Form I Rubisco is one of the most abundant enzymes on Earth. It is responsible for assimilating atmospheric CO2 into organic matter, but how it obtained its high CO2 specificity is unknown.
Researchers at the Max Planck Institute for Terrestrial Microbiology, SYNMIKRO and NTU Singapore explored this question, presenting their findings in a recent paper in Science: “Evolution of increased complexity and specificity at the dawn of Form I Rubiscos” .
A core tool of their research was mass photometry, a biophysical technique that measures the mass of biomolecules in solution. We spoke to Luca Schulz, the first author of the publication, to gain a deeper understanding of the findings and the role of mass photometry in obtaining them. Continue reading to learn more about this pioneering research and application of mass photometry.
You can also register for an upcoming webinar in which Luca discusses his research and learn more about this topic.
Luca is a graduate student at the Max Planck Institute for Terrestrial Microbiology in Marburg, Germany under shared supervision by Georg Hochberg and Tobias Erb. His research focuses on understanding the evolution of protein complexes, particularly the carboxylase Rubisco that assimilates most inorganic carbon from the atmosphere. Luca is also one of Refeyn’s 2021 travel grant winners.
On Twitter: @schulluc
The research questions: How and when did Rubisco evolve its specificity for CO2?
The research by Luca and his colleagues focuses on Rubisco’s evolution, a process that began billions of years ago – before there was free oxygen in the Earth’s atmosphere. As photosynthesis evolved, it produced atmospheric free oxygen, which acted as an undesirable substrate during Rubisco’s catalysis. In response, aerobic organisms evolved to use Rubiscos with higher specificity for CO2. Today, as Luca explains, Rubisco “is the protein that fixes carbon in algae, plants and many other microorganisms”.
How and when Rubisco evolved its high CO2 specificity, which all modern Form 1 Rubiscos have has been unknown. One hypothesis is that a set of eight small subunits (SSUs) is at least partly responsible. Modern Form I Rubisco is a 16mer, assembling into a complex of eight catalytic large subunits (LSUs) and eight noncatalytic small subunits (SSUs) – L8S8 (Figure 1).
The SSU represents the most obvious structural difference between Form I and ancestral Rubiscos. “Ancestrally, Rubisco was only composed of the large subunits,” Luca says. “Over the course of its evolution, the protein complex started forming higher-order oligomers, as the large subunit started interacting with the small subunit”, he adds.
The aim of the team’s research was to find out when and how the SSU was gained during the protein’s evolution, and why it became pivotal for Rubisco’s function. As Luca explains, “We are basically trying to decipher the evolutionary interval over which this SSU was recruited into the complex, why it was recruited and why the present Rubisco now fully depends on the SSU, even though it previously worked without it”.
Figure 1: Rubisco form I assembles into an L8S8 hexadecamer. 3D image created with data obtained using X-ray diffraction.
Figure source: 
The experimental challenge
The hypothesis that the SSU conferred Rubisco’s high CO2 specificity could not be tested previously since the SSU is essential for the enzyme’s solubility and catalytic activity. This meant that simply removing it was not an option. The team instead used a combination of ancestral sequence reconstruction and mass photometry to retrace the path of Rubisco’s evolution.
The role of mass photometry: Measuring the oligomerization status of ancestral Rubiscos
To determine when Rubisco gained the SSU, Luca and his colleagues identified and generated ancestral Rubisco LSU variants that do not bind to SSUs. Mass photometry enabled them to measure the oligomerization states of those ancestral Rubiscos and determine which of them interacted with an SSU. As Luca explains, “Since we are trying to decipher the evolutionary step over which a protein became more complex and started forming higher-order oligomers, mass photometry was vital for us. Mass photometry allowed us to quickly screen protein complexes in solution.”
Additionally, mass photometry helped in determining how Rubisco gained the SSU. Ancestral Rubiscos that possessed an SSU had several genomic substitutions in comparison to non-SSU-interacting Rubiscos. Introducing some of these substitutions to the non-SSU-interacting ancestral Rubisco enabled SSU binding and thereby the formation of an L8S8 complex. The team measured this complex formation with mass photometry.
Finally, again with the help of mass photometry, they discovered why Rubisco depends on the SSU for solubility. They found that in the absence of an SSU, the LSU self-assembles into fibers, making the Rubisco enzyme insoluble. Luca comments that mass photometry was key to this discovery. “Using [mass photometry], we were able to see very regular peaks of the Rubisco’s core complex (LSU), corresponding to the fibroid formation. With other techniques, we would have dismissed this finding as aggregated protein or void peaks.”
After successfully using mass photometry to determine how the modern Rubisco developed its high CO2 specificity, Luca plans to continue using the technology for his future experiments. He says,
“For any biochemical experiments that I will do in the future, I will always use mass photometry as a technique that either complements other techniques or replaces them entirely. I will try to keep mass photometry as integrated in my work as possible because it is a very honest technique that gives you a direct picture of your sample and there is not a lot of room for interpretation.
He adds that, with mass photometry, “In a way, you can visualize your protein, which you cannot do with other techniques.”
Read our technical blog explaining the principle behind mass photometry and why mass photometry is useful.
Here you can learn how mass photometry directly measures the relative concentrations of all protein populations in a sample, in a single-molecule fashion.
In this webinar, Dr. Margaret Stratton (UMass Amherst) talks about her work on the role of CaMKII in different tissues. Mass photometry has been used to quantitatively determine the stoichiometry of different variants of this crucial oligomeric enzyme.
 L. Schulz et al., “Evolution of increased complexity and specificity at the dawn of form I Rubiscos,” Science, vol. 378, no. 6616, pp. 155–160, Oct. 2022, doi: 10.1126/science.abq1416.
 RCSB PDB - 7QSW: L8S8-complex forming RubisCO derived from ancestral sequence reconstruction of the last common ancestor of SSU-bearing Form I RubisCOs. https://www.rcsb.org/structure/7QSW (accessed Oct. 13, 2022).