Breaking protein nanocages' symmetry - Part II

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Breaking protein nanocages' symmetry

Today, we will continue our series about protein nanocages! I started it last issue, and today we have another very cool paper, so stick around.

In case someone was not paying attention, let’s have a fast recap. Protein nanocages are symmetrical, wireframe-like nanostructures made entirely of proteins. They are similar to natural constructs like viral capsids, and just like them the nanocages self-assembly from smaller subunits to create ordered shapes. Researchers like them because of their many promises: for example, in drug delivery and vaccine development, but they could also be building blocks for nanomaterials or scaffolds for advanced enzymatic catalysis. But as we saw last time, a lot of these applications depend on creating more complex nanocages. The mainstream design approaches are focused on symmetric assemblies: easier to use, but limiting for size and complexity.

Okay, so now we are ready to jump into today’s paper. This paper is the sibling of last week’s study (the labs even collaborated on a back-to-back publication, as far as I understand), and, just like siblings, they have a lot in common. In both papers, the authors used programmed pseudosymmetry to achieve more complex nanocages. Pseudosymmetry uses slightly different, but structurally compatible, subunits to create their assemblies. This is more or less where the similarities end: I think it’s interesting how the two papers start from the same premise, but they go into very different directions. And the aims of the authors here seem also different: while the other researchers were focused on making bigger structures, here the precision and the modularity of the new nanocages takes center stage. Okay, let’s get into the meat of the paper!

Their design strategy can be divided into three steps:

1. Base cage design: their approach starts by designing a standard nanocage using homotrimers (units formed by three identical subunits), based on already published subunits. These homotrimers can be imagined like little pyramids made with your fingers: they have three edges and can be combined to create bigger structures. Depending on the length of the edges, the homotrimers can self-assemble into different geometries: a bigger pyramid, a cube or an icosahedron (a soccer ball-like shape).

2. Introducing heterotrimers: In the next step, the authors introduced heterotrimers, formed by three distinct but compatible subunits. Each subunit is engineered with specific interfaces to control the self-assembly, and only some of the subunits can interact with the others. The heterotrimers look like crowns, and they are the second type of building block needed for the formation of more complex nanocages.

3. Complex assembly: Finally, the two types of building blocks are mixed, and they assemble into complex (and pretty cool) new geometries.

Okay, this was the general idea! Of course, this entire strategy is based on a back and forth between computational tools and wet lab experiments. AI design tools and in silico docking are used to create and modify the protein subunits, and they are then synthetized and evaluated in the lab, using a variety of techniques, including electron microscopy. This iterative approach ensured that the final nanocages were not only theoretically sound but also experimentally validated for stability and functionality.

And what were the results of this strategy? Glad you asked. They designed and synthetized 3 new nanocages, with different levels of complexity: two of these geometries are not even present in nature! Super cool.

• The first nanocage they created had a tetrahedral symmetry (imagine a tripod, but extended on the top), composed of 48 subunits with an internal diameter of 33-nanometer.

• The second nanocage has a octahedral symmetry, with a sort of 3D-cross shape going on. It is formed by 96 (!) subunits and has an internal diameter of 43 nm. This structure demonstrated the scalability of the structure and, interestingly, also exhibits quasisymmetry, where the same subunits adopts different conformations, a common feature in viral capsids.

• The last nanocage was the biggest and most complex one. It’s an icosahedral structure composed of 240 (!!) subunits with a 75-nanometer diameter! This nanocage resembles a spiky ball in shape, with the spikes being the crown-like structures. If we don’t count the nanocages we covered last week, this is the largest nanocage ever created!

The researchers also spent some time modifying this last nanocage to show that by simply adding a ligand to the subunits, the nanocage can specifically target cells. This, together with the huge internal diameter of the nanocages, shows that they could be incredibly helpful for delivery of targets, such as nucleic acids or other molecules.

This is a very cool paper! And as always, it would be worth reading it only for the very cool figure: but trust me, there is a lot more in it than I didn’t cover!

I am very happy I did this mini-series! Protein design is a field I don’t really know, and it’s very cool to know it more. I think that protein design is poised to revolutionize medicine, nanotechnology, and synthetic biology, and the future looks incredibly bright. Stay tuned for more, and as always, thanks for reading!!

In other news:

• Nanopore sequencing of… Proteins? While everyone is familiar with DNA sequencing, protein sequencing is less understood. But protein sequencing is essential for understanding protein structure and function, and has evolved a lot, with single-molecule technologies being the latest innovation. This review covers nanopore sensing, which has emerged as a reliable tool, advancing amino acid recognition, peptide differentiation, and sequence reading.

• Bringing histones into the (nanotech) fold: Histones are the OG when it comes to interacting and folding with DNA: these protein complexes bind to DNA and twist it around themselves. So why not combining them with DNA nanotech? This paper shows how to use histones to create hybrid protein-DNA structures with precision and in a scalable way, without being constrained by sequences. Pretty interesting!

• Fixing DNA origami with entropy: Entropy is heavily involved in the self assembly of biomolecules, including proteins and RNAs. So why not studying using DNA origami? This study presents a DNA origami system that uses conformational entropy to drive predictable self-assembly. By varying scaffold loop parameters, the equilibrium distribution of isomers can be tuned, with results matching model predictions. Very cool! I need to read it more in details.

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