scholarly journals Atomic force microscopy visualizes mobility of photosynthetic proteins in grana thylakoid membranes

2018 ◽  
Author(s):  
Bibiana Onoa ◽  
Shingo Fukuda ◽  
Masakazu Iwai ◽  
Carlos Bustamante ◽  
Krishna K. Niyogi
Keyword(s):  
High Speed ◽  
Spinacia Oleracea ◽  
Protein Complexes ◽  
Rotational Diffusion ◽  
Thylakoid Membranes ◽  
Spatiotemporal Resolution ◽  
Force Microscopy ◽  
Photosynthetic Complexes ◽  
Protein Density ◽  
Photosynthetic Protein Complexes

ABSTRACTThylakoid membranes in chloroplasts contain photosynthetic protein complexes that convert light energy into chemical energy. Photosynthetic protein complexes are considered to undergo structural reorganization to maintain the efficiency of photochemical reactions. A detailed description of the mobility of photosynthetic complexes in real-time is necessary to understand how macromolecular organization of the membrane is altered by environmental fluctuations. Here, we used high-speed atomic force microscopy to visualize and characterize the in situ mobility of individual protein complexes in grana thylakoid membranes isolated from Spinacia oleracea. Our observations reveal that these membranes can harbor complexes with at least two distinctive classes of mobility. A large fraction of grana membranes contained proteins with quasi-static mobility, exhibiting molecular displacements smaller than 10 nm2. In the remaining fraction, the protein mobility is variable with molecular displacements of up to 100 nm2. This visualization at high-spatiotemporal resolution enabled us to estimate an average diffusion coefficient of ∼1 nm2 s-1. Interestingly, both confined and Brownian diffusion models could describe the protein mobility of the second group of membranes. We also provide the first direct evidence of rotational diffusion of photosynthetic complexes. The rotational diffusion of photosynthetic complexes could be an adaptive response to the high protein density in the membrane to guarantee the efficiency of electron transfer reactions. This characterization of the mobility of individual photosynthetic complexes in grana membranes establishes a foundation that could be adapted to study the dynamics of the complexes inside the intact and photosynthetically functional thylakoid membranes to be able to understand its structural responses to diverse environmental fluctuations.STATEMENT OF SIGNIFICANCEWe characterized the dynamics of individual photosynthetic protein complexes in grana thylakoid membranes from Spinacia oleracea by high-speed atomic microscopy (HS-AFM). Direct visualization at high spatiotemporal resolution unveils that the mobility of photosynthetic proteins is heterogeneous but governed by the confinement effect imposed by the high protein density in the thylakoid membrane. The photosynthetic complexes display rotational diffusion, which might be a consequence of the crowded environment in the membrane and a mechanism to sustain an efficient electron transfer chain.

scholarly journals Probing the biogenesis pathway and dynamics of thylakoid membranes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tuomas Huokko ◽  
Tao Ni ◽  
Gregory F. Dykes ◽  
Deborah M. Simpson ◽  
Philip Brownridge ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Photosystem I ◽  
Spatial Organization ◽  
Electron Flux ◽  
Protein Complexes ◽  
Thylakoid Membranes ◽  
Thylakoid Biogenesis ◽  
Photosynthetic Complexes ◽  
Photosynthetic Machinery ◽  
Pcc 7942

AbstractHow thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.

scholarly journals Regulatory factors for the assembly of thylakoid membrane protein complexes

2012 ◽  
Vol 367 (1608) ◽  
pp. 3420-3429 ◽  
Author(s):  
Wei Chi ◽  
Jinfang Ma ◽  
Lixin Zhang
Keyword(s):  
Membrane Protein ◽  
Thylakoid Membrane ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Protein Complexes ◽  
Thylakoid Membranes ◽  
Regulatory Factors ◽  
Photosynthetic Organisms ◽  
Photosynthetic Complexes ◽  
Membrane Protein Complexes

Major multi-protein photosynthetic complexes, located in thylakoid membranes, are responsible for the capture of light and its conversion into chemical energy in oxygenic photosynthetic organisms. Although the structures and functions of these photosynthetic complexes have been explored, the molecular mechanisms underlying their assembly remain elusive. In this review, we summarize current knowledge of the regulatory components involved in the assembly of thylakoid membrane protein complexes in photosynthetic organisms. Many of the known regulatory factors are conserved between prokaryotes and eukaryotes, whereas others appear to be newly evolved or to have expanded predominantly in eukaryotes. Their specific features and fundamental differences in cyanobacteria, green algae and land plants are discussed.

scholarly journals Single-Molecule Dynamics of the DNA−EcoRII Protein Complexes Revealed with High-Speed Atomic Force Microscopy

