RESUMO
Excitatory amino acid transporters (EAATs) are secondary transport proteins that mediate the uptake of glutamate and other amino acids. EAATs fulfil an important role in neuronal signal transmission by clearing the excitatory neurotransmitters from the synaptic cleft after depolarization of the postsynaptic neuron. An intensively studied model system for understanding the transport mechanism of EAATs is the archaeal aspartate transporter GltPh. Each subunit in the homotrimeric GltPh supports the coupled translocation of one aspartate molecule and three Na(+) ions as well as an uncoupled flux of Cl(-) ions. Recent crystal structures of GltPh revealed three possible conformations for the subunits, but it is unclear whether the motions of individual subunits are coordinated to support transport. Here, we report the direct observation of conformational dynamics in individual GltPh trimers embedded in the membrane by applying single-molecule fluorescence resonance energy transfer (FRET). By analysing the transporters in a lipid bilayer instead of commonly used detergent micelles, we achieve conditions that approximate the physiologically relevant ones. From the kinetics of FRET level transitions we conclude that the three GltPh subunits undergo conformational changes stochastically and independently of each other.
Assuntos
Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas de Transporte de Glutamato da Membrana Plasmática/química , Modelos Moleculares , Sódio/química , Transferência Ressonante de Energia de Fluorescência , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Bicamadas Lipídicas/metabolismo , Estrutura Terciária de Proteína , Pyrococcus horikoshii/química , Pyrococcus horikoshii/metabolismoRESUMO
Processive DNA synthesis by the αεθ core of the Escherichia coli Pol III replicase requires it to be bound to the ß2 clamp via a site in the α polymerase subunit. How the ε proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of ε. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel ß-binding site in ε that, in conjunction with the site in α, maintains a closed state of the αεθ-ß2 replicase in the polymerization mode of DNA synthesis. The ε-ß interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.
Assuntos
DNA Polimerase III/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Replicação do DNA/genética , Replicação do DNA/fisiologia , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/metabolismo , Estabilidade Enzimática/genética , Escherichia coli/genética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Multimerização Proteica/genética , Multimerização Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Homologia de Sequência de AminoácidosRESUMO
Fluorescence microscopy studies have shown that many proteins localize to highly specific subregions within bacterial cells. Analyzing the spatial distribution of low-abundance proteins within cells is highly challenging because information obtained from multiple cells needs to be combined to provide well-defined maps of protein locations. We present (to our knowledge) a novel tool for fast, automated, and user-impartial analysis of fluorescent protein distribution across the short axis of rod-shaped bacteria. To demonstrate the strength of our approach in extracting spatial distributions and visualizing dynamic intracellular processes, we analyzed sparse fluorescence signals from single-molecule time-lapse images of individual Escherichia coli cells. In principle, our tool can be used to provide information on the distribution of signal intensity across the short axis of any rod-shaped object.
Assuntos
Escherichia coli/citologia , Microscopia de Fluorescência , Membrana Celular/metabolismo , Citosol/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Processamento de Imagem Assistida por Computador , Transporte ProteicoRESUMO
ATP-binding cassette (ABC) transporters form the largest class of active membrane transport proteins. Binding and hydrolysis of ATP by their highly conserved nucleotide-binding domains drive conformational changes of the complex that mediate transport of substrate across the membrane. The vitamin B12 importer BtuCD-F in Escherichia coli is an extensively studied model system. The periplasmic soluble binding protein BtuF binds the ligand; the transmembrane and ATPase domains BtuCD mediate translocation. Here we report the direct observation at the single-molecule level of ATP, vitamin B12 and BtuF-induced events in the transporter complex embedded in liposomes. Single-molecule fluorescence imaging techniques reveal that membrane-embedded BtuCD forms a stable complex with BtuF, regardless of the presence of ATP and vitamin B12. We observe that a vitamin B12 molecule remains bound to the complex for tens of seconds, during which several ATP hydrolysis cycles can take place, before it is being transported across the membrane.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Vitamina B 12/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Conformação ProteicaRESUMO
Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of membrane transport proteins with fluorescent probes at the single-molecule level. First, we explain how to produce membrane-reconstituted transporters with fluorescent labels. Next, we detail the construction of a microfluidic flow cell to image immobilized proteoliposomes on a total internal reflection fluorescence microscope. We conclude by describing the methods that are needed to analyze fluorescence movies and obtain useful single-molecule data.