For some more fun biochemistry (& a lot of cool evolutionary implications) I recommend that you check out the original publication and/or message me!

A huge thanks to my mentors Georg Hochberg and Tobi Erb, as well as all co-authors, who have been crucial in every step of this project. :)

Focusing on substitutions that occurred in such regions, we identified historical changes that drastically reduced Rubisco’s activity but whose negative effect is buffered by the presence of the SSU.

Inspired by NMR-guided directed evolution, we used hydrogen-deuterium exchange experiments to identify structural features of Rubisco that change their deuterium uptake behavior (which, in very condensed form, can be indicative of altered protein dynamics) upon inhibitor binding.

Intriguingly, some Rubisco variants we created were soluble and octameric, yet catalytically inactive without the SSU. This suggested that historical substitutions can render catalysis SSU-dependent. To find such substitutions, we focused on active site responsive regions.

This suggested a direct link between Rubisco’s active site and the octamer interface. Indeed, addition of tight-binding inhibitors reconstituted oligomerization into octamers (see figure above) and pre-incubating Rubisco with a weak-binding inhibitor recovered activity in assays.

We found that the Rubiscos we worked with could not efficiently catalyze their reaction when they did not form octamers. This contrasts the many Rubiscos that exist as simple dimers of large subunits. But how can we prove that the loss of oligomerization really was the cause?

We found that historical substitutions at the dimer-dimer interface made octamer formation SSU-dependent. The SSU bridges adjacent dimers, which stabilizes the dimer-dimer interface. This allows evolution to explore a less stable interface. But why is octamer formation important?

We were interested in the biochemical mechanisms by which Rubisco started to depend on the SSU (and generally how proteins start to depend on new subunits). To investigate these mechanisms, we introduced historical substitutions into a Rubisco that could bind the new subunit but did not yet need it.

Form I Rubiscos assemble into an octamer of large subunits (purple) that is capped by small subunits (SSU) at the bottom and top of the complex (beige). This stoichiometry evolved from a simple octamer of large subunits and the SSU subsequently became essential for the complex.