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

Abstract

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 (<i>Erithacus rubecula</i>), namely <i>Er</i>Cry4a and <i>Er</i>Cry4b. <i>Er</i>Cry4a 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 <i>Er</i>Cry4b isoform. We therefore characterize the properties of <i>Er</i>Cry4b to determine whether it fulfils the prerequisites to be a radical-pair magnetic sensor molecule. Our results show that <i>Er</i>Cry4b protein does not bind FAD <i>in vitro</i>. Computational simulations reveal that the lack of FAD binding in <i>Er</i>Cry4b is probably due to protein structural dynamics. Furthermore, immunoprecipitation assays coupled with mass spectrometry suggest that <i>Er</i>Cry4b protein is undetectable in the robin retina, cerebellum, and liver. Meanwhile, transcript analyses show that <i>ErCRY4b</i> mRNA abundance is 10 times lower than that of <i>ErCRY4a</i> in the retina. In conclusion, <i>Er</i>Cry4b 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.