Characterization Of Light-Signaling In The Circadian Photoreceptor Proteins Vivid And Cryptochrome

dc.contributor.authorVaidya, Ananden_US
dc.contributor.chairCrane, Brianen_US
dc.contributor.committeeMemberEalick, Steven Edwarden_US
dc.contributor.committeeMemberCerione, Richard Aen_US
dc.description.abstractBiological clocks are ubiquitous in nature; observed in almost all life forms. The role of clock is not only to synchronize the various biological activities of an organism, but also to synchronize the organism to the day/night cycle and gain the most out of sunlight. At the molecular level, a negative feedback loop is the basis of eukaryotic clocks and light triggers activity within the feedback loop affecting either the negative or the positive arm of the loop. In the current study, both these modes of light input to the clock have been investigated. Vivid (VVD) is a photoreceptor protein in the fungus Neurospora crassa and is important for photoadaptation in continuous light. VVD senses light via a flavin cofactor incorporated in a light, oxygen and voltage sensing (LOV) domain. Light induces the formation of a covalent bond between the flavin and a conserved cysteine, which causes conformational changes with VVD that lead to its dimerization. This dimer is the signaling state of VVD and it was crystallized in this study using a Met135I:Met165I variant that increases the lifetime of the light state. The structure reveals the mechanism of light-signaling where in light induced bond formation alters hydrogen bond pattern, leading to the release of the N-terminal loop of the protein, which ultimately docks into the other subunit to form the dimer. The dimeric structure was confirmed in s olution and in the organism. Based on similarities in sequence alignments, this mechanism appears to be conserved among all fungal LOV proteins. Cryptochrome (CRY) is the principal photoreceptor in the fruit fly Drosophila melanogaster that senses light via a flavin cofactor. Determination of the conformational changes associated with light exposure and the role of the flavin redox state in these conformational changes was the aim of the investigation. A trypsin based limitedproteolysis assay was developed to monitor the light state conformation of CRY. This assay in combination with mass spectral analysis, kinetic studies and mutational analysis reveals structural rearrangements in and around the C-terminal extension, which are strongly coupled to the light-induced redox changes of the flavin. Chemically reducing CRY in the dark was sufficient to replicate the structural rearrangements observed in the presence of light, which strongly indicates that the role of light is to reduce the flavin.en_US
dc.identifier.otherbibid: 7959684
dc.subjectcircadian clocken_US
dc.subjectLOV domainen_US
dc.subjectlight sensingen_US
dc.titleCharacterization Of Light-Signaling In The Circadian Photoreceptor Proteins Vivid And Cryptochromeen_US
dc.typedissertation or thesisen_US and Chemical Biology Universityen_US of Philosophy D., Chemistry and Chemical Biology


Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
4.59 MB
Adobe Portable Document Format