| Summary: | <p>The ability of migratory birds to sense and use the Earth’s magnetic field during migration is hypothesised to involve cryptochrome proteins located in their retinae. It is proposed that photo-initiated reactions within these proteins generate radical pairs (RPs) as transient intermediates, which have product yields that are sensitive to the magnitude and direction of the external magnetic field, and ultimately allow birds to visualize the geomagnetic field. Cryptochromes are currently the only candidate chemical compass magnetoreceptor for this radical pair mechanism (RPM)- based hypothesis (Chapter 1) and thus it is important to take steps towards more fully understanding the photochemistry and photosignalling mechanisms occurring.</p>
<p>This thesis has two main themes. Firstly, a highly sensitive fluorescence-based spectroscopic technique has been developed to further increase sensitivity and make it more suitable for studying precious biological samples. Whilst many (absorption- based) spectroscopic techniques have investigated magnetic field effects (MFEs) on isolated cryptochrome proteins, these experiments have typically been performed in vitro and thus are still very far removed from biological conditions. This thesis describes the first attempts towards studying cryptochromes in living cells and thus observing an MFE in vivo (Chapter 2). A very small, negative MFE is observed, however this was attributed to the RP chemistry occurring in the growing medium rather than the cryptochromes. The exquisite sensitivity of the experiment is still highlighted, however, and the re-designed fluorescence technique is further used to explore the photochemistry of RPs in restricted environments (micelles) to investigate how altering the RP environment can affect the photochemistry of a system (Chapter 3). A negative MFE is observed in a thionine/DABCO system which is vastly increased when the system is constricted to an SDS micelle. The relationship between quencher concentration and MFE was shown to be biphasic, with dynamic quenching changing to static quenching with increasing DABCO concentration, and the importance of the micelle environment was demonstrated as altering the size or charge of the micelle affected the MFE.</p>
<p>Secondly, the relationship between the photochemistry and signal transduction mechanism in cryptochromes has been explored. Currently, very little is known about the photosignalling pathway in cryptochromes (specifically animal cryptochromes), however one widely held model proposes the involvement of a light-induced conformational change which initiates the signalling cascade. By using a technique known as Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), a blue-light induced conformational change has been observed on DmCry WT and been localised to a region of the protein known as the C-terminal tail (CTT). Further, by comparing the behaviour of DmCry WT with that of its mutants, DmCry W342F and W394F, in which the electron transfer (ET) chain is interrupted at the third and fourth tryptophan position respectively (by replacing the tryptophan with a redox inert phenylalanine), altering the photochemistry has been shown to have a direct effect on the magnitude of the conformational change, highlighting the importance of these tryptophans for the signalling state of the protein (Chapter 4).</p>
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