Partial Conversion of an Animal-Like Cryptochrome into a Plant Cryptochrome

Area of Science:

• Biochemistry
• Photobiology
• Molecular Biology

Background:

• Cryptochromes are flavin-binding photoreceptors found across diverse organisms.
• The green alga *Chlamydomonas reinhardtii* possesses both plant-like (pCRY) and animal-like (aCRY) cryptochromes with distinct photochemical properties.
• aCRY functions as a (6-4) photolyase and photoreceptor, utilizing 8-hydroxy-5-deazaflavin (8-HDF) as a light-harvesting pigment, and features an asparagine residue where pCRY has an aspartic acid for proton donation.

Purpose of the Study:

• To investigate the impact of substituting asparagine with aspartic acid (aCRY-N395D) on aCRY's photochemistry and conformational response.
• To elucidate the role of the proton donor residue and the light-harvesting pigment (8-HDF) in aCRY function.
• To understand the evolutionary divergence of cryptochrome and photolyase functions.

Main Methods:

• Nanosecond time-resolved UV-vis spectroscopy.
• Fourier-transform infrared (FTIR) difference spectroscopy.
• Comparative analysis of wild-type and mutant aCRY, with and without 8-HDF.

Main Results:

• The N395D mutation in aCRY induced pCRY-like ultrafast proton transfer to the flavin and hypsochromic shifts, with flavin neutral radical formation occurring within 100 ns.
• This single amino acid exchange accelerated photoactivation for DNA repair compared to wild-type aCRY.
• Conformational changes characteristic of pCRY were observed in aCRY-N395D, underscoring the critical role of aspartate in cryptochrome signaling.

Conclusions:

• The substitution of a single amino acid (Asn to Asp) profoundly alters aCRY's photochemical and signaling capabilities, making it resemble pCRY.
• Aspartate is crucial for initiating signaling pathways in cryptochromes.
• These findings provide significant insights into the functional diversification within the cryptochrome/photolyase protein superfamily.