Rubisco enzyme molecule
Rubisco. Molecular model of the enzyme rubisco (ribulose bisphosphate carboxylase oxygenase) complexed with 2-carboxyarabinitol biphosphate. Rubisco is thought to be the most abundant and important protein found in nature. It occurs in all plants and fixes carbon dioxide during photosynthesis.

Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO), the world’s most abundant protein, adopts multiple assemblies, although the origins and distribution of its different oligomeric states remain cryptic. Now, researchers show how evolution of the enzyme involved in photosynthesis can lead to a surprising diversity of molecular assemblies and that proteins can change their structural arrangement more easily than previously thought.

This work is published in Science Advances in the paper, “Structural plasticity enables evolution and innovation of RuBisCO assemblies.

“The big finding from this paper is that there’s a lot of structural plasticity,” said Patrick Shih, PhD, assistant professor at UC Berkeley. “Proteins may be much more flexible, across the field, than we’ve believed.”

The team studied a type of RuBisCO (form II) found in bacteria and a subset of photosynthetic microbes using traditional crystallography—a technique capable of atomic-level resolution—combined with another structure-solving technique, small-angle X-ray scattering (SAXS), that has lower resolution but can take snapshots of proteins in their native form when they are in liquid mixtures. SAXS has the additional advantage of high-throughput capability.

Previous work had shown that the better-studied type of RuBisCO found in plants (form I) always takes an “octameric core” assembly of eight large protein units arranged with eight small units, whereas form II was believed to exist mostly as a dimer with a few rare examples of six-unit hexamers. After using the complementary techniques to examine samples of RuBisCO from a diverse range of microbe species, the authors observed that most form II RuBisCO proteins are actually hexamers, with the occasional dimer, and they discovered a never-before-seen tetrameric (four unit) assembly.

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

The reconstruction suggests that the gene for form II RuBisCO has changed over its evolutionary history to produce proteins with a range of structures that transform into new shapes or revert back to older structures quite easily. In contrast, during the course of evolution, selective pressures led to a series of changes that locked form I RuBisCO in place (structural entrenchment) which is why the octameric assembly is the only arrangement seen. According to the authors, it was assumed that most protein assemblies were entrenched over time by selective pressure to refine their function, like we see with form I RuBisCO. But this research suggests that evolution can also favor flexible proteins.

After completing the ancestral sequence reconstruction, the team conducted mutational experiments to see how altering the rubisco assembly, in this case breaking a hexamer into a dimer, affected the enzyme’s activity. Unexpectedly, this induced mutation produced a form of RuBisCO that is better at utilizing CO2. All naturally occurring RuBisCO frequently binds the similarly sized O2 molecule on accident, lowering the enzyme’s productivityThere is a great deal of interest in genetically modifying the RuBisCO in agricultural plant species to increase the protein’s affinity for CO2, in order to produce more productive and resource-efficient crops. To this end, there has been a lot of focus on the protein’s active site.

“This is an interesting insight to us because it suggests that in order to have more fruitful results engineering RuBisCO, we can’t just look at the simplest answer, the region of the enzyme that actually interacts with CO2,” said Albert Liu, a graduate student in Shih’s lab. “Maybe there are mutations outside of that active site that actually participate in this activity and can potentially change protein function in a way that we want. So that’s something that really opens doors to future avenues of research.”

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