Evolution of molecular innovations in cyanobacterial light-perceiving systems

Novel functional features are prominent throughout evolution, yet paradoxical: how does evolution create something innovative when all it can work with is variation of established biology? Neo-functionalization of proteins after gene duplication is one common explanation, driven by natural selection for a new, potentially innovative function. However, groundbreaking novelty may not be explained by adaptive diversification of existing proteins. In this thesis, we tackled the paradox of molecular innovation with molecular phylogenetics in two original research publications. The first article examined the evolution of cyanobacteriochromes (CBCRs), a class of phytochromes found exclusively in cyanobacteria. CBCRs gained the innovative ability to collectively sense the entire spectrum of visible light with a single-domain protein, in contrast to canonical tri-domain phytochromes that respond primarily to red- and far-red light signals. Using ancestral sequence reconstruction (ASR) and biochemical verification of resurrected proteins, we showed that the last common ancestor of CBCRs responded reversibly to green- and red-light signals. Latent blue-light perception and the ability to bind alternative chromophores, coupled with the minimalistic domain architecture may have enabled the vast diversification of CBCRs. This indicates that molecular innovation can potentially be achieved by reducing protein complexity, which may open up sequence space for new functions, such as broader color perception. The second article focused on the evolution of a novel allosteric regulation in cyanobacterial photoprotection by direct protein-protein interaction. It is unclear whether such required protein surface compatibilities can only be built by selection in small incremental steps, or whether they can also emerge fortuitously. Here, we used ASR and biophysical protein characterization to retrace the evolution of the allosteric interaction between the orange carotenoid protein (OCP) and its unrelated regulator, the fluorescence recovery protein (FRP). This interaction evolved when a precursor of FRP was horizontally acquired by cyanobacteria. FRP’s precursors could already interact with and regulate OCP even before these proteins first encountered each other in an ancestral cyanobacterium. The OCP–FRP interaction exploits an ancient dimer interface in OCP, which also predates the recruitment of FRP into the photoprotection system. This shows how evolution can easily fashion complex regulatory systems from pre-existing components, even without prior gene duplication. Together, we have shown that chance events may play an underestimated role in protein evolution and can indeed lead to groundbreaking innovations in biology

Fortuitously compatible protein surfaces primed allosteric control in cyanobacterial photoprotection

Highly specific interactions between proteins are a fundamental prerequisite for life, but how they evolve remains an unsolved problem. In particular, interactions between initially unrelated proteins require that they evolve matching surfaces. It is unclear whether such surface compatibilities can only be built by selection in small incremental steps, or whether they can also emerge fortuitously. Here, we used molecular phylogenetics, ancestral sequence reconstruction and biophysical characterization of resurrected proteins to retrace the evolution of an allosteric interaction between two proteins that act in the cyanobacterial photoprotection system. We show that this interaction between the orange carotenoid protein (OCP) and its unrelated regulator, the fluorescence recovery protein (FRP), evolved when a precursor of FRP was horizontally acquired by cyanobacteria. FRP’s precursors could already interact with and regulate OCP even before these proteins first encountered each other in an ancestral cyanobacterium. The OCP–FRP interaction exploits an ancient dimer interface in OCP, which also predates the recruitment of FRP into the photoprotection system. Together, our work shows how evolution can fashion complex regulatory systems easily out of pre-existing components