PhD defense: Structural insights into the photoactivation mechanism of the orange carotenoid protein

By Rory Munro (IBS/Dynamics and kinetics of molecular processes Group)

Cyanobacteria have significantly shaped the history of life on the planet in several ways, and their success is largely credited to their ability to adapt to transient environmental conditions, particularly solar irradiation. As such, photosynthetic regulation is of the utmost importance. Light harvesting Phycobilisomes (PBS) utilize a variety of pigments to capture and funnel light energy into photosynthetic reaction centers, maximizing the availability of excitation to drive this important photochemical reaction. However, under strong irradiation, the over-excitation of PBS pigments can block the energy transfer cascade, leading to triplet-state excitation, which if not neutralized, generates singlet oxygen, threatening cellular survival. Thus, the Orange Carotenoid Protein (OCP) acts as a photoprotective agent to regulate the flow of incoming excitation at the core of the PBS. Structurally, the OCP comprises the N- and C-terminal domains (NTD and CTD, respectively), which share a tunnel within which the keto-carotenoid chromophore embeds. The pigment is mainly stabilized by two conserved hydrogen bonds, anchoring its keto-oxygen to the CTD.

In the presence of strong blue-green light, the dark-adapted OCPO will transition to the active, metastable OCPR, which binds to the PBS to dissipate excess excitation into the environment as heat. The OCP photoactivation mechanism is scarcely understood and actively debated, however it is generally accepted that absorption of a blue-green photon: i) excites the carotenoid to one of three possible excited states ii) ruptures the H-bonds tethering the keto-carotenoid to the CTD, which iii) instigates a 12 Å translocation of the carotenoid into the NTD permitting the dissociation of the two domains which exposes the PBS-binding epitope. However, upon single photon absorption, the first photo-productive intermediate has an incredibly low yield, which has so far greatly complicated the study of the photoactivation mechanism by structural methods. Recently, it was proposed based on spectroscopic studies that depending on the functionalizing carotenoid, effective OCP photoactivation requires either one photon, or the consecutive absorption of two, making the understanding and structural study of the photoactivation mechanism even more complex.

In this work we utilized a variety of biochemical, biophysical, and structural methods to elucidate the OCP photoactivation mechanism. Notably, we used room-temperature crystallography to show that the one-photon intermediate, thus far only characterized spectroscopically, can be trapped and accumulated in the crystal. We then verified the importance of this photo-intermediate and the structural dynamics therein using mutagenesis, temperature-resolved fluorimetry, and absorption spectroscopy – both steady-state and transient. The functionalization of different co-factors was also examined, offering a structural basis for the two-photon mechanism. Finally, the photo-induced structural changes associated with the dark-state and the one-photon intermediate were characterized using time-resolved serial femtosecond crystallography on the atomic scale from femto- to milliseconds. The findings obtained from this multi-technique approach offer a clearer view of the photoactivation mechanism, illustrating how otherwise extremely transient states can be stabilized over several decades of time by select structural changes in the architecture of the protein. This work thus addresses multiple unanswered questions regarding OCP photoactivation, paving the way for the rational design of tailored variants suited for optogenetics applications or the regulation of bio-inspired artificial photosynthetic systems.