Investigations into the membrane architecture of cyanobacterial thylakoid membranes and nanopatterning of cyanobacterial photosystems

Synechocystis sp. PCC6803 is a cyanobacterium used as a model organism to study photosynthesis. Thylakoid membranes are specialised invaginated areas of membrane that are enriched in the light harvesting antenna complexes and reaction centres. Energy is directed towards PSI and PSII by the phycobilisome complexes to drive charge separation. The native organisation of the photosynthetic complexes in cyanobacterial thylakoid membranes is still relatively unclear.

Procedures were developed to isolate and treat thylakoid membranes from Synechocystis to make them suitable for AFM imaging. Methods to remove contaminating material from thylakoid membranes were trialled and assessed by EM and AFM. Different approaches were used to induce the fragmentation of thylakoid membranes to produce flat, single layered lipid bilayers that were ideal for AFM imaging.

To determine the supramolecular organisation of photosynthetic protein complexes in cyanobacterial thylakoid membranes; AFM was used to image the membrane fragments produced by the methods that have been developed in this study. It was possible to identify protein complexes present in thylakoid membrane fragments initially from Synechocystis and subsequently from Thermosynechococcus elongatus. In membrane fragments from both cyanobacteria it was possible to identify PSII complexes in addition to complexes with lower topology than PSII that had dimensions consistent with the cytochrome b6f complex. In membrane fragments from T.elongatus it was possible to image PSI; which was found in densely packed, highly ordered arrays which have not previously been reported. Membrane fragments from T.elongatus were also imaged which contained PSI in a more disordered organisation.

In light of recent advances in lithographic techniques; it is now possible to produce nanopatterns of immobilised photoactive protein complexes. Such nanopatterns can be used to investigate the functionality and the energy transfer properties of immobilised protein complexes. When Synechocystis is grown in low iron conditions the PSI trimer forms a complex with 18 copies of the IsiA protein called the IsiA-PSI supercomplex. Methods for producing a highly purified preparation of this complex for the purposes of nanopatterning were developed and the purified IsiA PSI supercomplex was analysed by EM and AFM. Purified IsiA-PSI supercomplexes from Synechocystis and PSII complexes from Thermosynechococcus elongatus were immobilised on nanopatterns produced using reverse nanoimprint lithography. The binding specificity of protein complexes to the nanopatterns was determined using AFM. The dimensions of the nanopatterns were assessed with fluorescence microscopy and the spectroscopic properties of the immobilised complexes were investigated using fluorescence emission spectroscopy and fluorescence lifetime imaging. The fluorescence emission spectrum and the measured fluorescence lifetime of immobilised PSII complexes was comparable to that of active PSII complexes in solution. The fluorescence emission spectrum of immobilised IsiA-PSI supercomplexes was consistent with that of IsiA-PSI supercomplexes in solution. The measured fluorescence lifetime of the immobilised supercomplex was however significantly longer than that of supercomplexes in solution.