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Cells have evolved specific machineries in a defined cellular environment to boost metabolic activities. The Liu Lab aims to explore the molecular basis underlying the self assembly and functional regulations of biological machineries. Emerging questions to be addressed include:
(1) How does nature develop functional machineries to enhance cellular metabolism?
(2) How do the structures of biological machineries ensure their physiological functions?
(3) How are the structure and function of the machineries regulated in response to the changing environment?

Advanced knowledge of the biological machinery will underpin the synthetic engineering of new biological “devices” to power cellular metabolism, and provide biotechnological solutions to grand challenges such as global food and energy security. The funding is kindly provided by UK Royal Society, Biochemistry Society, Biotechnology and Biological Sciences Research Council (BBSRC), China Scholarship Council, Marie Curie Fellowship.

 

1. Bacterial microcompartments
Bacterial microcompartments (BMCs) are proteinaceous organelles widespread among bacterial phyla. They compartmentalize enzymes within a selectively permeable shell and play important roles in carbon fixation, pathogenesis, and microbial ecology. Among the distinct types of bacterial microcompartments, carboxysomes are the central carbon-fixation organells evolved in cyanobacteria and some chemoautotrophs. Their physiological significance, self-assembly and modularity features have attracted increasing interest in synthetic engineering to build new nanoreactors and scaffolding systems for metabolic enhancement.

We combine molecular biology, biochemistry, synthetic biolgy, and microscopy to characterize, at molecular resolution, the structure and dynamics of BMC shell facet assembly (Nano Letters 2016]. The research shows that preformed hexamers assemble into uniformly oriented shell layers, a single hexamer thick. Shell hexamers can dissociate from and incorporate into assembled sheets, indicating a flexible intermolecular interaction. Furthermore, we demonstrate that the self-assembly and dynamics of shell proteins are governed by specific contacts at the interfaces of shell proteins. Our study provides novel insights into the formation, interactions, and dynamics of BMC shell facets, which are essential for the design and engineering of self-assembled biological nanoreactors and scaffolds based on BMC architectures.

We also use live-cell confocal fluorescence imaging to explore the biosynthesis and subcellular localization of β-carboxysomes within a model cyanobacterium, Synechococcus elongatus PCC7942, in response to light variation [Plant Physiol 2016]. β-carboxysome biosynthesis is accelerated in response to increasing light intensity, thereby enhancing the carbon fixation activity of the cell. Inhibition of photosynthetic electron flow impairs the accumulation of carboxysomes, indicating a close coordination between β-carboxysome biogenesis and photosynthetic electron transport. Likewise, the spatial organization of carboxysomes in cells correlates with the redox state of photosynthetic electron transport chain. This study provides essential knowledge for modulating β-carboxysome biosynthesis and function in cyanobacteria.

We characterize the macromolecular architecture and inherent physical mechanics of single β-carboxysomes using electron microscopy, atomic force microscopy (AFM) and proteomics [Nanoscale 2017]. Intact β-carboxysome comprises three structural domains, a single-layered icosahedral shell, an inner layer and paracrystalline arrays of interior Rubisco. Furthermore, the topography and intrinsic mechanics of functional β-carboxysomes are determined in native conditions using AFM and AFM-based nanoindentation, revealing the flexible organization and soft mechanical properties of β-carboxysomes compared to rigid viruses. The research provides clues into the natural characteristics of β-carboxysome organization and nanomechanics, which can be extended to diverse bacterial microcompartments and are important considerations for the design and engineering of functional carboxysomes in other organisms to supercharge photosynthesis. It offers an approach for inspecting the structural and mechanical features of synthetic organelles and protein scaffolds.

References:
Nanoscale, 2017, 9(30): 10662-10673
Nano Letters, 2016, 16(3): 1590-1595
Plant Physiol, 2016, 171(1): 530-541


2. Photosynthetic machinery
"Without photosynthesis, no complex ecosystems and higher life forms including man would exist." The thylakoid membrane is the site for photosynthetic reactions and responses in cyanobacteria, algae and higher plants [BBA 2016]. We performed direct visualization of the native organization and mobility of photosynthetic complexes in cyanobacterial thylakoid membranes, using atomic force, confocal, and total internal reflection fluorescence microscopy. Knowledge of the cyanobacterial thylakoid membrane could be extended to chloroplast and mitochondrial membranes [Trends Plant Sci 2013].

