Biology subjects

Hungerland, J.

Publications and source records attributed to Hungerland, J..

4 recordsLinked to original sources

bioRxiv2026

Non-equilibrium vibrational dynamics govern ultrafast electron transfer in avian cryptochrome

Photoactivated intermolecular electron transfer (ET) in cryptochromes proceeds along chains of aromatic residues and creates a spatially separated pair of radical electrons. Ultrafast time-dependent spectroscopy can provide experimental insight into this process and theoretical estimates of charge transfer rates are commonly obtained via Marcus theory. Here, we present a new perspective on the ET in European robin cryptochrome 4a (ErCry4a) that synthesizes insights from real-time ET calculations, ultrafast spectroscopic measurements and analytical derivations. The simulations exemplify that molecular vibrations play an essential role in enabling the ET dynamics, which was further rationalized through analytical derivations. Ultrafast pump-probe spectroscopy provided experimental access to the first 1.5 ns of the ET cascade, where multiple radical pair recombination rates arise due to the dynamic equilibrium along the ET chain. We show that the motions of the protein environment and the ET dynamics are inseparably coupled, violating the timescale separation required for Marcus theory. The presented results highlight that non-equilibrium coupling between electronic and nuclear motion dominates ET kinetics in ErCry4a during the first nanosecond after photo-excitation. The findings exemplify the limits of Marcus theory and refine the interpretation of ultrafast spectroscopic signatures in cryptochromes.

biophysics↗

bioRxiv2026

AlphaFold3 predicted LWO G-protein complex from European robin features active-state biased Gα

Avian phototransduction and magnetoreception have been proposed to involve shared retinal proteins, including interactions between long-wavelength opsin (LWO), the cone-specific heterotrimeric G protein (Gt), and cryptochrome 4a (Cry4a), yet structural information on avian phototransduction complexes is lacking. Here we present and critically assess two atomistic models of the European robin LWO-Gt complex generated by distinct modelling strategies. A full-complex prediction using AlphaFold3 yields a tightly packed, structurally stable interface but exhibits pronounced activation-like conformational features of the Gt-subunit that persist in simulations of the isolated protein, revealing a strong bias toward the active state. In contrast, a template-guided assembly based on single-chain predictions and an experimental rhodopsin-Gt reference structure forms a weaker interface and shows no intrinsic activation bias, while still displaying subtle activation-related dynamics. These results demonstrate that machine-learned complex prediction can encode functional states independently of the local interaction environment, thereby limiting its interpretability for signalling mechanisms that hinge on activation equilibria. Our findings highlight the need for explicit assessment of conformational-state bias when modelling regulatory protein assemblies and provide a structural framework for future studies of Cry4a-dependent modulation of retinal G-protein signalling in avian magnetoreception.

biophysics↗

bioRxiv2026

Absence of 8-HDF and MTHF Antenna Chromophore Binding in ErCRY4a Suggests a Possible Flavin-Only Cofactor State: Insights from Biochemical and Computational Analyses

Cryptochromes and photolyases are blue-light-sensitive flavoproteins that generally bind flavin adenine dinucleotide (FAD) and have distinct functions. Cryptochrome 4a (CRY4a) is a protein expressed in the double-cone photoreceptors of the retina in migratory songbirds like European robin (Erithacus rubecula) and is hypothesized as the primary sensor for avian magnetoreception. In addition to FAD, most photolyases and some cryptochromes bind antenna chromophores such as 8-hydroxy-5-deazaflavin (8-HDF) or 5,10-methenyltetrahydrofolate (MTHF) to enhance light absorption. Here, we investigated whether Erithacus rubecula Cryptochrome 4a (ErCRY4a) also binds 8-HDF and/or MTHF. 8-HDF binding was studied by co-expressing ErCRY4a with the fbIC gene that encodes for 8-HDF synthase and thus for production of 8-HDF in E. coli. As a positive control for 8-HDF binding, we expressed Xenopus laevis 6-4 photolyase (Xl6-4PL) which is known to bind both FAD and 8-HDF. This experiment resulted in successful binding of 8-HDF to Xl6-4PL, but not to ErCRY4a. We studied the binding of MTHF using in vitro reconstitution followed by UV-Vis spectroscopy and isothermal titration calorimetry (ITC) assays. No interaction was observed between MTHF and ErCRY4a. To theoretically understand the binding of potential antenna chromophores to ErCRY4a, we performed computational analyses. We found no similarity at the relevant binding sites between the sequences of ErCRY4a with proteins shown to bind MTHF or 8-HDF. This suggests that the binding pocket is not conserved. Our study proposes that ErCRY4a only harbor one light-sensitive cofactor, which in turn suggests a functional specialization different from most photolyases.

biochemistry↗

bioRxiv2025

Cryptochrome 4b protein is likely irrelevant for the 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 were reported in European robin (Erithacus rubecula), namely ErCry4a and ErCry4b. It is known that ErCry4a protein exhibits electron transfer between the flavin adenine dinucleotide (FAD) cofactor and tryptophan residues that generates magnetically sensitive radical pairs for magnetoreception. However, little is known about the ErCry4b isoform. We therefore characterized the properties of ErCry4b to see whether it fulfills prerequisites to be a radical pair magnetic sensor molecule. Our results show that ErCry4b protein does not bind FAD in vitro. Computational structure simulations revealed that the FAD non-binding in ErCry4b is likely due to protein structure dynamics. Furthermore, ErCry4b protein abundance in the robin retina, cerebellum and liver is below the detection limit of immunoprecipitation assays coupled with mass spectrometry. Meanwhile, transcript analyses show that ErCRY4b mRNA abundance is 10 times less than ErCRY4b in the retina. In conclusion, ErCry4b does not fulfill 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.

biochemistry↗