Cryptochrome and the Role of the Radical Pair Mechanism in Animal Magnetoreception

This is a paper I wrote for a Biochemistry class while getting my undergraduate degree. It's a mock grant proposal, which means in real life you would write something similar when trying to get funding for whatever project you want to study. In this case, the class was an overview of the field of biophysical chemistry. I had to choose a biological system of interest and develop a realistic (read: fundable) project around it using the techniques we studied in class. I'm releasing this draft under the standard Devtome CC license, so feel free to use or adapt it for actual research purposes - I'd love to see this idea become a reality.


Models and implications of animal magnetoreception

Magnetoreception is the ability to sense the Earth’s magnetic field, and is most familiar as the mechanism by which migratory birds are able to navigate accurately. In birds – the magnetosensing organism that have received the most study - two models of magnetoreception have been proposed: the first proposes the use of magnetite (Fe3O4) particles in the beak, and the second proposes a magnetosensitive radical pair reaction in the retina (2,6,7,10). In recent years, there has accumulated significant evidence for the radical pair model over the magnetite model. The two observations best in support of this model are that magnetoreception, both in birds and in drosophila, is light-dependent, suggesting a photo-activated electron transfer mechanism, and that magnetoreception is responsive to the inclination, but not the polarity of the magnetic field (1,7).

The protein cryptochrome (Cry) has been implicated as the source of this radical pair mechanism, with significant evidence pointing to it as the only possible protein suitable (1-3). Knockout studies in drosophila have shown magnetoreception to be dependent on both Cry and blue-light, and magnetoreception is able to be recovered in knockouts with transgenic Crys from a wide variety of sources, including other insects and humans (3,11). There are two types of cryptochromes, characterized by their biological role: type I cryptochromes (as in drosophila) function as circadian photoreceptors, while type II cryptochromes (as in the monarch butterfly and in humans) function as a component of the circadian clock in a light-independent manner (9). In humans, cryptochrome plays an important role as a component of the main molecular clock, and due to its potential sensitivity to magnetic fields, has important implications for human health – in fact, Cry has been proposed as a likely mechanism in which exposure to strong magnetic fields can cause cancer (8). Further, Cry may play a role in the visual acuity of humans, by “providing a spherical coordinate system for integrating spatial position” (8). Understanding the mechanism by which cryptochrome is sensitive to magnetic fields will help elucidate this protein’s remarkable involvement in human health and sensory perception.

The radical pair mechanism

Magnetically sensitive reactions almost always involve molecules with unpaired electrons. (1) The short-lived reaction intermediate that is formed during an electron transfer reaction is called a radical pair. The unpaired electrons can exist in either a singlet (S) or a triplet (T) state. Each electron spin has an associated magnetic moment, which allows an external magnetic field to influence the interconversion between the S and T states (1). For the equilibrium and kinetics of a reaction to be influenced by a magnetic field, the reaction therefore must be restricted to using just one of the S or T state (1). Because the Earth’s magnetic field has a relatively low strength of about 0.5G (~50 μT), there are significant addition limitations on reactions that could be sensitive to its influence (2). Specifically of interest are the interactions of the electron spins with each other (hyperfine interaction) and with the magnetic field (Zeeman interaction). To be sensitive to the Earth’s magnetic field, the strength of the hyperfine interaction must be of a similar order to that of the Zeeman interaction such that the field can influence the interconversion between S and T states (1,2). The period of a 50 μT field is approximately 1 μs, thus, for the Earth’s magnetic field to influence the reaction, the radical pair must be stable enough to persist for at least 1 μs (1). To achieve a radical pair with this stability, the edge-to-edge separation must be less than 1.5nm, while the center-to-center separation must be around 2nm (1). This significantly limits possible molecules that could be involved in a radical pair. Further, for the reaction to act as a magnetic compass, it must interact differently depending on the direction of the magnetic field. This means that there must be some rotational constraints on the radical pair, either on the Cry protein through anchoring to the cytoskeleton (1,2) or through photoselection effects induced by the directionality of the light entering the eye (14).

**Figure 1**. A reaction scheme forming the basis of a magnetoreceptor. In one model of Cry, A = Trp and B = FAD.

Several models have been proposed for the components of the radical pairs, all of which involve electron transfer from cryptochrome’s cofactor FAD to varying electron donors. In animals, FAD’s ground state is either an anionic or neutral radical state (FAD•– or FADH•) (1,2,6,10). This is in contrast to the studies done on plant cryptochromes and in photolyases (an evolutionary ancestor and ortholog of Cry), where FAD is found in its fully oxidized state, FADox (4,6). As the other cofactor, amino acids in the Cry protein, such as the terminal tryptophan in the highly conserved “Trp-triad” (6,13), a tyrosine residue (4,13), or a radical electron donator in solution such as superoxide (2) have been proposed.

The more popular models involving the Trp-triad have been shown to be sensitive to strong magnetic fields (4,5,9), but there is limited evidence suggesting that they are sensitive to weaker magnetic fields. Further, mutation studies have shown that the Trp-triad is not actually necessary for magnetoreception in drosophila, and dependence only on light less than 420nm casts doubt on the idea that an intra-protein amino acid is the final electron donor, since 450nm light is required to cause electron transfer to a Trp or Tyr residue (3). Studies on Cry sensitivity to magnetic fields have largely carried out on bacterial and plant Cry, which have been shown to have significant differences in the radical states of the FAD cofactor than animal Cry (12). With the exception of (13), studies have animal Cry have been limited to computer modeling.

