How to crystallize spaghetti
Folding a wiggly piece of spaghetti into a distinct three-dimensional shape is not an easy task. Fortunately, PhD student Alana Gudinas was up for the challenge. In a recently published study, they detailed the folding of a spaghetti-like bacterial protein into a structure that researchers then visualized with X-ray crystallography. By understanding how and why the spaghetti folds in the presence of some metals but not others, the team hopes to develop new technologies for sustainably sourcing and recycling different metals from the environment.
Gudinas, a physicist by training, works in the lab of Danielle Mai, Assistant Professor of Chemical Engineering. As a biomaterials engineer, they are interested in taking nature-inspired protein designs and tweaking them for use in environmental clean-up or metal recycling. Some naturally occurring proteins preferentially bind to toxic metals, like mercury and lead, and could be repurposed to remove harmful metal toxins. Another potential application is capturing rare earth metals, like neodymium and dysprosium, from mine drainage. These elements are critical for many sustainable energy technologies but can be difficult to source.
“Mine drain-off is a huge secondary source of rare earth elements,” said Gudinas. “There are trace amounts that aren’t economically viable to capture using traditional industrial techniques but might be possible to capture with something like a protein. That’s the grand vision.”
For now, their focus is on understanding the fundamental biophysics of protein folding in the presence of different metal ions. Their favorite model protein comes from a surprising source: Bordetella pertussis, the bacterium that causes whooping cough. The bacterium produces a protein known as RTX. RTX is a wiggly protein that flops around in solution until it encounters a calcium ion. In the presence of calcium, RTX undergoes a drastic shape change from spaghetti to something rigid and accordion-like. This shapeshifting occurs when the bacterium is infecting its host, part of how it causes disease.
RTX, like other proteins, is made up of a linear sequence of amino acids. Gudinas was excited about trying to engineer RTX because it has a repetitive sequence that is relatively easy to manipulate for binding other types of metals besides calcium, once you figure out the rules.
“Really small differences in sequence can have a really big impact, especially on something as complicated as metal affinity,” said Gudinas.
Learning crystallography at the Nucleus
To learn the rules for metal binding, Gudinas systematically studied RTX with magnesium, strontium, and barium, a series of metals that have similar properties to calcium but with different diameters. They used a variety of techniques to provide valuable information about the ensemble of different shapes that RTX can adopt with each of these metals. But in order to get the high-resolution data that could reveal the subtle structural differences of single atoms, they needed X-ray crystallography, a technique in which Alana had no previous experience.
So, the Mai lab turned to the Nucleus at Sarafan ChEM-H, a collection of resources that provide access to high-end instrumentation, hands-on training, and expert consultation in a variety of areas that are not often found in academia, together with a C-ShaRP Experiential Learning Grant that provided additional funding for instrument use and training in shared facilities. Through this program, Gudinas began coming to the Nucleus once or twice a week to learn crystallography with Daniel Fernandez, Director of the Macromolecular Structure group, a collaboration that lasted nearly a year due to the floppy nature of RTX and its propensity to clump up in solution, a major problem for crystallography.
“In most cases, it didn’t work,” Fernandez said. “The proteins are very unstable. When we had a pure protein sample, and we mixed it with metal ions, it immediately crashed out of solution. But I was very fortunate to work with Alana who was quickly able to get the concepts of crystallography despite not coming from a structural biology background.”
Even when the data were slow to materialize (or should I say, crystallize?), Gudinas enjoyed the time purifying protein, setting up screening trays, and even scanning through hundreds of images, looking for crystals.
“I can’t even tell you how much fun I had. I loved coming to the Nucleus.”
“I can’t even tell you how much fun I had. I loved coming to the Nucleus. The instruments in the facility—I’ve never seen anything like that. You can set up so many experiments relatively quickly in a way that makes something daunting turn into something very possible.”
Solving an elusive crystal structure of a disordered protein
After much trial and error, Gudinas and Fernandez eventually obtained a high-resolution structure of RTX bound to strontium, the first structure of the protein bound to any metal other than calcium. Surprisingly, the structures were nearly identical, even though strontium is significantly larger, distinguished only by a few key residues in the most flexible region of the protein that very slightly shift to accommodate the larger ion. Gudinas hopes that others will continue to build on these findings to design better materials and proteins for a variety of environmental, and potentially therapeutic, applications.
“Proteins are the future,” said Gudinas. “We still don’t know everything they can do. There is still an immense need for rigorous experiments to understand this interplay between protein sequence, structure, folding, and metal interactions. I’m optimistic that this will be an exciting platform for material design.”
Reflecting on the time spent in the Nucleus, Gudinas feels lucky to have found themselves in the right place at the right time to be able to work on this project and for the training opportunities and resources that were available to see it through to completion, despite the challenges of working with such a finicky protein.
“Working at the Nucleus gave me the sense of scientific discovery and collaboration that I always dreamed about. Daniel is one of those experts that you’re very lucky to meet as a young student. Someone who truly just loves science and has bottomless curiosity. Someone who can come up with new ideas or insights at the drop of a hat and has such an immense wealth of knowledge. Getting that kind of insight from an expert who has been in the field for so long, who has seen crystallography evolve over time, and just working in the lab together is, I think, why we all do science. Honestly, I’ve just been enormously grateful that I got the chance to do that.”
Danielle Mai is also an Assistant Professor, by courtesy, of Materials Science and Engineering, a member of Bio-X, and a member of the Wu Tsai Human Performance Alliance. Other Stanford co-authors include Master’s student Gatha Shambharkar, PhD student Marina Chang, and Tsutomu Matsui, Research Engineer at the SLAC National Accelerator Laboratory. Additional support with preparing protein samples and conducting crystallographic screens was provided by Olivia N. Pattelli at the Macromolecular Structure Group, Nucleus at Sarafan ChEM-H. Additional support with crystallographic data collection was provided by beamline staff at the Stanford Synchrotron Radiation Lightsource at SLAC National Accelerator Laboratory stations BL9-2 and BL12-2. This research is also supported by the Air Force Office of Scientific Research and the National Science Foundation Graduate Research Fellowship Program.
