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Mechanistic and structural studies of Light-Oxygen- Voltage (LOV) domain proteins

Kalvaitis, Mindaugas Edvardas 2019. Mechanistic and structural studies of Light-Oxygen- Voltage (LOV) domain proteins. PhD Thesis, Cardiff University.
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In this work, structural and mechanistic understanding of LOV domain proteins has been provided by applying biophysical techniques including protein X-ray crystallography, nuclear magnetic resonance (NMR) and circular dichroism (CD). LOV domains are photosensory modules where exposure to blue light triggers the formation of a reversible cysteinyl-flavin covalent photoadduct. The formation of this chemical bond has been studied extensively, but how this covalent adduct governs residue rearrangements within the flavin binding pocket and the allosteric regions of the LOV domain core remained elusive. By capturing Ochromonas danica Aureochrome1a LOV (OdAu1aLOV) domain in three distinct states, structural evidence for a novel residue rearrangement has been provided. X-ray crystal structures of dark, illuminated (where dark grown crystals were photoactivated prior crystal harvesting) and light grown OdAu1aLOV revealed three distinct conformations of a highly conserved glutamine 293 residue. Reflecting on the three distinct states, a mechanism proposing how this residue links the chromophore binding pocket to the allosteric A’a helix has been proposed. This is the first record of glutamine 293 swinging away from the flavin binding pocket being captured by X-ray crystallography. This transient swing changes the hydrogen bond network formed upon photoadduct formation and could be crucial to facilitate large scale structural changes. This key glutamine residue is highly conserved in other LOV domain proteins, hence understanding the transition between these three distinct conformations provides novel insights to assist the rational design of novel optogenetic tools. Furthermore, native flavin mononucleotide (FMN) cofactor was replaced with 5- deazaflavin mononucleotide (5dFMN) analogue to understand OdAu1aLOV structural changes further. The current standing hypothesis suggests that the formation of the covalent cysteinyl-FMN photoadduct upon illumination results in the protonation of the FMN-N5. It has been suggested that this protonation state change, from unprotonated in the dark state and protonated in the lit state, triggers structural changes via a conserved glutamine residue. Considering that 5dFMN is a structural FMN analogue where N5 is replaced with a carbon (C5), no protonation is possible in this position and 5dFMN should be inactive if N5 protonation is crucial for the activation. Upon photoactivation of 5dFMN OdAu1aLOV, a thermally stable cysteinyl-5dFMN covalent adduct was generated and highresolution crystal structures provide conclusive evidence that, as for FMN, this adduct forms at the C4a position also triggering some residue rearrangements within the flavin binding pocket. Nonetheless, CD and NMR spectra revealed only partial light IV responsiveness, yet no major structural changes were evident for 5dFMN reconstituted OdAu1aLOV post photoactivation. These results therefore suggest that N5 protonation is important to lock the protein in the lit state conformation. To probe functionality of 5dFMN further, the cofactor was introduced into Avena sativa LOV2 (AsLOV2), another LOV domain protein. Similarly to OdAu1aLOV, partial light responsiveness was evident by CD studies suggesting that 5dFMN displayed light dependent structural changes. This was also studied further by 19F NMR through labelling AsLOV2 with 5-fluoro-L-tryptophan, a fluorine (19F) probe, allowing direct observation of Ja unfolding. NMR results suggested that 5dFMN AsLOV2 experienced changes in the cofactor chemistry including interruption of conjugated isoalloxazine ring system, which from these observations was hypothesised to contribute in achieving the active lit state conformation. Finally, conformational changes of OdAu1aLOV were studied by 19F NMR in order to investigate the importance of allosteric A’a and Ja helices in achieving the lit state conformation. Results highlighted that the removal of A’a results in insoluble protein, indicating that this auxiliary helix is crucial, whilst the truncation of the Ja helix resulted in a functional protein, indicating that this extension is not required.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Chemistry
Date of First Compliant Deposit: 1 June 2020
Last Modified: 08 Jun 2021 08:49

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