Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.634971
Title: The photobiology of cyanobacteriochrome Tlr0924
Author: Hauck, Anna
ISNI:       0000 0004 5353 556X
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2015
Availability of Full Text:
Access from EThOS:
Access from Institution:
Abstract:
Several different classes of photoreceptors have evolved to support function of organisms in all kingdoms of life. Cyanobacteriochromes (CBCRs) are photoreceptors unique to cyanobacteria and have been an area of great interest in recent years. Analogously to the phytochrome superfamily, they harbour a covalently ligated, linearised bilin chromophore, which undergoes light-driven E/Z isomerisation around the C15=C16 double bond to convert between a ground state and a photoactive state. CBCRs have developed unique tuning mechanisms for their spectral sensitivities and collectively cover the entire visible region. The CBCR Tlr0924 of Thermosynechoccocus elongatus belongs to a blue/green light sensitive subgroup with a conserved cysteine in a DXCF motif. This residue is involved in the transient formation of a second thioether linkage with the chromophore, which is vital to the formation of the blue-absorbing state and hence the functionality of the protein. Full-length Tlr0924 protein has been subjected to detailed spectroscopic characterisation in this thesis in order to provide a comprehensive understanding of the timescales and mechanisms of the molecular processes that take place during the photocycle of this CBCR. Initial stationary spectroscopic techniques confirmed the presence of two chromophore populations with blue ground state absorbance (Pb) and green- (Pg, phycoviolobilin) or red light (Pr, phycocyanobilin) sensitive photoproduct states. Photoproduct states also absorbed blue light according to the S0 to S2 transitions. Pb, Pg and Pr showed high thermal stability with only low dark reversion rates of Pg and Pr to Pb, and slow phycocyanobilin (PCB) to phycoviolobilin (PVB) isomerisation. The importance of the second thioether linkage was demonstrated by chemical modification of the cysteine, which prevented Pb state formation. Instead, illumination revealed a photoisomerised PVB reaction intermediate at 564 nm. The photoconversions were subsequently studied in greater detail by employing time-resolved absorbance spectroscopy techniques in the visible- and infrared spectral range and low-temperature trapping techniques. Pb and Pg states were demonstrated to photoisomerise to their respective blue- and red-shifted intermediates within picoseconds. In the infrared region of the spectrum this transition was accompanied by a shift of the D-ring C=O stretching mode, representative of the altered hydrogen bonding environment after the D ring flip. These isomerisations proceeded at cryogenic temperatures implying only local structural changes are involved. In the Pb to Pg/r activation reaction no further intermediates were resolved across the ps to s time scales. The isomerised chromophore was observed to lose the second thioether linkage with a life-time of 937 ms for PVB and 3.1 s for PCB. Characterisation of the reverse reaction mainly focussed on the PVB chromophore due to low signal-to-noise ratios in PCB data. The photoreaction concluded faster (23.6 ms) than the forward reaction despite proceeding via an additional red-shifted intermediate (5.3 μs), which corresponded to the species that was chemically trapped previously. Since photoproduct formation for both chromophores was only possible above 220 K, it could be concluded that larger protein domain motions were required in the process. This work therefore shows unprecedented detail in the characterisation of a CBCR photoreceptor. It fills gaps especially regarding secondary reactions whilst complementing data on the truncated protein.
Supervisor: Not available Sponsor: BBSRC ; TgK Scientific
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.634971  DOI: Not available
Share: