Shaking up proteins with engineering

Proteins, as Brent Nannenga describes them, are “little machines inside life that make things happen.” They play many critical roles in all living organisms, acting as the building materials for cells, antibodies that guard against infection and messengers that ferry information between cells, tissues and organs.

Proteins are composed of amino acids strung together, which fold into intricate three-dimensional structures. These structures — tangled tubes, spherical bundles, recurring spirals folded into themselves and more — determine what the proteins do.

“Their function is intimately tied to their structure, so to understand their function, you must know their structure,” says Nannenga, an assistant professor of chemical engineering at Arizona State University.

Nannenga, a faculty member in the Ira A. Fulton Schools of Engineering, has embarked on two ambitious projects to expand our knowledge of protein structures and put it to use. Using his engineering background coupled with experience in structural biology, he aims to leverage the roles of existing proteins for new purposes as well as design and construct new proteins with novel properties and functions.

Nannenga plays with proteins the way a precocious child might play with Lego bricks — dutifully studying how they fit together before dumping the box all over the floor to see what cool stuff he can make.

Factories for tiny tools
Nannenga is working to use proteins to create inorganic nanomaterials in a more precise and environmentally friendly way.

Nanomaterials are materials with internal or surface structures in the nanoscale. Objects on the nanoscale are measured in nanometers, which is a millionth of a millimeter. A millimeter is already quite small to begin with — approximately the diameter of lead in a wooden pencil. Proteins measure about four nanometers, so they serve as an excellent starting point to craft nanomaterials.

Nannenga plays with proteins the way a precocious child might play with Lego bricks — dutifully studying how they fit together before dumping the box all over the floor to see what cool stuff he can make.

Nanomaterials possess unique optical, mechanical or electronic properties. They are used for specialized coatings and enhancements in electronics, and they even hold promise for use in smart pharmaceuticals and drug delivery systems.

The physical and chemical techniques used in fabricating these tiny materials are costly, requiring toxic compounds and high temperatures. However, biological creation only requires water, salt proteins and room temperature, according to Nannenga.

“Biology has been doing this for millions and millions of years,” says Nannenga. “More recently, in the last couple decades, people have been trying to use proteins to template and grow inorganic nanomaterials. While people have found methods that have worked, there hasn’t been a lot of fundamental knowledge of what’s actually happening or how it’s working.”

Through this natural approach, there’s the possibility of dropping the engineered proteins into cells and transforming that cell into a nanomaterial factory.

“Biology in general is just very good at organizing things very specifically and precisely,” he adds. “The promise of using proteins-driven nanomaterial synthesis is environmentally friendly and has the potential to be very specific and directed and controllable.”

“It’s the next step of biomimicry. Here, we’re developing an understanding of the fundamentals and making new things instead of just mimicking.”

Through genetic engineering, proteins allow for greater control over functionality, allowing designers to dictate multiple tasks. Nannenga posits the possibility of bi-functional proteins, in which half produces a certain nanomaterial while the other seeks out and binds to a specific cell or tissue to deliver its nanomaterial cargo to a designated location. A cell could even be directed to produce two different materials to create higher order assemblies.

“It’s the next step of biomimicry,” says Nannenga of the project, which is supported by the Air Force Office of Scientific Research. “Here, we’re developing an understanding of the fundamentals and making new things instead of just mimicking.”

What Nannenga discovers will be added to the Protein Data Bank, a global, open initiative to catalog 3-D structures of proteins, nucleic acids and other complex assemblies.

“It has all the hi-res protein structures in the world — hundreds of thousands of them,” says Nannenga. “But there’s not one structure of a protein-inorganic nanomaterial complex in the whole databank. It’s a big gap.”

Identifying drug targets
Another focus of Nannenga’s work is the identification of protein structures, specifically G-protein-coupled receptors (GPCRs).

GPCRs are the largest family of membrane proteins and an integral part of many biological processes. They act as mailboxes for cells, taking in signals and mediating physiological responses to hormones, neurotransmitters and environmental stimulants.

“They’re one of the most important classes of membrane proteins because a large fraction of drugs target GCPRs,” says Nannenga.

