Purification and characterization of the Danaus plexippus cryptochromes

Cryptochromes are flavoproteins that function as blue-light photoreceptors to regulate growth in plants and to entrain the circadian clock in animals. The photochemical and signaling mechanisms by which cryptochromes function are still undefined. Two cryptochromes identified in Danaus plexippus have shown distinct in vivo activities where DpCry1 is similar to Drosophila melanogaster cryptochrome and DpCry2 is similar to mammalian cryptochromes. I purified and biochemically characterized both D. plexippus cryptochromes. DpCry1 is the first animal cryptochrome to contain near-stoichiometric flavin. Photoreduction of FADox to FAD°¯ and fluorescence lifetime measurements showing an extremely short lifetime of flavin in DpCry1 both suggest a novel photochemical mechanism for DpCry1. DpCry2 contained only trace amounts of flavin and both proteins contained sub-stoichiometric MTHF. Purification of DpCry1 as the first animal cryptochrome with near-stoichiometric flavin will enable crucial in vitro biochemical and biophysical experiments that have never been conducted on an animal cryptochrome.