly to another rather than to re-fabricate a new one.

"All these challenges require to work on the 'cement' that brings and holds the nanoparticles together," says Dujardin. "Forces – e.g. electrostatic, hydrophobic, hydrogen bonding – between nanoparticles and molecular ligands tethered to the nanoparticle surface are what scientists have been designing to create smart 'cement' over the past decades. Supramolecular chemistry has provided many such examples but nature is using this everywhere, too. Take for example DNA strands that interact smartly which each other and with proteins; or proteins that form assemblies that are both complex, specific and reconfigurable."

Scientists have used DNA to create architectures – 'origamis' – of predetermined geometries at the one-nanometer scale and this has been a tremendously active research field led by the pioneering work of Nadrian Seeman at New York University (for instance, read more here: "The gripping potential of DNA nanotechnology" and "Nanorobotic arm to operate within DNA sequence").

"Yet the chemistry of DNA makes it excellent as a structuring cement, but much less so as a functional material: DNA is a perfectly designable scaffold – the shape of folded DNA strand can be predicted with a household computer – but it has very poor intrinsic properties," Dujardin points out. " In contrast, proteins combine the ability to create scaffolds and to foster complex chemistry with organic molecules but, more importantly, with inorganic materials such as for example tooth enamel or bones."

He notes that the strongest biomolecule pair is the streptavidin/biotin couple, which has long been used as a strong cement to assemble nanoparticles (this was the pioneering work of Stephen Mann at the University of Bristol).

Unfortunately proteins are extremely difficult to engineer.

The most common approach consists in isolating a natural protein and modifying it in painstakingly small steps: one or few amino acids at a time and then see whether it has an effect on its properties.

An alternative is to consider simpler polypeptides. Although easier to work with and quite efficient, as demonstrated early on by Derek Woolfson (also at the University of Bristol), it lacks the potential for the robust 3D structure that DNA and proteins have (Biochemistry, "Sticky-End Assembly of a Designed Peptide Fiber Provides Insight into Protein Fibrillogenesis").

"This is where Philippe Minard's work (Trends in Biotechnology, "Artificial proteins from combinatorial approaches") at Université Paris-Sud blew my mind when I discovered it almost a decade ago," says Dujardin. "He could design fully artificial proteins (alpha-repeat proteins). Not one at a time, but 1 billion together; all of them sharing a same robust 3D scaffold. Each of them different from the neighbor for a small (∼15%) fraction of the total sequence. Their size, about 5 nm, was close the one of nanoparticles with known interesting properties, (for example gold or silver nanoparticles with plasmonic properties, fluorescent semiconducting quantum dots, magnetic particles used in MRI, other metal nanoparticle with catalytic properties, etc.)."

With such a population of similar yet different proteins, you can apply Darwin's evolution principle as was demonstrated by Stanley Brown in the early 1990s ("Engineered iron oxide-adhesion mutants of the Escherichia coli phage lambda receptor"): You expose this population of proteins to a target (for example one chosen protein) and you fish out of the 1 billion individuals the few that adhere to the target. They are then multiplied by a standard phage display method. By repeating this selection test three times, it is possible to identify the few artificial proteins that exhibit super-affinity for the target.

And now you have created a pair of proteins with very high affinity for each other.

Minard's team can produce as many pairs of proteins as they want: they just need to change the target and start over again.

"This is when Philippe and I started working together because I wanted to drive the assembly of particles using the protein pair formation.," notes Dujardin. "To do so we attach one protein to one nanoparticle type and the other protein to another nanoparticle. This is done using standard coupling chemistry. We also made sure that the proteins would not be perturbed by the nanoparticle surface and remain active for pair formation."

Specifically, the researchers wanted to have several pairs, so that in the end they could chose to assemble A with B or C with D, for example. While this is straightforward with DNA, it has been out of reach with proteins.

However, in 2013, Minard and his team succeeded in creating proteins pairs (PLOS One, "Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)").

With new work published in the February 10, 2016 online edition of ACS Nano ("Nanoparticles Self-Assembly Driven by High Affinity Repeat Protein Pairing"), Dujardin and Minard have now taken a first step towards the goal of protein-driven assembly of nanoparticles.