We study the native arrangement and dense packing of photosystem I (PSI), photosystem II (PSII), and cytochrome (Cyt) b6f within thylakoid membranes at the molecular level. By functionally tagging PSI, PSII, Cyt b6f , and ATP synthase with fluorescent proteins, we characterize the heterogeneous distribution of these four photosynthetic complexes and determined their dynamic features within the crowding membrane environment using live-cell fluorescence imaging. Red light-induced clustering localization and adjustable diffusion of photosynthetic complexes in thylakoid membranes, representative of the reorganization of photosynthetic apparatus in response to environmental changes [Mol Plant 2017].

F1Using fluorescent tagging and live-cell confocal fluorescence microscopy imaging, we investigated the distribution of key respiratory electron donors, type-I NAD(P)H dehydrogenase (NDH-1) and succinate dehydrogenase (SDH), in live cells of the cyanobacterium Synechococcus elongatus PCC 7942 [PNAS 2012]. We found that, when cells are grown under low light, both complexes form discrete patches in the thylakoid membranes. Exposure to moderate light leads to redistribution of complexes, and they become evenly distributed in thylakoid membranes. The distribution of the complexes within the thylakoid membranes is under redox-regulated physiological control. Redistribution of the complexes correlates with a major change in the relative probability of the two electron transport pathways. Our research indicates that the sub-micron scale distribution of respiratory complexes governs the partitioning of reducing power and that redistribution of electron transport complexes on these scales is a physiological mechanism to regulate electron flow.

We also studied the spatial organization of the bacterial photosynthetic apparatus, and the strategies employed for efficient harvesting and trapping of solar energy, as well as the long-distance quinone pathways. The quantitative measurements of the unfolding process and structural stability of membrane proteins (LH2) in native biological membrane revealed forces and energies that assure structural and functional integrity of LH2, as well as how LH2 interact with other proteins in the supramolecular architecture [PNAS 2011]. It suggests explicitly the importance of LH2 ring-shaped architecture, and that the complex stability is supported and depends on their specific molecular environment in native membrane. The acquired information gives hints how molecular interactions drive photosynthetic protein assembly.

F3Moreover, we examined the supramolecular architectures of phycobilisomes (PBsomes) on the thylakoid membranes from the unicellular red alga Porphyridium cruentum using atomic force microscopy (AFM) and electron microscopy (EM)[JBC 2008]. On the basis of the crowding organisation of PBsome on thylakoid membrane, we further investigated the diffusion dynamics of PBsomes in red algal cell using fluorescnece recovery after photobleaching (FRAP)[PLoS ONE 2009]. The mobility of PBsomes in cells were estimated in depth. Additionally, we applied single-molecule spectroscopy to perform the first evaluation on the fluorescence dynamics of individual PBsomes of P. cruentum [PLoS ONE 2008]. The observations strongly indicate an energetic decoupling occurring in the light-harvesting complex and the photoprotection role of PBsomes to prevent photodamage of the photosynthetic reaction centers.

References:
Mol Plant, 2017, 10(11): 1434–1448
BBA - Bioenergetics, 2016, 1857(3): 256-265
Trends Plant Sci, 2013, 18(5): 277-286
Proc. Natl. Acad. Sci. USA, 2012, 109(28): 11431-11436
Proc. Natl. Acad. Sci. USA, 2011, 108(23): 9455-9459
J Struct Biol, 2011, 173(1): 138-145
J Mol Biol, 2009, 393(1): 27-35
J Biol Chem, 2008, 283(50): 34946-34953
PLoS ONE, 2009, 4(4): e5295
Photosynth Res, 2008, 95(2-3): 169-174
PLoS ONE, 2008, 3(9): e3134