Red blood cells are amazing. They take oxygen from our lungs and carry it all our body to keep us alive. The hemoglobin molecule in red blood cells transports oxygen by changing its shape in no way whatsoever. Four copies of the same protein in hemoglobin open and close as a petal, structurally coupled to respond to each other. Using supercomputers, researchers have just started designing proteins that self-assemble to combine and resemble life-giving molecules such as hemoglobin. The researchers say their methods could be used for useful technologies such as pharmaceutical targeting, artificial energy harvesting, "smart" sensing and building materials and much more.
A science team did this by leaving proteins, meaning they changed the subunits of proteins, the amino acids, to give the proteins an artificially high positive or negative charge. Using proteins derived from jellyfish, the researchers were able to assemble a complex sixteen protein structure composed of two stacked octamers by supercharging alone. Results reported in January 201
The team then used supercomputer simulations to validate and inform these experimental results. Supercomputer awards at Stampede2 at the Texas Advanced Computing Center (TACC) and Comet at the San Diego Supercomputer Center (SDSC) were awarded to the researchers through the XSEDE, the Extreme Science and Engineering Discovery Environment funded by the National Science Foundation (NSF).  "We found that by taking proteins that do not normally interact with each other, we can make copies that are either highly positive or very negatively charged," says study author Anna Simon, a postdoctoral researcher at Ellington Lab of UT Austin. "By combining the very positive and negatively charged copies, we can let the proteins accumulate in very specific structured collections," Simon said. The researchers call their "supercharged protein assembly" strategy where they run defined protein interactions by combining engineered supercharged variants.
"We took advantage of a very well-known and fundamental principle from nature that opposite charges attract", study co-author Jens Glaser added. Glaser is an assistant researcher at Glotzer Group, the Department of Chemical Engineering at the University of Michigan. "Anna Simon & # 39; s group found out that when they blend these charged variants of green fluorescent protein, they get highly ordered structures. It was a real surprise," Glaser said.
The stacked octave structure resembles a braided ring. It consists of 16 proteins – two interwoven eight-ring rings that interact in very specific, discrete patches. "The reason why it is so difficult to construct proteins that interact synthetically is to make these interactive patches and make them all line up so that they allow the proteins to accumulate in larger, regular structures, is really difficult," explained Simon. They got the problem by adding many positive and negative charges to construct variants of green fluorescent protein (GFP), a well-researched lab mouse protein derived from Aequorea victoria jellyfish. The positively charged protein which the cerulean fluorescent protein (Ceru) +32 called had additional possibilities for interacting with the negatively charged protein GFP-17. "By giving these proteins all these possibilities, these different places where they potentially interact could be able to choose the right ones," Simon said. "There were certain patterns and interactions there, available and energetic beneficiaries that we did not necessarily predict in advance that would allow them to accrue in these specific forms."
To get the engineered charged fluorescent proteins, Simon and co-authors Arti Pothukuchy, Jimmy Gollihar and Barrett Morrow encoded their genes, including a chemical label used for purification on portable pieces of DNA called plasmids in E. coli, and then harvested the label protein, as E. coli grew. The researchers blended the proteins together. They first believed that the proteins could only interact to form large, irregularly structured lumps. "But then it was what we continued to see, this weird, fun top about 12 nanometers, it was much smaller than a large lump of protein, but significantly larger than the single protein," Simon said. They measured the size of the particles formed using a Zetasizer instrument at the Texas Materials Institute of UT Austin, and verified that the particles contained both cerulean and GFP proteins Förster Resonance Energy Transfer (FRET), which measure energy transfer. between different colored fluorescent proteins produces fluorescence in response to different energies of light to see if they are close together. Negative spot electron microscopy identifies the specific structure of the particles performed by the group of David Taylor, Assistant Professor of Molecular Bioscience at UT Austin. It showed that the 12 nm particle consisted of a stacked octamer consisting of sixteen proteins. "We found out they were these beautifully shaped flower-like structures," Simon said. Co-author Yi Zhou of Taylor's group UT Austin increased the resolution even more by cryo-electron microscopy to reveal atomic level details of the stacked octamer.
Computational modeling refined the measurements of how the proteins were arranged to a clear picture of the beautiful, flower-like structure, according to Jens Glaser. "We had to come up with a model that was complex enough to describe the physics of the charged green fluorescent proteins and present all the relevant atomic details, but was effective enough to allow us to simulate this in a realistic timetable. a fully atomistic model, it would have taken us over a year to get a single simulation out of the computer, but quickly the computer was, "Glaser said.
The simplified model by reducing the solution without sacrificing important details of the interactions between proteins. "Therefore, we used a model where the shape of the protein is accurately represented at a molecular surface, like that measured from the crystallographic structure of the protein," Glaser added.
"What really helped us turn this around and improve what we could get from our simulations was cryo-EM data," says Vyas Ramasubramani, a graduate student in Chemical Engineering at the University of Michigan. "It really helped us find the optimal configuration to put into these simulations, which then helped us validate the stability arguments we made and hopefully predict future ways we can destabilize or change that structure. "Ramasubramani said.
The researchers demanded a lot of computational power to make the calculations on the scale they wanted.
"We used XSEDE to basically take these big systems where you have many different pieces interacting with each other and calculating all of it instantly, so when you start moving your system forward for a certain time, You can get an idea of how it will evolve in some real time, "Ramasubramani said. "If you were trying to make the same kind of simulation as we did on a laptop, it would have taken months if not years to really go into understanding if some kind of structure would be stable. Do not use XSEDE where you could use essentially 48 cores, 48 calculate units at one time to make these calculations very parallel, we would have made it much slower. "
Supercomputer Stampede2 at TACC contains 4,200 Intel Knights Landing and 1,736 Intel Skylake X calculate nodes. Each Skylake node has 48 cores, the basic unit of a computer processor. "The Skylake nodes in the Stampede2 supercomputer were instrumental in achieving the performance needed to effectively calculate these electrostatic interactions that act between the oppositely charged proteins," says Glaser. "The availability of the Stampede2 supercomputer was at the right time for us to perform these simulations."
Initially, the science team tested their simulations on the Comet system on the SDSC. "When we first found out what type of model should be used and whether this simplified model would give us reasonable results, Comet was a good place to try those simulations," Ramasubramani said. "Comet was a great test bed for what we do."
Based on the larger scientific picture, the researchers hope that this work promotes understanding of why so many proteins in nature will oligomerize or stay together to form more complex and interesting structures.
"We showed that there is no need to be a very specific, advanced set of plans and interactions for these structures to form," Simon said. "This is important because it means we may and probably can take other sets of molecules as we want to make oligomerize and generate both positively charged and negatively charged variants, combining them and specifically ordering structures for them."  Natural biomaterials such as bones, feathers and shells can be hard but light. "We believe that supercharged protein assembly is an easier way to develop the kind of materials that have exciting synthetic properties without having to spend so much time or knowing exactly how to get together beforehand," said Simon. . "We believe it will accelerate the ability to construct synthetic materials and to discover and explore these nanostructured protein materials."
The study "Supercharging enables organized assembly of synthetic biomolecules" was published in the journal Nature Chemistry in January 2019.
Researchers coaxinate proteins to form synthetic structures with mimicking nature
Anna J. Simon et al. Supercharging enables organized assembly of synthetic biomolecules, Nature Chemistry (2019). DOI: 10,1038 / s41557-018-0196-3
University of Texas in Austin
Supercomputers help supercharge protein assembly (2019, March 29)
March 30, 2019
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