| Summary: | <p>Avian magnetoreception, the ability of birds to perceive and navigate using the Earth’s magnetic field, is a phenomenon whose basis has intrigued scientists for decades. One leading hypothesis is the radical pair mechanism (RPM) which has been proposed as a possible magneto-sensing mechanism in biological systems such as the magnetoreception of night migratory songbirds (<strong>Chapter 1</strong>). In particular, the retinal cryptochrome 4a proteins of these birds are believed to exhibit the key features necessary for magnetosensing. That is, the formation of photoinduced radical pairs (RPs) whose interaction with magnetic fields, due to their quantum spin dynamics, causes variations in the yields of their photoproducts, known as a magnetic field effect (MFE). The quantum mechanics of spin, the RPM and its manifestation in cryptochrome 4a proteins is introduced in <strong>Chapter 2</strong>.</p>
<p>The RPM in cryptochrome 4a proteins occurs over a time scale spanning several orders of magnitude (ps - min) with the photoproducts formed exhibiting significant spectral overlap in their absorption (<strong>Chapter 2</strong>). Consequently, a variety of spectroscopic techniques are required to probe the entirety of the photocycle. Furthermore, the MFEs generated in these proteins tend to be small and as such require specialised sensitive spectroscopic techniques to reliably detect them. One such set of techniques are optical cavity experiments which provide significant sensitivity gains over traditional single-pass measurements. These methods, in particular the technique known as broad-band cavity enhanced absorption spectroscopy (BBCEAS), form the bulk of experimental results in this thesis and are introduced in <strong>Chapter 3</strong>.</p>
<p>Thanks to recent advances in the expression of cryptochrome 4a proteins, their photochemistry (<em>in vitro</em>) and efficacy as a potential magnetoreceptor can now be explored. In <strong>Chapter 4</strong> the ability of these proteins to develop MFEs is demonstrated for a variety of avian species prior to exploration, in <strong>Chapters 6</strong> and <strong>7</strong>, of the role of particular amino acid residues which are highly evolutionarily conserved in the night-migratory passerines.</p>
<p>Historically, the use of the reoxidant potassium ferricyanide (FC) has been used in the spectroscopic study of proteins (in <strong>Chapter 4</strong> for example) to aid in sample recovery following irradiation. <strong>Chapter 5</strong> presents a study on the photochemistry of FC itself and highlights some potential problems it can cause when studying the RPM in cryptochrome 4a. Furthermore, <strong>Chapter 5</strong> demonstrates that MFEs in these proteins can be studied using the more biologically relevant molecular oxygen as an alternative reoxidant.</p>
<p>Due to the small MFEs produced in crpytochrome proteins, their relatively poor stability and time-consuming and costly expression, new techniques with improved sensitivity and which allow faster data acquisition or reduced sample consumption are highly sought after. Therefore, in a shift of focus, <strong>Chapters 8</strong> and <strong>9</strong> introduce two newly developed techniques, optical cavity enhanced transient absorption spectroscopy (OCTAS) and microdroplet fluorescence magnetically altered reaction yield (μDF-MARY) spectroscopy. The former offers improved sensitivity and spectral resolution in absorption measurements over the ns-μs time scale while the latter significantly reduces the amount of sample required to measure continuous fluorescence MFEs and minimises laser induced sample degradation.</p>
|
|---|