Schematic flowchart of the Au NPs self-assembly driven by the αRep protein pair formation. Step I: anionic thiopeptide surface capping of citrate-stabilized Au nanoparticles. Step II: protein functionalization by ligand exchange. Step III: nanoparticle self-assembly by protein pair recognition. Inset: TEM image of a massive nanoparticle film formed between A3- and α17-functionalized Au nanoparticles. Scale bar 2 µm. (Reprinted with permission by American Chemical Society)

In this ground-breaking work, they show that gold nanoparticles with a diameter of 10nm can be assembled using two different protein pairs. The novelty lies in several aspects as the researchers

work with fully folded proteins, not peptides or DNA; do not serendipitously modify existing proteins by 1 or two amino acids but rather create fully artificial proteins, the size of which can be varied and the chemistry of which (at least on one out of six sides) can be randomly modified; select the most suitable proteins to address a particular objective (e.g. direct the strong self-assembly of nanoparticles); and work at the interface between biomolecule (used as a smart cement) and nanomaterials (that bring physical properties), which is still like a virgin land.

They further demonstrate that the strength of the nanoparticle assembly is the same as the pure protein pair formation, which implies that the proteins do the job and are not denatured or distorted by their attachment onto the nanoparticle surface.

Finally, the scientists found that they can disassemble the nanoparticles again by using a trick: injecting a large quantity of free proteins unattached to a particle results in the unpairing of the nanoparticle aggregates.

(a-d) TEM images of Au nanoparticles? self-assembly induced by the affinity pairing of the conjugates A3•α17 (a and c) or A3•α2 (b and d) at a nanoparticle:protein molar ratio 1:30 (a and b) or 1:20 (c and d). (e) Schematic of affinity competition tests where the addition of a 10-fold excess of free αRep A3 bearing no cysteine tag induces the disassembly of the nanoparticle conjugates. (f and g) TEM images of the disassembled Au-A3• α2-Au (f) and Au-A3•α17-Au (g) conjugates after incubation in the presence of free A3. (Reprinted with permission by American Chemical Society)

"An interesting observation we made is that the more proteins you attach on the nanoparticle surface, the larger the super-assembly gets," says Dujardin. "We observed the formation of large (many tens of microns and possibly up to millimeter size) free-standing films made of a single layer of particles. When only few (1, 2 or 3) proteins are attached, then you produce clusters of only a few particles (∼20)."

While this work has shown that protein design leads to assemblies with very different topology (film vs clusters), the team now wants to demonstrate that the strength of the protein-protein interaction can be transferred to the assembly of functional nanomaterials.

Specifically, they want to show that they now can assemble nanoparticles of different types. For example, combine plasmonic gold nanoparticles with fluorescent quantum dots to benefit from the enhanced light field near the gold nanoparticle to promote more intense fluorescence.

It also would be feasible bringing together proteins with enzymatic properties and catalytic nanoparticles for 'green chemistry' applications.

As Dujardin points out, "what we have demonstrated with our simple system of designable cement based on artificial proteins could now be made as complex as any material scientist could wish in order to combine nanoparticle properties."

Besides multiple-component assemblies, another important step going forward will be the ability to control the organization of nanoparticles in three dimensions.

"For this, the rigid scaffold of the alpha-repeat proteins will be very useful," says Dujardin. "Then, we can not only decide to put nanoscale building block A (proteins, inorganic nanoparticles, organic nanoparticles, etc.) close to B but also A at the center, B on the left of A, C on the top of A, D below A, and so on."

The other direction the team is currently working on is to use targets other than proteins when they select the individuals among the 1-billion protein library. Dujardin, along with another team lead by Clement Nizak, has already demonstrated this with antibodies (The Journal of Physical Chemistry, "Selection of Arginine-Rich Anti-Gold Antibodies Engineered for Plasmonic Colloid Self-Assembly").

The ability to create hybrid organic-inorganic interfaces with a rigid protein on a solid surface would open a whole new realm in designing the metal interface, which is so important in catalysis, colloidal synthesis, optical properties of nanoparticles, but also bio-imaging, hyperthermia therapeutic, etc.

Virtually any application that depends on positioning a nanoparticle in a specific location could benefit from this work.