Over the past century, scientists have developed and deployed amazing techniques such as X-ray crystallography and cryo-electron microscopy to determine the structure of proteins and thereby answer a myriad of important questions. But new research shows that understanding protein structure can be more complicated than we think.

A group of researchers at the Lawrence Berkeley Lab studied the world’s most abundant protein, an enzyme involved in photosynthesis called rubisco, and how evolution has enabled molecular assemblies to accomplish the same task. We have shown how this can lead to an amazing diversity ofFindings released today scientific progressrevealing that many of the proteins we thought we knew may actually exist in other, unknown forms.

Historically, when scientists solved a structure and determined, for example, that a protein is a dimer (composed of two units), they assumed that similar proteins also exist in dimer form. there was a possibility. However, small sample size and sampling bias are unavoidable factors given that it is very difficult to convert naturally liquid proteins into solid, crystallized forms that can be interrogated by X-ray crystallography. and was hiding reality.

“If you’re walking outside and you see someone walking their dog, you’ve never seen a dog before and you see a wiener dog, you’re like, ‘OK, this is what all dogs look like. But what you need to do is go to a dog park and see the diversity of all the dogs there,” says lead author, Biological Sciences. “One lesson from this paper, which extends beyond rubisco to all proteins, is that the The question is, are we looking at the true extent of the structure of nature, or are these biases making everything look like a wiener dog?”

Wanting to explore all the different Rubisco arrangements in a metaphorical dog park and find out where they came from, Shih’s lab used Berkeley Lab’s Advanced Light Source to study structures in the bioscience field. We worked with biology experts. The team collaborated on classical crystallography (a technique capable of atomic resolution) and another structural analysis technique, small-angle X-ray scattering (SAXS). Although the resolution is lower, it is possible to take snapshots of proteins in liquid mixtures in their native form. SAXS has the added advantage of high throughput capabilities. This means that dozens of individual protein assemblies can be processed in rapid succession.

Previous studies have shown that the more well-studied type of rubisco (Form I) found in plants always adopts an “octameric core” assembly of eight large protein units interspersed with eight smaller units. It was done. Rare examples of 6-unit hexamers are rare. After examining Rubisco samples from a variety of microbial species using these complementary techniques, the authors found that most Form II Rubisco proteins are actually hexamers and sometimes dimeric. , and discovered a previously unseen tetramer (4 units). assembly.

Combining this structural data with the respective protein-coding gene sequences allowed the team to perform ancestral sequence reconstructions. This is a computer-based molecular evolution method that can deduce what ancestral proteins looked like based on the sequence and appearance of modern proteins that evolved from them. .

The rearrangements suggest that the gene of form II rubisco changed during its evolutionary history to produce proteins with a variety of structures that changed to new shapes or reverted to old structures very easily. I’m here. In contrast, during evolution, selective pressure led to a series of changes that locked Form I rubisco in place. This is a process called structural anchorage. This is why the octameric assembly is the only arrangement currently seen. According to the authors, it was assumed that most protein assemblies were anchored over time by selective pressure to refine their function, as seen in Form I rubisco. However, this study suggests that evolution may favor flexible proteins.

“The big finding from this paper is that there is a lot of structural plasticity,” said Shih, who is also an assistant professor at the University of California, Berkeley. “Proteins may be much more flexible than we believed.”

After completing the reconstruction of the ancestral sequence, the team performed mutation experiments to see how alterations in Rubisco assembly (in this case breaking the hexamer into a dimer) affect the activity of the enzyme. Did. Unexpectedly, this induced mutation produced a form of Rubisco that better utilized its target molecule, CO.₂All naturally occurring rubiscos frequently combine Os of similar size.₂ Accidental Molecules, Decreased Enzyme Productivity. There is considerable interest in genetically engineering the agricultural plant species Rubisco to increase the protein’s affinity for CO.₂, to produce more productive and resource efficient crops. However, much attention has focused on the active site of the protein, the region of the protein where CO resides.₂ or O₂ Knead.

“This is an interesting insight for us, because to engineer Rubisco to fruitful results, we cannot look at the simplest answer: just the region of the enzyme that actually interacts with CO. because it suggests₂Lead author Albert Liu, a graduate student in Shih’s lab, said, “Maybe we can actually participate in this activity and suddenly find ourselves outside that active site that could alter protein function in the way we want.” There are mutations. That really opens the door for future research avenues.”

Co-author Paul Adams, associate lab director of biosciences and vice president of technology at JBEI, added: Structural biology techniques to study one of the most important problems in biology and reach some unexpected conclusions. “

Structural biology experiments were performed at the Advanced Light Source (ALS) at Berkeley Lab, a Department of Energy (DOE) Office of Science user facility. The SYBILS beamline is funded in part by DOE’s Office of Bioenvironmental Research. X-ray crystallography was performed at the Berkeley Center for Structural Biology. JBEI is a bioenergy research center managed by Berkeley Lab. This work was funded by the DOE Office of Science and the David and Lucile Packard Foundation.