RESUMO
Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain "layer" of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs.
Assuntos
Receptores de AMPA/química , Receptores de AMPA/metabolismo , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Fluoruracila/análogos & derivados , Fluoruracila/metabolismo , Técnicas de Inativação de Genes , Ácido Caínico/metabolismo , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Ratos , Receptores de AMPA/agonistas , Receptores de AMPA/genéticaRESUMO
Iron (Fe) availability is a major limiting factor for primary production in aquatic environments. Cyanobacteria respond to Fe deficiency by derepressing the isiAB operon, which encodes the antenna protein IsiA and flavodoxin. At nanomolar Fe concentrations, a PSI-IsiA supercomplex forms, comprising a PSI trimer encircled by two complete IsiA rings. This PSI-IsiA supercomplex is the largest photosynthetic membrane protein complex yet isolated. This study presents a detailed characterization of this complex using transmission electron microscopy and ultrafast fluorescence spectroscopy. Excitation trapping and electron transfer are highly efficient, allowing cyanobacteria to avoid oxidative stress. This mechanism may be a major factor used by cyanobacteria to successfully adapt to modern low-Fe environments.
Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/fisiologia , Água Doce/microbiologia , Ferro/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo , Adaptação Biológica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cianobactérias/enzimologia , Cianobactérias/genética , Água Doce/análise , Estresse Oxidativo , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/genéticaRESUMO
The supramolecular organization of photosystem II (PSII) complexes in the photosynthetic membrane of the cyanobacterium Synechocystis 6803 was studied by electron microscopy. After mild detergent solubilization, crystalline PSII arrays were extracted in which dimeric PSII particles associate in multiple rows. Image processing of the arrays shows that the PSII dimers are tightly packed at distances of 12.2 and 16.7 nm. The domains are considered to be an important type of association for preventing either spill-over energy from PSII towards photosystem I (PSI) or direct energy flow from phycobilisomes to PSI, because the latter can only be at periphery of the arrays.
Assuntos
Proteínas de Bactérias/química , Complexo de Proteína do Fotossistema II/química , Synechocystis/enzimologia , Dimerização , Microscopia Eletrônica , Estrutura Terciária de Proteína , Solubilidade , Tilacoides/enzimologia , Tilacoides/ultraestruturaRESUMO
The larger protein complexes of the cyanobacterial photosynthetic membrane of Thermosynechoccus elongatus and Synechocystis 6803 were studied by single particle electron microscopy after detergent solubilization, without any purification steps. Besides the "standard" L-shaped NDH-1L complex, related to complex I, large numbers of a U-shaped NDH-1MS complex were found in both cyanobacteria. In membranes from Synechocystis DeltacupA and DeltacupA/cupB mutants the U-shaped complexes were absent, indicating that CupA is responsible for the U-shape by binding at the tip of the membrane-bound arm of NDH-1MS. Comparison of membranes grown under air levels of CO(2) or 3% CO(2) indicates that the number of NDH-1MS particles is 30-fold higher under low-CO(2).
Assuntos
Proteínas de Bactérias/metabolismo , Synechococcus/metabolismo , Synechocystis/metabolismo , Tilacoides/metabolismo , Proteínas de Bactérias/química , Microscopia Eletrônica , Synechococcus/ultraestrutura , Synechocystis/ultraestruturaRESUMO
Bioenergetic reactions in chloroplasts and mitochondria are catalyzed by large multi-subunit membrane proteins. About two decades ago it became clear that several of these large membrane proteins further associate into supercomplexes and since then a number of new ones have been described. In this review we focus on supercomplexes involved in light harvesting and electron transfer in the primary reactions of oxygenic photosynthesis and on the mitochondrial supercomplexes that catalyze electron transfer and ATP synthesis in oxidative phosphorylation. Functional and structural aspects are overviewed. In addition, several relevant technical aspects are discussed, including membrane solubilization with suitable detergents and methods of purification. Some open questions are addressed, such as the lack of high-resolution structures, the outstanding gaps in the knowledge about supercomplexes involved in cyclic electron transport in photosynthesis and the unusual mitochondrial protein complexes of protists and in particular of ciliates.
Assuntos
Cloroplastos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/ultraestrutura , Cloroplastos/ultraestrutura , Transporte de Elétrons , Microscopia Eletrônica , Mitocôndrias/química , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/química , Proteínas Mitocondriais/isolamento & purificação , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Fosforilação Oxidativa , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/isolamento & purificaçãoRESUMO
A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different cellular processes. Typical membrane domains are found in bacteria where a specific membrane protein is abundantly expressed. Lipid rafts form another example. Despite the rareness of conventional organelles as found in eukaryotes, some bacteria are known to have an intricate internal cell membrane organization. Membrane proliferation can be divided into curvature and invaginations which can lead to internal compartmentalization. This study discusses some of the clearest examples of bacteria with such domains and internal membranes. The need for membrane specialization is highest among the heterogeneous group of bacteria which harvest light energy, such as photosynthetic bacteria and halophilic archaea. Most of the highly specialized membranes and domains, such as the purple membrane, chromatophore and chlorosome, are found in these autotrophic organisms. Otherwise the need for membrane differentiation is lower and variable, except for those structures involved in cell division. Microscopy techniques have given essential insight into bacterial membrane morphology. As microscopy will further contribute to the unraveling of membrane organization in the years to come, past and present technology in electron microscopy and light microscopy is discussed. Electron microscopy was the first to unravel bacterial morphology because it can directly visualize membranes with inserted proteins, which no other technique can do. Electron microscopy techniques developed in the 1950s and perfected in the following decades involve the thin sectioning and freeze fractioning of cells. Several studies from the golden age of these techniques show amazing examples of cell membrane morphology. More recently, light microscopy in combination with the use of fluorescent dyes has become an attractive technique for protein localization with the natural membrane. However, the resolution problem in light microscopy remains and overinterpretation of observed phenomena is a pitfall. Thus, light microscopy as a stand-alone technique is not sufficient to prove, for instance, the long-range helical distribution of proteins in membrane such as MinD spirals in Bacillus subtilis. Electron tomography is an emerging electron microscopy technique that can provide three-dimensional reconstructions of small, nonchemically fixed bacteria. It will become a useful tool for studying prokaryotic membranes in more detail and is expected to collect information complementary to those of advanced light microscopy. Together, microscopy techniques can meet the challenge of the coming years: to specify membrane structures in more detail and to bring them to the level of specific protein-protein interactions.