Biochemistry ◽  
2009 ◽  
Vol 48 (44) ◽  
pp. 10492-10498 ◽  
Author(s):  
Jamie L. Gilmore ◽  
Yuki Suzuki ◽  
Gintautas Tamulaitis ◽  
Virginijus Siksnys ◽  
Kunio Takeyasu ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
High Speed ◽  
Protein Complexes ◽  
Force Microscopy ◽  
Atomic Force ◽  
Single Molecule Dynamics ◽  
Molecule Dynamics

scholarly journals Characterizing the supercomplex association of photosynthetic complexes in cyanobacteria

2021 ◽  
Vol 8 (7) ◽  
pp. 202142
Author(s):  
Zimeng Zhang ◽  
Long-Sheng Zhao ◽  
Lu-Ning Liu
Keyword(s):  
Rational Design ◽  
Binding Proteins ◽  
Close Association ◽  
Photosynthetic Apparatus ◽  
Thylakoid Membranes ◽  
Efficient Energy Transfer ◽  
Force Microscopy ◽  
Photosynthetic Complexes ◽  
Artificial Photosynthetic ◽  
Pcc 7942

The light reactions of photosynthesis occur in thylakoid membranes that are densely packed with a series of photosynthetic complexes. The lateral organization and close association of photosynthetic complexes in native thylakoid membranes are vital for efficient light harvesting and energy transduction. Recently, analysis of the interconnections between photosynthetic complexes to form supercomplexes has garnered great interest. In this work, we report a method integrating immunoprecipitation, mass spectrometry and atomic force microscopy to identify the inter-complex associations of photosynthetic complexes in thylakoid membranes from the cyanobacterium Synechococcus elongatus PCC 7942. We characterize the preferable associations between individual photosynthetic complexes and binding proteins involved in the complex–complex interfaces, permitting us to propose the structural models of photosynthetic complex associations that promote the formation of photosynthetic supercomplexes. We also identified other potential binding proteins with the photosynthetic complexes, suggesting the highly connecting networks associated with thylakoid membranes. This study provides mechanistic insight into the physical interconnections of photosynthetic complexes and potential partners, which are crucial for efficient energy transfer and physiological acclimatization of the photosynthetic apparatus. Advanced knowledge of the protein organization and interplay of the photosynthetic machinery will inform rational design and engineering of artificial photosynthetic systems to supercharge energy production.

scholarly journals The Particle Filter Method to Integrate High-Speed Atomic Force Microscopy Measurement with Biomolecular Simulations

2020 ◽  
Author(s):  
Sotaro Fuchigami ◽  
Toru Niina ◽  
Shoji Takada
Keyword(s):  
Atomic Force Microscopy ◽  
Particle Filter ◽  
Structural Dynamics ◽  
High Speed ◽  
Md Simulations ◽  
Coarse Grained ◽  
Filter Method ◽  
Spatiotemporal Resolution ◽  
Force Microscopy ◽  
Atomic Force

ABSTRACTThe high-speed atomic force microscopy (HS-AFM) can observe structural dynamics of biomolecules at single-molecule level in real time near physiological condition, but its spatiotemporal resolution is limited. Complementarily, molecular dynamics (MD) simulations have higher spatiotemporal resolutions albeit with some artifact. Here, in order to integrate the HS-AFM data and coarse-grained (CG)-MD simulations, we develop a particle filter method, one of the sequential Bayesian data assimilation approaches. We tested the method in a twin experiment. We first made a reference HS-AFM movie from a CG-MD trajectory of a test molecule, a nucleosome, which serves as an “experimental measurement”. Then, we performed the particle filter simulation with 512 particles that captured large-scale nucleosome structural dynamics compatible with the AFM movie. Comparing the particle filter simulations with 8 - 8192 particles, we found that the use of more particles consistently results in larger likelihood for the whole AFM movie. By comparing the likelihoods from different ionic concentrations and from different timescales, we found that the “true” concentration and timescale can be inferred as the largest likelihood of the whole AFM movie, but not that of each AFM image. The particle filter method provides a general approach to integrate the HS-AFM data with MD simulations.

scholarly journals High-Speed Atomic Force Microscopy: Membrane Deformation Protein Complexes in Action

2021 ◽  
Vol 120 (3) ◽  
pp. 2a
Author(s):  
Simon Scheuring ◽  
Grigory Tagiltsev ◽  
Nebojsa Jukic ◽  
Alma P. Perrino
Keyword(s):  
Atomic Force Microscopy ◽  
High Speed ◽  
Protein Complexes ◽  
Membrane Deformation ◽  
Force Microscopy ◽  
Atomic Force

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

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