methanethiosulfonate and 4-maleimido-2-2-6-6-tetramethylpiperidinooxyl

To identify interaction sites we measured the rotational motion of a spin label covalently bound to the side chain of a cysteine genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, and 279. Upon the addition of F-actin, the mobility of all the spin labels, especially at position 13, 271, or 279, of Tm was inhibited significantly. Slow spin-label motion at the C-terminus (at the 230th and 271st residues) was observed upon addition of troponin. The binding of myosin-head S1 fragments without troponin immobilized Tm residues at 146, 160, 190, 209, 230, 271, and 279, suggesting that these residues are involved in a direct interaction between Tm and actin in its open state. As immobilization occurred at substoichiometric amounts of S1 binding to actin (a 1:7 molar ratio), the structural changes induced by S1 binding to one actin subunit must have propagated and influenced interaction sites over seven actin subunits.

Topics: Actin Cytoskeleton; Actins; Animals; Cyclic N-Oxides; Disulfides; Electron Spin Resonance Spectroscopy; Mesylates; Muscle, Skeletal; Protein Binding; Rabbits; Rotation; Spin Labels; Time Factors; Tropomyosin; Troponin

Ligand-gated membrane channels selectively facilitate the entry of iron into prokaryotic cells. The essential role of iron in metabolism makes its acquisition a determinant of bacterial pathogenesis and a target for therapeutic strategies. In Gram-negative bacteria, TonB-dependent outer membrane proteins form energized, gated pores that bind iron chelates (siderophores) and internalize them. The time-resolved operation of the Escherichia coli ferric enterobactin receptor FepA was observed in vivo with electron spin resonance spectroscopy by monitoring the mobility of covalently bound nitroxide spin labels. A ligand-binding surface loop of FepA, which normally closes its transmembrane channel, exhibited energy-dependent structural changes during iron and toxin (colicin) transport. These changes were not merely associated with ligand binding, but occurred during ligand uptake through the outer membrane bilayer. The results demonstrate by a physical method that gated-porin channels open and close during membrane transport in vivo.

Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; Biological Transport; Carrier Proteins; Colicins; Cyclic N-Oxides; Cysteine; Electron Spin Resonance Spectroscopy; Enterobactin; Escherichia coli; Escherichia coli Proteins; Ferric Compounds; Indicators and Reagents; Ion Channel Gating; Ligands; Membrane Proteins; Mesylates; Porins; Protein Conformation; Receptors, Cell Surface; Spin Labels