Supramolecular Structure, Function And Physiological Acclimation Of Light-harvesting Complex And Photosynthetic Membrane In Red Algae

Phycobilisomes(PBsomes)are the major light-harvesting antennae complexes in cyanobacteria and red algae.They are aggregations of water-soluble phycobiliproteins(PBPs)and linker polypeptides,and serve as external antenna macrocomplexes associated to the stromal surfaces of thylakoid membranes.PBPs are a distinctively colored group of disk-shaped macromolecular proteins bearing covalently attached open-chain tetrapyrroles,known as phycobilins(bilins),orderly assembled into PBsomes.Four spectral groups of PBPs are commonly identified: phycoerythrins(PEs),phycocyanins(PCs),allophycocyanins(APCs)and sometimes phycoerythrocyanins(PECs).In the absence of photosynthetic reaction centers(RCs), the PBsomes are highly fluorescent.Solar energy is initially absorbed by the pigments of PEs(λ_max=545～565 nm)and transferred by nonradiative transfer in turn via PCs (λ_max=620 nm),APCs(λ_max=650 nm),and eventually to chlorophylls(Chls)with a high efficiency.In this thesis,I will present the detailed investigations on the properties of PEs, the spectral feature and the topography of PBsomes,the supramolecular architecture and photoacclimation of entire photosynthetic membrane in red algae using microscopic imaging in nano scale,the diffusion dynamics of PBsomes upon the thylakoid membrane,and the fluorescence dynamics of PBsomes at single molecule level.In addition,the packing organization of LH2s,the light-harvesting complexes from photosynthetic bacterium Rhodobacter sphaeroides,in artificially created 2D crystals is characterized.Introduction of photosynthesisPhotosynthesis is an essential conversion of solar light to biological energy in photosynthetic organisms.This highly efficient process starts from the light capturing by light-harvesting antenna of photosynthetic RCs.In Chapter 1,I provide a general introduction about the biological roles,diversity and evolution of photosynthesis. Then I focus on the photosynthesis in cyanobacteria and red algae,and their photosynthetic elements including the PBsomes,PSs,Cyt b₆f complexes and ATPase. In particular,the studies of light-harvesting antenna complexes,the PBsomes,are overviewed,consisting of its components,spectral properties,structures,and protein-protein interactions.The interactions of individual photosynthetic complexes, as well as the supramolecular architecture and the physiological photoacclimation of the overall thylakoid membrane network are summarized.Isolation of pure PEsPEs have been widely used in food,cosmetics,immunodiagnostics and analytical reagents.An efficient one-step chromatography method for purification of R-PEs from Polysiphonia urceolata was described in Chapter 2.Pure R-PEs were obtained with an absorbance ratio A565/A280 of 5.6 and a high recovery yield of 67.33%using a DEAE-Sepharose Fast Flow chromatography with a gradient elution of pH,alternative to common gradient elution of ionic strength.Such an effective methodology greatly reduces the traditional processing steps as well as the possibility of protein loss and denaturation during the overall operation,and a high recovery could thus be obtained.The absorption spectrum of R-PE was characterized with three absorbance maxima at 565 nm,539 nm and 498 nm,respectively.The fluorescence emission spectrum at room temperature was measured to be 580 nm.The results of native-PAGE,and SDS-PAGE showed no contamination by other proteins in the PE solution,which suggests an efficient method for the separation and purification of R-PEs from P urceolata for further accurate analysis.Active conformation and function of R-PEsX-ray crystallography of proteins has revealed high-resolution peptide conformations and amino acid organizations.However,investigations on the structural and functional stability of proteins in response to the environmental variations are limited in terms of this methodology.On the basis of the previous developed separating methodology of R-PEs,in Chapter 3,we explore the pH-induced conformational and functional dynamics of R-PEs isolated from P urceolata.The spectroscopic and structural variations of R-PEs monitored by means of absorption,fluorescence and circular dichroism(CD)spectra are investigated, together with analysis of the crystal structure of R-PE.R-PEs present a spectroscopic stability in pH range between 3.5 and 10,and relative structural sensitivity in pH range between 5 and 9,in response to the pH variations.Structural analysis allows us to better understand the assembly pattem of R-PE complexes.The tertiary structure of R-PE hexamer is fixed by specific interactions between several key anchoring residues,providing a stable protein environment for the chromophores to perform physiological energy migration.Local flexibility of protein peptide arrangement is allowed in response to the environmental disturbance.Our data further reveal that the charged amino acids and aromatic amino acid residues are highly involved in the association of R-PE complex.More specifically,aromatic amino acids,especially Tyr residues,are found to be capable to modify the interprotein energy transfer by close contacts with neighboring chromophores.This study combining analysis on the available crystal structure with active structural and functional investigations will provide new insights into the conformation and function of protein of interest,in addition to R-PEs.Single-particle structural inspections on the PBsomesThe structure of PBsomes from Porphyridium cruentum has been studied before with electron microscopy(EM).In Chapter 4,EM combining with single particle averaging was performed for the first time to investigate the supramolecular architecture of PBsomes from P.cruentum.Isolated PBsomes are found to have a relatively flexible conformation.In contrast,PBsome-thylakoid vesicles provide relatively uniform PBsome structure,and allow us to acquire a spatial view of hemiellipsoidal structure.A three-dimensional model of the hemiellipsoidal PBsome is proposed.Under low-light growth conditions,the PBsomes on the membrane are mostly arranged in ordered domains.Whereas at higher light intensities,the distribution of PBsomes is largely disordered.It is the first time to observe the variety of PBsome arrangements upon isolated thylakoid membranes.We suggest that one PBsome likely lines up