Certain proteins, known as photoreceptor proteins, are sensitive to light and undergo reversible light-induced conformational changes to effect basic intracellular signalling pathways or gene regulation in response to it. For instance, UVR8, a photoreceptor protein for UV-B light, is a homodimer in the dark that dissociates into monomers upon absorption of a UV-B photon. Interestingly, the UVR8 C-terminal tail is thought to extend from the protein bulk (core) structure after UV-B-induced monomerisation and associated conformational changes, promoting its interaction with a partner protein known to be a major factor in UV-B signalling. Another photoreceptor, transcription factor CarH, assembles into a homotetramer in the dark following adenosylcobalamin (AdoCbl) binding. Exposure to light (green, in particular) leads to tetramer disassembly. UVR8 uses an intrinsic cluster of tryptophan residues at the dimer interface as the light-sensing chromophore, whereas CarH uses AdoCbl. Due to their light-induced conformational changes, these photoreceptor proteins can be exploited as the core components of photoswitches and optogenetic tools, which can be used in several science fields. In order to investigate their natural functions, and to develop and optimise these proteins as tools, their structural changes must be understood. For that, a technique that both maintains proteins in their native form and that allows distinguishing the different conformations they undergo is required. This thesis presents data from novel, native photo-initiated ion mobility-mass spectrometry approaches that have been successfully tested with both UVR8 and CarH, and have provided valuable new insight into their light-induced structural changes. Fluorescence stopped-flow investigations were also conducted. Results show that core UVR8 dimer exists in a single conformational state in the dark, and cleanly dissociates into one monomer conformation upon UV-B activation. In contrast, full-length UVR8 exists in two conformational states in the dark, compact and extended conformations. Upon photoactivation, the two dimeric conformations dissociate into compact and extended monomer conformations, respectively. These results suggest that it is the presence of the terminal tails that give rise to the two different conformational states in the dark, and that these depend on whether the terminal tails are folded close to the core of the protein (compact conformation) or are more disordered (extended conformation). Regarding CarH, the stopped-flow fluorescence results suggest that binding of AdoCbl occurs at the same rate in the WT and the G192Q variant (this mutation impairs tetramerisation, with the variant only assembling into a dimer). Dimer formation also appears to occur at the same rate in both proteins, which suggests that the WT tetramerisation event does not affect the rate of dimerisation, and thus must occur after it. A 1â2â4 (monomer to dimer to dimer-of-dimers) mechanism for WT assembly following AdoCbl binding is therefore proposed. The MS results, in turn, show that AdoCbl-bound CarH dark state encompasses two tetramer conformations: a homotetramer, and a tetramer comprising three AdoCbl-bound CarH monomers and one AdoCbl-bound CarH monomer lacking its DNA-binding domain. The redundancy of the fourth DNA-binding domain suggests cooperativity between the other three DNA-binding domains. Depletion of both tetramers with concomitant appearance of Cbl-bound CarH monomer adducts after photoconversion confirms AdoCbl-bound photolysis and consequent tetramer dissociation.
|Date of Award||1 Aug 2018|
- The University of Manchester
|Supervisor||Sam Hay (Supervisor), Alex Jones (Supervisor) & Perdita Barran (Supervisor)|