Award details

Light controlled photosynthesis in the metabolically versatile bacterium Rps palustris

Reference	BB/E002609/2
Principal Investigator / Supervisor	Dr Miroslav Papiz
Co-Investigators / Co-Supervisors	Dr Anthony Fordham-Skelton, Dr Theonie Georgiou
Institution	University of Liverpool
Department	Sch of Biological Sciences
Funding type	Research
Value (£)	334,191
Status	Completed
Type	Research Grant
Start date	09/03/2009
End date	08/04/2011
Duration	25 months

Abstract

Bphs and CBD protein samples will be produced by over expressing in E coli. Protocols have been developed to purify these proteins. LH4 is prepared from Rps. palustris cells grown under low-light conditions and harvested from photosynthetic membranes. pucAB-a deletion mutants, that are devoid of LH2 contaminating complex, will be used to further improve the purity of the sample Crystallisation trials of Bphs, CBD and LH4 will be undertaken using the Screenmaker 96+8 crystallisation robot. Customised crystallisation screens will be made with a Hamilton STARlet liquid handling robot. X-ray diffraction data will be collected at the SRS or DLS and structures determined using MAD or MR techniques. SAXS data will be collected on Bph4, Bph3 and CBD at the SRS station 2.1. Data will be collected in the Pr and Pfr states established by illuminating the samples with red or far-red light. Time resolved spectroscopy data will be measured on Bphs, CBD and LH4 at the CCLRC Central Laser Facility and preliminary THz measurements will be carried out at the CCLRC ERLP when it is available. 2D-Spectroscopy will be carried out at the University of California, Berkeley in collaboration with Prof. G. Fleming.A collaboration with Prof. J.T. Beatty and Prof. J. C. Liao will look at Bphs and response regulator interactions that control photosynthesis. WT and deletion mutants will be used for spectroscopic characterisation and RNA microarray analysis to correlate patterns of gene expression. Phenotypes will be characterised under different light growth conditions by near IR absorption spectroscopy of whole cells monitoring the 800 to 850 nm absorption ratio as an indicator of LH4 expression. For more detailed determination of the proportions of light-harvesting complexes, peptides isolation by HPLC, will be identified by N-terminal sequencing or by ESI-MS analysis. An arrangement has been made to use the ESI-MS facility at the Biosciences Department in Liverpool University.

Summary

The bacterium Rps. palustris is a metabolically versatile photosynthetic organism that can assimilate a variety of carbon sources including the greenhouse gas CO2 and the important plant polymer lignin. It is therefore important for understanding how carbon is recycled in the environment. It is also of interest as a source of biofuels. The bacterium has light-harvesting complexes that capture solar energy and which are designed to be optimal in the near IR between 800-880 nm. The bacteria can experience low-light conditions because the water they live in attenuates significantly in the near IR and because of light absorption by other photosynthetic organisms. It has an unusual light-harvesting complex (LH4), that is expressed under low-light conditions, with unique spectroscopic properties. The organism is also unusual in containing 6 bacteriophytochromes which are two-component systems that are driven by light and control gene expression. Three of the Bphs have been shown to be involved in controlling LH4 expression while Bph3 is responsible for controlling a large cluster of genes responsible for photosynthesis. These include LH1/RC peptides and several enzymes responsible for the biosynthesis of chromaphores. Bphs contain a biliverdin chromaphore that undergoes a Z to E isomerisation on red light illumination producing the Pfr form which reverts to the Pr state on far-red light illumination. The movement is transduced through the PHY domain which changes the efficiency of autophosphorylation in the histidine kinase domain and also affects the rate of release of the phosphate to an Asp residue of a response regulator. Bph3 has a very different C-terminal domain which does not involve phosphorylation and is believed to directly compete with the DNA for binding of the repressor PpsR2. Three spectroscopically active Bphs and a reduced protein containing only the chromaphore binding domain (CBD) have been expressed and characterised. A low resolution structure of LH4has been determined and the complex has been characterised by near IR absorption, CD and single molecule spectroscopy techniques. High resolution structures of Bphs and LH4 are required to understand the details of biological action. In particular how light is being used for signalling and at the same time as a source of energy to optimally photosynthesise in low-light conditions. Light induced shape changes will be investigated by Small Angle X-ray Scattering and the dynamics of light-transduction and energy transfer will be characterised by time resolved spectroscopy and interpreted in the light of the structure. One of the objectives of using time resolved spectroscopy techniques is to understand the requirements of ultra fast IR, Raman and 2D-Spectroscopy methods and the potential of these methods on fourth generation light sources such as 4GLS.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

Associated awards:

BB/E002609/1 Light controlled photosynthesis in the metabolically versatile bacterium Rps palustris

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