with one PSII dimer in red algae under low-light conditions is hypothesized because the red algal PSâ…¡is enlarged by a possible membrane-bound peripheral antenna which is absent in cyanobacteria.Native architecture and dynamics of thylakoid membrane of red algaeThe architecture of the entire photosynthetic membrane network determines,at the supramolecular level,the physiological roles of the photosynthetic protein complexes.So far,a precise picture of the native configuration of red algal thylakoids is still lacking.In Chapter 5,we investigate the supramolecular architectures of native thylakoid membranes from red alga P.cruentum,for the first time,using atomic force microscopy(AFM).The topography of individual PBsomes is characterized to be spatially hemiellipsoidal.Furthermore,the native organization of thylakoid membranes presented variable arrangements of PBsomes,either a random arrangement,or rather ordered arrays of PBsomes,depending on light conditions.In particular,PBsomes were organized crowdingly in both cases.The packing of PBsomes is studied to determine not only the organizations of PBsomes,but also those of PSs in the thylakoid membrane.Furthermore,such crowding effects may restrict the large-scale lateral mobility of PBsomes on the surface of thylakoids. The dynamics of PBsomes studied using fluorescence recovery after photobleaching(FRAP)The lateral mobility of PBsomes on the surface of thylakoid membranes in cyanobacteria has been proposed.However,the structural inspections imply that the rapid diffusion of PBsomes may be greatly inhibited upon the crowding membrane surface.In Chapter 6,we examine for the first time the dynamic of photosynthetic membrane in red alga P cruentum with FRAP.Our data obtained from native cell showed the existence of partial fluorescence recovery,similar to that visualized in cyanobacteria.However,FRAP also occurs in the glutaraldehyde(GA)-fixed cell in vivo and ensemble PBsomes in vitro.Therefore,FRAP of red algal cell is ascribed to an intrinsic photophysics of the bleached PBsomes in situ,rather than the rapid diffusion of PBsomes on thylakoids in vivo,which has been proposed to be involved in excitation energy redistribution between photosystemâ… (PSI)and photosystemâ…¡(PSâ…¡).There should be other mechanisms for the PBsomes-related energy redistribution in red algae.In addition,we selectively monitor the fluorescence of PE instead of that of the entire PBsome in FRAP.The results of in vivo and ensemble experiments show that the bleaching laser applied in FRAP could result in the fluorescence increase of PE.Furthermore,the comparative data from GA-treated PBsomes and cells elucidate the energetic decoupling of PEs in the PBsome rods.Due to this decoupling,part of the fluorescence of PBsomes is dissipated from PE.It can presumably explain the partial fluorescence recovery observed in FRAP.Single-molecule spectroscopic study on isolated PBsomesAccording to the ensemble results,in Chapter 7,single-molecule spectroscopy is applied for the first time on the PBsomes of red alga P.cruentum to detect the fluorescence emissions of PEs and PBsome terminal emitters(APB)simultaneously, and the real-time detection could greatly characterize the fluorescence dynamics of individual PBsomes in response to intense light.Our data reveal that strong green-light can induce the fluorescence decrease of APB,as well as the fluorescence increase of PE at the first stage of photobleaching.It strongly indicated an energetic decoupling occurring between PE and its neighbor.The fluorescence of PE was subsequently observed to decrease,showing that PE could be photobleached when energy transfer in the PBsomes was disrupted.In contrast,the energetic decoupling was not observed in either the PBsomes fixed with GA,or the mutant PBsomes lacking B-PE and remaining b-PE.It was concluded that the energetic decoupling of the PBsomes occurs at the specific association between B-PE and b-PE within the PBsome rod.In addition,this process is demonstrated to be power- and oxygen-dependent.Photosynthetic organisms have developed multiple protective mechanisms to prevent photodamage in vivo under high-light conditions.In cyanobacteria,the orange carotenoid protein(OCP)has been demonstrated to play roles in the photoprotective mechanism.However,the direct PBsome-related energy dissipation mechanism in red algae is still unclear.Such a decoupling process is proposed to be a strategy corresponding to the PBsomes to prevent photodamage of the photosynthetic RCs. Furthermore,our results implied a novel photoprotective role ofγ-subunit-containing PE in red algae.Packing of LH2s studied by AFMUnlike PBsomes in cyanobacteria and red algae,the peripheral photosynthetic LH2 complexes from the bacterium Rhodobacter sphaeroides are embedded in the photosynthetic membranes,transferring energy to LH1 and RCs.Microscopic and light spectroscopic investigations on the supramolecular architecture of bacterial photosynthetic membranes have revealed the photosynthetic protein-complexes to be arranged in a densely packed energy-transducing network.Protein packing may play a determinant role in the formation of functional photosynthetic domains and membrane curvature.To further investigate in detail the packing effects of like-protein photosynthetic complexes,in Chapter 8,I report an AFM investigation on artificially created 2D-crystals of LH2s from R.sphaeroides.Instead of the usually observed 1 or 2 different crystallization lattices for one specific preparation protocol we find 7 different packing lattices.The most abundant crystal types all show a tilting of the LH2 complex.Most surprisingly,although the LH2 complex is a monomeric protein-complex in vivo,we find a LH2 dimer packing motif.I further characterize two different dimer configurations:in Type 1 the LH2 complexes are tilted inwards,in Type 2 outwards.Closer inspection of the lattices surrounding the LH2 dimers indicates their close resemblance to those LH2 complexes that constitute a lattice of zig-zagging LH2.In addition,analyses of the tilt of the LH2 complexes within the zig-zag lattice and that observed within the dimers corroborate their similar packing-motif.The Type 2 dimer configuration exhibits a tilt that,in absence of up-down packing,could bend the lipid bi-layer leading to the strong curvature of the LH2 domains as observed in R.sphaeroides photosynthetic membranes in vivo.