Because GPCRs regulate so many processes within our bodies, they’re targeted by drugs that treat everything from hypertension and asthma to allergies and acid reflux. A study from the National Center for Biotechnology Information estimated that approximately 700 drugs focus on GPCRs, accounting for about 35 percent of medications approved by the Food and Drug Administration.

Because of their pharmaceutical versatility, GPCRs have long been an area of interest for the scientific community. In 2012, the Nobel Prize in Chemistry was awarded to two American scientists for their work to understand GPCRs and their functions.

Now, Nannenga and his collaborator Wei Liu, a structural biologist in ASU’s School of Molecular Sciences, aim to fully map the structure of GPCRs. They are using an electron crystallography technique Nannenga developed during his postdoctoral work at the Howard Hughes Medical Institute. The method, which shoots electrons through crystallized proteins to identify their structure, is called micro-electron diffraction, or MicroED.

“To achieve crystallization needed for structural analysis, purified proteins are screened for tens of thousands of conditions that allow for the formation of well-ordered crystals,” explains Nannenga.

Without those ideal conditions, protein production will occur chaotically, rendering the molecules unusable for characterization. When the proteins crystallize, they maintain their original structure, but are oriented into a highly ordered, three-dimensional array.

“Once you have that crystal, you can put it in an X-ray beam — or in our case, electrons — and you get diffraction patterns, which you can use to solve the structure,” says Nannenga.

The resulting diffraction patterns simply appear as points of light. The location and intensity of each reveals information about the protein’s structure.

“It maps out where all the atoms are in the crystal, each spot telling you something about each atom,” says Nannenga. “But it’s only the sum of those spots that reveals its structure.”

The technique allows for the collection of data from very small crystal structures. It caught the attention of the National Institutes of Health, which is funding the refinement of MicroED.

“Traditionally in X-ray crystallography you need big crystals, because they get bombarded with so much radiation. They need to withstand that damage to give you enough data,” says Nannenga.

“To achieve crystallization needed for structural analysis, purified proteins are screened for tens of thousands of conditions that allow for the formation of well-ordered crystals."

Since GPCRs usually form very small crystals, MicroED holds promise for mapping their structure. Unfortunately, GPCR crystals are grown in a special matrix called a lipidic cubic phase. LCP is a viscous, gooey substance with a texture like toothpaste.

“That’s a problem, because electrons interact so strongly with matter that they give you a lot of information off a small sample,” says Nannenga. “But what that also means is that if your sample is really thick, you’re not going to see anything.”

Currently, Nannenga is figuring out a way to grow GPCR crystals in the gooey, opaque LCP while keeping the sample thin enough for electrons to interact with for structural characterization. If his effort is successful, it holds promise for a host of other proteins that are grown in LCP.

“It could open up this technique to a number of proteins that, as of right now, really aren’t accessible,” says Nannenga. “There’s a huge backlog of specifically membrane proteins that are too small for X-ray diffraction and they’re in this goo so you can’t use electron microscopy.”

An oddity in his field
While he’s not the only chemical engineer in the world dabbling in structural biology, Nannenga is one of the few. 

During his doctoral studies at the University of Washington, Nannenga had his first brush with structural biology. He was working on a project to engineer bacteria to better express membrane proteins for structural analysis.

“Everyone told me that the protein structure was super important to function, so I started looking these up… and it would just look like a blob,” he recalls. “So, I started to get really interested in it, and thought it would be helpful to know what it all meant.”

Nannenga's transdisciplinary focus left some universities at a loss. "They didn’t know where to put me. Once I got to ASU, it was never questioned. It’s just what we do here.”

As Nannenga completed his doctoral degree, his advisor, Tamir Gonen, was hiring postdocs for his lab. Before long, Nannenga had developed the new electron crystallography method, while getting a crash course in structural biology and transmission electron microscopy from Gonen along the way.

While interviewing for professorships at different universities following his post-doc work, however, Nannenga had a little trouble finding a niche.

“The fact that my expertise was structural biology and chemical engineering — not solely chemical engineering — they didn’t know where to put me,” he says.

While his transdisciplinary focus isn’t undesirable, it is different for a lot of institutions. It wasn’t until he interviewed at ASU, his alma mater, that he felt like he’d found a home for his research.

“Once I got to ASU, it was never questioned,” he says. “It’s just what we do here.”