How cells respond to light or time is a fundamental question in biology. Cryptochromes (CRYs) are evolutionarily conserved blue light receptors or key components of the circadian oscillator found in major evolutionary lineages, from bacteria to human and have been intensively studied. However, the structure-function relationship of CRYs from evolutionary perspective is unclear. In this thesis, I interrogated the evolutionary roles of universally conserved residues (UCRs) of Arabidopsis thaliana cryptochrome 2 (CRY2) (Chapter 2) and developed optogenetic tools by engineering CRY2 using continuous directed evolution techniques (Chapter 3).

UCRs are invariable amino acids evolutionarily conserved among members of a protein family across diverse kingdoms of life. UCRs are considered important for stability and/or function of protein families, but it has not been experimentally examined systematically. In Chapter 2, I experimentally analyzed 51 UCRs of Arabidopsis CRY2 that are universally conserved in eukaryotic cryptochromes from Arabidopsis to human. Surprisingly, I found that UCRs required for stable protein expression of CRY2 in plants are not similarly required for stable protein expression of human hCRY1 in human cells. Moreover, 74% of the stably expressed CRY2 proteins mutated in UCRs retained wild-type-like activities for at least one of the photoresponses I analyzed. My finding suggests that the evolutionary mechanisms underlying conservation of UCRs or that distinguish UCRs from non-UCRs determining the same functions of individual cryptochromes remain to be investigated.

CRY2 mainly regulates plant photomorphogenesis through blue-light-specific interactions with numerous protein partners. Such blue-light-specific interactions have been exploited in optogenetics to manipulated biological events in a timely and precisely manner. Chapter 3 focused on the development of a novel pair of blue-light-dependent interacting proteins: CRY2-BIC1 (Blue-light Inhibitor of Cryptochromes 1) and applied PACE (Phage Assisted Continuous Evolution) to increase the dynamic range of CRY2-BIC1 blue-light dependent interaction. I isolated variants of CRY2 with stronger interactions with BIC1 and developed soluble expression and protein-dissociating PACE to facilitate further engineering of CRY2, which could give hints on characteristics of UCRs and non-UCRs.