Cryptochrome 4b protein is probably irrelevant for radical pair-based magnetoreception in the European robin.

Avian cryptochrome 4 (Cry4) protein is a putative magnetosensitive molecule facilitating precise long-distance navigation in migratory birds. Two splice variants of Cry4 have been reported in the European robin (Erithacus rubecula), namely ErCry4a and ErCry4b. ErCry4a protein is known to generate magnetically sensitive radical pairs for magnetoreception through electron transfer between the flavin adenine dinucleotide (FAD) cofactor and tryptophan residues within the protein. However, little is known about the ErCry4b isoform. We therefore characterize the properties of ErCry4b to determine whether it fulfils the prerequisites to be a radical-pair magnetic sensor molecule. Our results show that ErCry4b protein does not bind FAD in vitro. Computational simulations reveal that the lack of FAD binding in ErCry4b is probably due to protein structural dynamics. Furthermore, immunoprecipitation assays coupled with mass spectrometry suggest that ErCry4b protein is undetectable in the robin retina, cerebellum, and liver. Meanwhile, transcript analyses show that ErCRY4b mRNA abundance is 10 times lower than that of ErCRY4a in the retina. In conclusion, ErCry4b does not fulfil the prerequisites to be a radical pair-based magnetic sensing molecule due to the lack of FAD binding, and it might not even be expressed as a functional protein in the European robin.