Cross-linking and electron microscopy studies of the structure and functioning of the Escherichia coli ATP synthase

2000 ◽  
Vol 203 (1) ◽  
pp. 29-33 ◽  
Author(s):  
R.A. Capaldi ◽  
B. Schulenberg ◽  
J. Murray ◽  
R. Aggeler
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Oxidative Phosphorylation ◽  
Atp Synthase ◽  
Proton Gradient ◽  
Cross Linking ◽  
Combination Of Methods ◽  
Chemical Cross Linking

ATP synthase, also called F(1)F(o)-ATPase, catalyzes the synthesis of ATP during oxidative phosphorylation. The enzyme is reversible and is able to use ATP to drive a proton gradient for transport purposes. Our work has focused on the enzyme from Escherichia coli (ECF(1)F(o)). We have used a combination of methods to study this enzyme, including electron microscopy and chemical cross-linking. The utility of these two approaches in particular, and the important insights they give into the structure and mechanism of the ATP synthase, are reviewed.

scholarly journals Ionophore activity of sarcotoxin I, a bactericidal protein of Sarcophaga peregrina

1985 ◽  
Vol 229 (2) ◽  
pp. 453-458 ◽  
Author(s):  
M Okada ◽  
S Natori
Keyword(s):  
Escherichia Coli ◽  
Oxidative Phosphorylation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Bactericidal Effect ◽  
Proton Gradient ◽  
Wild Type ◽  
Atp Pool

When Escherichia coli was treated with sarcotoxin I, a potent bactericidal protein of Sarcophaga peregrina (fleshfly), K+ inside of the cells leaked out rapidly and the ATP pool of the cells rapidly decreased. These results suggested that the bactericidal effect of sarcotoxin I was due to its ionophore activity, and that it blocked the generation of ATP by inhibiting formation of the proton gradient essential for oxidative phosphorylation. This was confirmed by use of an uncA mutant, which was much less susceptible than the wild-type strain to sarcotoxin I under fixed ionic conditions.

scholarly journals Bacteriocin Release Protein Triggers Dimerization of Outer Membrane Phospholipase A In Vivo

1999 ◽  
Vol 181 (10) ◽  
pp. 3281-3283 ◽  
Author(s):  
Niek Dekker ◽  
Jan Tommassen ◽  
Hubertus M. Verheij
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Lag Time ◽  
Phospholipase A ◽  
Cross Linking ◽  
Outer Membrane Phospholipase A ◽  
Bacteriocin Release Protein ◽  
Chemical Cross Linking

ABSTRACT Bacteriocin release protein is known to activate outer membrane phospholipase A (OMPLA), which results in the release of colicin fromEscherichia coli. In vivo chemical cross-linking experiments revealed that the activation coincides with dimerization of OMPLA. Permeabilization of the cell envelope and dimerization were characterized by a lag time of 2 h.

15:30 STRUCTURAL ELUCIDATION OF DISC1 PATHWAY PROTEINS USING ELECTRON MICROSCOPY, CHEMICAL CROSS-LINKING AND MASS SPECTROSCOPY

2012 ◽  
Vol 136 ◽  
pp. S74 ◽  
Author(s):  
Nicholas I. Bradshaw ◽  
Dinesh C. Soares ◽  
Juan Zou ◽  
Christopher K. Kennaway ◽  
Zhou Angel Chen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectroscopy ◽  
Structural Elucidation ◽  
Cross Linking ◽  
Chemical Cross Linking

scholarly journals Oligomeric Sensor Kinase DcuS in the Membrane of Escherichia coli and in Proteoliposomes: Chemical Cross-linking and FRET Spectroscopy

2010 ◽  
Vol 192 (13) ◽  
pp. 3474-3483 ◽  
Author(s):  
Patrick D. Scheu ◽  
Yun-Feng Liao ◽  
Julia Bauer ◽  
Holger Kneuper ◽  
Thomas Basché ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Living Cells ◽  
Full Length ◽  
Cross Linking ◽  
Oligomeric State ◽  
Bacterial Membranes ◽  
Chemical Cross Linking

ABSTRACT DcuS is the membrane-integral sensor histidine kinase of the DcuSR two-component system in Escherichia coli that responds to extracellular C4-dicarboxylates. The oligomeric state of full-length DcuS was investigated in vitro and in living cells by chemical cross-linking and by f luorescence r esonance e nergy t ransfer (FRET) spectroscopy. The FRET results were quantified by an improved method using background-free spectra of living cells for determining FRET efficiency (E) and donor fraction {fD = (donor)/[(donor) + (acceptor)]}. Functional fusions of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) variants of green fluorescent protein to DcuS were used for in vivo FRET measurements. Based on noninteracting membrane proteins and perfectly interacting proteins (a CFP-YFP fusion), the results of FRET of cells coexpressing DcuS-CFP and DcuS-YFP were quantitatively evaluated. In living cells and after reconstitution of purified recombinant DcuS in proteoliposomes, DcuS was found as a dimer or higher oligomer, independent of the presence of an effector. Chemical cross-linking with disuccinimidyl suberate showed tetrameric, in addition to dimeric, DcuS in proteoliposomes and in membranes of bacteria, whereas purified DcuS in nondenaturing detergent was mainly monomeric. The presence and amount of tetrameric DcuS in vivo and in proteoliposomes was not dependent on the concentration of DcuS. Only membrane-embedded DcuS (present in the oligomeric state) is active in (auto)phosphorylation. Overall, the FRET and cross-linking data demonstrate the presence in living cells, in bacterial membranes, and in proteoliposomes of full-length DcuS protein in an oligomeric state, including a tetramer.

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