Folding kinetics of recognition loop peptides from a photolyase and cryptochrome-DASH

Cryptochromes (CRY) and photolyases (PL) use a common flavin adenine dinucleotide cofactor and homologous protein scaffold to accomplish numerous, seemingly dissimilar functions. PL repairs UV-damaged DNA in a mechanism requiring light and DNA base flipping. CRY cannot repair DNA, and instead function in core biological processes including plant photomorphogenesis, circadian rhythm, and magnetoreception. One subclass, CRY-DASH, does catalyze repair of single-stranded DNA; compromised base flipping may deactivate its tight binding to duplex DNA substrates. We recently demonstrated that the a "recognition loop" involved in DNA binding by both PL and CRY-DASH is among the most flexible regions in the two proteins, and exhibits especially heightened dynamics in CRY-DASH. Here, we establish that these distinct dynamics are encoded by the loop sequences: we quantify the flexibility of the isolated loop peptides through the kinetics and activation parameters for their folding. Mirroring the dynamics within the proteins, the CRY-DASH recognition loop peptide folds 2.5-fold faster than its counterpart in PL, predominantly due to a lower enthalpy of activation. We propose that these distinct dynamics are functionally significant in DNA recognition. Binding duplex DNA in the catalytically-active base-flipped conformation imposes significant order on the recognition loop, and a corresponding entropic penalty. This may be surmounted by the more preorganized PL recognition loop, but may impose too large a barrier for the more dynamic loop in CRY-DASH. These results suggest that evolution of protein dynamics, through local sequence tuning in the recognition loop, may be an important mechanism for functional diversification in PL and CRY.