Edward P. Gogol

Dynamic assembly of MinD on phospholipid vesicles regulated by ATP and MinE

Selection of the division site in Escherichia coli is regulated by the min system and requires the rapid oscillation of MinD between the two halves of the cell under the control of MinE. In this study we have further investigated the molecular basis for this oscillation by examining the interaction of MinD with phospholipid vesicles. We found that MinD bound to phospholipid vesicles in the presence of ATP and, upon binding, assembled into a well-ordered helical array that deformed the vesicles into tubes. Stimulation of the MinD ATPase by addition of MinE led to disassembly of the tubes and the release of MinD from the vesicles. It is proposed that this MinE-regulated dynamic assembly of MinD underlies MinD oscillation.

The stalk connecting the F1 and F0 domains of ATP synthase visualized by electron microscopy of unstained specimens

E. coli F1 F0 ATP synthase has been reconstituted into membranes and visualized by electron microscopy of unstained samples preserved in thin layers of amorphous ice. Unlike previous observations in negative stain, these specimens are not exposed to potentially denaturing or perturbing conditions, having been rapidly frozen from well‐defined conditions in which the enzyme is fully active. The structures visualized in views normal to the lipid bilayer clearly show the presence of a narrow stalk approx. 45 Å long, connecting the F1 to the membrane‐embedded F0.

Structure of theEscherichia coli ATP synthase and role of the γ and ε subunits in coupling catalytic site and proton channeling functions

The structure of theEscherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the α and β subunits of the F1 part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 Å at its widest point. The α and β subunits are interdigitated in side view for around 60 Å of the 90 Å length of the molecule. The F1 narrows and has three-fold symmetry at the end furthest from the F0 part. The F1 is linked to F0 by a stalk approximately 45 Å long and 25–30 Å in diameter. The F0 part is mostly buried in the lipid bilayer. The γ subunit provides a domain that extends into the central cavity of the F1 part. The γ and ε subunits are in a different conformation when ATP+Mg2+ are present in catalytic sites than when ATP+EDTA are present. This is consistent with these two small subunits switching conformations as a function of whether or not phosphate is bound to the enzyme at the position of the γ phosphate of ATP. We suggest that this switching is the key to the coupling of catalytic site events with proton translocation in the F0 part of the complex.

Epitope mapping of monoclonal antibodies to the Escherichia coli F1 ATPase α subunit in relation to activity effects and location in the enzyme complex based on cryoelectron microscopy

The interaction of Escherichia coli F1 ATPase (ECF1) with several different monoclonal antibodies (mAbs) specific for the alpha subunit has been examined. The epitopes for each of the mAbs have been localized by using molecular biological approaches to generate fragments of the alpha subunit. The binding of several of the mAbs has also been examined by cryoelectron microscopy of ECF1 Fab complexes. One of the mAbs, alpha II, bound in the region Asn 109-Val 153 without affecting ATPase activity. Most of the mAbs bound in the C-terminal third of the alpha subunit. MAb alpha 1 bound between residues Gln 443 and Trp 513. This mAb activated ATPase activity and was visualized in cryoelectron microscopy, superimposed on the alpha subunit, indicating that the epitope was on the top or bottom of ECF1 in the hexagonal projection. Other mAbs to the C-terminus, including alpha D which also activated the enzyme, reacted between Gly 371 and Trp 513 but failed to bind to small overlapping fragments within this sequence. The epitopes for these mAbs are probably formed by the folded polypeptide which occurs only in Western analysis when long stretches of the alpha subunit are present, suggesting that the C-terminus of alpha is a self-folding domain. In cryoelectron microscopy, Fab fragments for alpha D were seen extending from the sides of the ECF1 complex in hexagonal projection.

Structure of the Escherichia Coli ATP Synthase from Electron Microscopy Studies

ATP synthesis in both prokaryotes and eukaryotes is carried out by a large membrane-bound complex made up of two functionally and structurally distinct parts. The catalytic part, the F1, is extrinsic to the membrane bilayer, and is attached to the integral membrane assembly, the F0, which provides the proton channel (Senior and Wise, 1983; Walker et al., 1984; Amzel and Pedersen, 1983).