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Proteins As Building Blocks

Peptides and proteins are being designed to fold or self-assemble into nanostructures

by Celia Henry Arnaud
May 27, 2013 | A version of this story appeared in Volume 91, Issue 21

Credit: Dek Woolfson
Coiled-coil peptide modules arrange themselves in a hexagonal array that folds into a cage. Here, an atomistic simulated model of 19 hexagons is overlaid on a diagram of a full cage.
19 hexagons from a self-assembled protein array overlaid on a rendering of the nanocage formed when the array is large enough.
Credit: Dek Woolfson
Coiled-coil peptide modules arrange themselves in a hexagonal array that folds into a cage. Here, an atomistic simulated model of 19 hexagons is overlaid on a diagram of a full cage.

Proteins are nature’s go-to material for many functions. Among their many talents, they serve as structural building blocks and reaction catalysts. Scientists would like to harness these diverse biological molecules to perform even more jobs, such as carrying cargo or catalyzing new reactions.

One way to do that is to figure out ways to use proteins as building blocks for precise structures that could make such new functions possible. This goal continues to be challenging, but chemists are making headway. Using different strategies, several independent research groups have recently shown that they can design proteins that form desired shapes.

One of the teams takes a page out of the DNA origami playbook. In that method, single-stranded DNA folds into desired shapes dictated by hydrogen bonding between the DNA bases. To make a corresponding protein nanostructure, Roman Jerala of the National Institute of Chemistry, in Ljubljana, Slovenia, and coworkers recently designed a single polypeptide chain that folds into a tetrahedron (Nat. Chem. Biol. 2013, DOI: 10.1038/nchembio.1248).

The chain consists of 12 helical segments connected by flexible loops that act as hinges. The helices pair up into six coiled coils—held together by hydrophobic and electrostatic interactions—that trace the edges of a tetrahedron. They produced the polypeptide in bacteria using a synthetic gene.

“The challenge was to design a polypeptide that traverses each edge of a tetrahedron exactly twice, forming coiled-coil dimers” Jerala says. To do that, they needed to “select orthogonal building elements that form pairs only with selected partners but not with other segments.”

They initially designed the tetrahedral path manually but also enlisted the help of Sandi Klavžar, a mathematician at the University of Ljubljana. He showed mathematically that forming a tetrahedron requires some dimers to be parallel and others to be antiparallel.

The order of the segments matters. Scrambling the sequence or leaving out a segment prevents the polypeptide from forming a tetrahedral shape. “This is very similar to native protein structures, where the order of amino acid residues defines the tertiary structure,” Jerala says.

In another method to make protein nanostructures, Derek (Dek) Woolfson and coworkers at the University of Bristol, in En­gland, also use coiled-coil motifs as building blocks (Science 2013, DOI:10.1126/science.1233936). Rather than using a single peptide chain, they started with two types of coiled-coil bundles—one with three identical coiled-coil peptides and the other with two different coiled-coil peptides. The bundles form hubs that assemble into an array of hexagons that can in turn form a sheet structure.

The growing hexagon sheet curves naturally. This curvature happens “because the peptides that make up the hubs are pinned together at one end and have positive charges at the other end,” Woolfson says. “The positive charges repel, but because they’re pinned at the other end they can’t push the whole assembly apart. They can only make it wedgelike.

“Eventually the sheet curves so much that the edges stick together and close,” Woolfson continues. The size of the cage depends on the “stickiness” of the edges, with stronger interactions resulting in smaller cages.

Todd O. Yeates and coworkers at the University of California, Los Angeles, use yet another approach to form protein nanostructures. They take protein-based oligomers and link them together in larger assemblies. Interactions between the linked oligomers result in the formation of protein cages through a symmetry-driven process.

Yeates’s team has been working on protein nanostructure formation for more than a decade. They began in 2001 by connecting a trimeric bromoperoxidase enzyme to a dimeric viral matrix protein with a helical linker. They expected that construct to self-assemble into a 16-nm, 12-subunit tetrahedral cage (Proc. Natl. Acad. Sci. USA 2001, DOI: 10.1073/pnas.041614998). The team wasn’t able to confirm cage formation at the time, but last year they obtained a crystal structure suggesting that the two proteins do assemble into a tetrahedral cage (Science 2012, DOI: 10.1126/science.1219351).

Credit: Nat. Chem. Biol.
A single polypeptide chain made of 12 helical segments folds into the six edges of a tetrahedron. Also shown are TEM images of tetrahedra and matching computer renderings.
Schematic representation of polypeptide chain with 12 coiled-coil segments that self-assemble into a tetrahedron.
Credit: Nat. Chem. Biol.
A single polypeptide chain made of 12 helical segments folds into the six edges of a tetrahedron. Also shown are TEM images of tetrahedra and matching computer renderings.

Now they have solved crystal structures of a series of variants of that first cage (J. Am. Chem. Soc. 2013, DOI: 10.1021/ja402277f). The observed structures were more flexible than they expected, with the dimer interface and linker being major sources of that flexibility. Yeates and coworkers think this flexibility points to the possibility of designing protein machines that change shape in a dynamic manner.

To develop such protein nanostructures into functional materials, they will most likely need to be chemically modified. “We see the nanocages as a scaffold,” Woolfson says. “These are bare molecules onto which you’ve got to put function, on either the outside or the inside. The challenge is going to be putting that functionality where you want it without disrupting the assembly you’ve made.”

Woolfson’s group plans to develop such nanocages as vaccine delivery vehicles. “Could you have a self-assembling kit that incorporates any vaccine you want?” he asks. “Rather than having to use cold storage to get virus particles from one part of the world to another, you could imagine making these small building blocks, getting them to the hospital where you need them, reconstituting them with water, and inoculating people with them.”

Another possible application is using peptide- or protein-based nanostructures as enzymes. Assembling structurally defined nanostructures from protein modules “is a shortcut to the formation of protein folds that are not yet as optimized as those in natural proteins,” Jerala says. “The approach allows us to form protein folds that do not exist in nature.”

Nanocages might also serve as gatekeepers for natural enzymes that let substrates in and products out through their holes. There is biological precedent for such constructs “because a lot of protein enzymes act as complexes,” Woolfson says.

No matter what applications people pursue, the next step is improving the success rate of designs, Yeates says. “If you read between the lines in a lot of papers in this area, the success rates are quite low.” As the design process becomes more routine, “it will become easier to make a variety of things,” he says.


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