Research Design and Specific Aims

In this study, we will identify and characterize the components involved in cryptochrome magnetoreception with cw-EPR and tr-EPR to develop a reasonable reaction scheme. We will then test the effects of a magnetic field on the reaction using TAS.

Specific Aim 1: Identify reaction components, characterize redox states, and develop a reaction scheme

To fully understand the mechanism behind magnetoreception in cryptochrome, we must be able to directly measure the effect of an applied magnetic field. We intend to characterize a pair of radicals that are created by a photo-induced electron transfer event. Therefore, it makes sense to use two techniques especially suited for studying light and unpaired electrons: absorption spectroscopy and electron paramagnetic resonance (EPR). Continuous wave EPR can be used to determine the exact redox states and identities of the molecules containing each electron of the radical pair (4). To study the relatively shorter-lived states created by the electron transfer, time resolved EPR can be used, also as described in (4). Once a reasonable reaction scheme is established, we can then study the effect of various strength magnetic fields on the reaction.

**Figure 2**. An example of the type of reaction scheme that will be developed. (8)

Specific Aim 2: Test the effects of an applied magnetic field on the reaction

Once a suitable reaction scheme is developed, we can then test the effects of the magnetic field in the reaction. Since FAD absorbs different wavelengths of light depending on its oxidation state, changes in the concentration of FAD in each state can be measured with absorption spectroscopy (5). By using time-resolved transient absorption spectroscopy, as was done on the Cry ortholog photolyases in (5), we can confirm our reaction scheme. Further, by applying a magnetic field while the reaction is happening, we can test the effects of the applied field on the equilibrium and kinetics of the reaction. Instead of testing the reaction at just one strength of magnetic field, as has been done in the past, we instead to test at several different strengths, making sure to include a field of 50 μT.

List of References

1. Rodgers, C. T., & Hore, P. J. (2009). Chemical magnetoreception in birds: the radical pair mechanism. Proceedings of the National Academy of Sciences,106(2), 353-360.
2. Solov'yov, I. A., & Schulten, K. (2009). Magnetoreception through cryptochrome may involve superoxide. Biophysical journal, 96(12), 4804-4813.
3. Gegear, R. J., Foley, L. E., Casselman, A., & Reppert, S. M. (2010). Animal cryptochromes mediate magnetoreception by an unconventional photochemical mechanism. Nature, 463(7282), 804-807.
4. Weber, S., Kay, C. W., Mögling, H., Möbius, K., Hitomi, K., & Todo, T. (2002). Photoactivation of the flavin cofactor in Xenopus laevis (6–4) photolyase: observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance. Proceedings of the National Academy of Sciences, 99(3), 1319-1322.
5. Henbest, K. B., Maeda, K., Hore, P. J., Joshi, M., Bacher, A., Bittl, R., … & Schleicher, E. (2008). Magnetic-field effect on the photoactivation reaction of Escherichia coli DNA photolyase. Proceedings of the National Academy of Sciences, 105(38), 14395-14399.
6. Solov’yov, I. A., Domratcheva, T., Moughal Shahi, A. R., & Schulten, K. (2012). Decrypting Cryptochrome: Revealing the Molecular Identity of the Photoactivation Reaction. Journal of the American Chemical Society, 134(43), 18046-18052.
7. Ritz, T., Adem, S., & Schulten, K. (2000). A model for photoreceptor-based magnetoreception in birds. Biophysical journal, 78(2), 707-718.
8. Maeda, K., Robinson, A. J., Henbest, K. B., Hogben, H. J., Biskup, T., Ahmad, M., … & Hore, P. J. (2012). Magnetically sensitive light-induced reactions in cryptochrome are consistent with its proposed role as a magnetoreceptor.Proceedings of the National Academy of Sciences, 109(13), 4774-4779.
9. Foley, L. E., Gegear, R. J., & Reppert, S. M. (2011). Human cryptochrome exhibits light-dependent magnetosensitivity. Nature communications, 2, 356.
10. Solov'yov, I. A., Mouritsen, H., & Schulten, K. (2010). Acuity of a cryptochrome and vision-based magnetoreception system in birds. Biophysical journal, 99(1), 40-49.
11. Vieira, J., Jones, A. R., Danon, A., Sakuma, M., Hoang, N., Robles, D., … & Ahmad, M. (2012). Human cryptochrome-1 confers light independent biological activity in transgenic Drosophila correlated with flavin radical stability. PloS one,7(3), e31867.
12. Öztürk, N., Song, S. H., Selby, C. P., & Sancar, A. (2008). Animal Type 1 Cryptochromes: Analysis Of The Redox State Of The Flavin Cofactor By Site-Directed Mutagenesis. Journal of Biological Chemistry, 283(6), 3256-3263.
13. Biskup, T., Paulus, B., Okafuji, A., Hitomi, K., Getzoff, E. D., Weber, S., & Schleicher, E. (2013). Variable Electron-Transfer Pathways in an Amphibian Cryptochrome: Tryptophan versus Tyrosine-Based Radical Pairs. Journal of Biological Chemistry.
14. Lau, J. C., Rodgers, C. T., & Hore, P. J. (2012). Compass magnetoreception in birds arising from photo-induced radical pairs in rotationally disordered cryptochromes. Journal of The Royal Society Interface, 9(77), 3329-3337.

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