Biomedical subjects

S Wilkens

Publications and source records attributed to S Wilkens.

24 records · Page 2Linked to original sources

PubMed1994

A cryoelectron microscopy study of the interaction of the Escherichia coli F1-ATPase with subunit b dimer.

A complex between the Escherichia coli F1-ATPase and a truncated form of the ECF0-b subunit was formed and examined by cryoelectron microscopy in amorphous ice. Image analysis of single particles in the hexagonal projection revealed that the polar domain of the b subunit interacts with a beta subunit different from the one which interacts with the epsilon subunit. The cavity in the enzyme, visible in the hexagonal projection, is not filled by the b polypeptide, therefore leaving enough room for extensive conformational changes of the gamma and epsilon subunits within the native F1F0 complex.

Amino Acid Sequence↗

PubMed1994

Structure of the gamma subunit of Escherichia coli F1 ATPase probed in trypsin digestion and biotin-avidin binding studies.

The arrangement and functional role of the gamma subunit of the Escherichia coli F1ATPase (ECF1) has been probed by protease digestion and avidin-biotin labeling experiments using wild-type enzyme and four mutants, gamma S8C, gamma T106C, gamma S179C, and gamma V286C, respectively. Trypsin was found to cleave the gamma subunit at four sites, Arg70, Lys199, Lys201, and Lys212. Cleavage at these four sites did not greatly reduce the high ATPase activity of the enzyme that is obtained when the epsilon subunit is removed by the protease treatment. However, prolonged trypsin cleavage led to loss of inhibition by epsilon subunit added back to the trypsin-treated enzyme. Endoproteinase-Lys-C cleaves the gamma subunit of ECF1 at three of the four sites, i.e. Lys199, Lys201, and Lys212, but not at Arg70. The enzyme was activated by treatment with this protease because of degradation and release of the epsilon subunit, but added pure epsilon subunit still caused inhibition of ATPase activity. Therefore, cleavage at Arg70 by trypsin is responsible for the loss of response to the epsilon subunit inhibition. Biotin was reacted with Cys residues at positions 8, 106, 179, and 286 in different gamma subunit mutants and the accessibility of the biotin to avidin monitored in the intact ECF1 from the different mutants. Avidin was able to react with biotin when incorporated at position 106, not at 8, 179, or 286. The four trypsin cleavage sites, Arg70, Lys199, Lys201, and Lys212, as well as Thr106 are in regions of the gamma subunit predicted to be mainly beta-sheet and beta-turn structures.

Amino Acid Sequence↗

PubMed1994

Coupling between catalytic sites and the proton channel in F1F0-type ATPases.

F1F0-type ATPases catalyse both ATP-driven proton translocation and proton-gradient-driven ATP synthesis. Recent cryoelectronmicroscopy and low-resolution X-ray studies provide a first glimpse at the structure of this complicated membrane-bound enzyme. The F1 part is roughly globular and linked to the membrane-intercalated F0 part by a narrow stalk domain, which contains the gamma-, delta- and epsilon-subunits along with domains of the b-subunit of the F0 part. Here, we review evidence that conformational and positional changes in the gamma- and epsilon-subunits provide the coupling between catalytic sites and proton translocation within the F1F0 complex.

Binding Sites↗

PubMed1994

Asymmetry and structural changes in ECF1 examined by cryoelectronmicroscopy.

The Escherichia coli ATPase (ECF1) has been studied by cryoelectronmicroscopy and an intrinsic asymmetry of the molecule in the hexagonal projection identified. The three beta subunits could be distinguished. One, which we have called beta 1, has a greater density in projection than the other two; the second, beta 2, is of intermediate density in projection, while the third, beta 3, is smeared out in density. These different features of the beta subunits were used to orient images, and the positions of the gamma and epsilon subunits then established. The location of the gamma subunit, as monitored by the central mass, was not fixed. This subunit could be found in positions that followed an arc from close to beta 2 to close to beta 3, a shift of around 10A, with respect to the center of the mass. The location of the epsilon subunit was monitored after reconstituting a complex of epsilon subunit-depleted ECF1 with a mutant epsilon subunit in which His at residue 38 had been replaced by Cys, and this Cys labeled with an approximately 14A gold particle. The epsilon subunit was found in positions described by an arc between an alpha subunit (alpha 1) and the neighboring beta subunit (beta 1), a shift of around 20A, with respect to the center of the gold particle. A nucleotide dependence of the position of the gamma subunit has been established by Gogol, E.P., Johnston, E., Aggeler, R. and Capaldi, R.A. (1990) Proc. Natl. Acad. Sci. USA 87, 9585-9589. A nucleotide dependence of the position of the epsilon subunit is shown here.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1992

Monomaleimidogold labeling of the gamma subunit of the Escherichia coli F1 ATPase examined by cryoelectron microscopy.

A novel approach for locating sites of interest in a protein complex has been developed using monomaleimidonanogold (MMN). The Escherichia coli F1 ATPase, when prepared without the delta subunit, contains only a single reactive cysteine on one of the three copies of the alpha subunit. This site was reacted with MMN and the gold cluster visualized on the protein complex by cryoelectron microscopy. Additional sites for modification with MMN were added by introducing cysteine residues through site-directed mutagenesis. Labeling of two mutants, gamma S8-C and gamma T106-C, in which Ser8 and Thr106, respectively, had been replaced by a cysteine, placed the gold cluster on the central mass that is seen in the hexagonal projection of the ECF1 complex. The results establish that the central mass contains the N-terminal part of the gamma subunit.

Electrophoresis, Polyacrylamide Gel↗

PubMed1992

Structure of the Escherichia coli ATP synthase and role of the gamma and epsilon subunits in coupling catalytic site and proton channeling functions.

The structure of the Escherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the alpha and beta subunits of the F1 part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 A at its widest point. The alpha and beta subunits are interdigitated in side view for around 60 A of the 90 A 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 A long and 25-30 A in diameter. The F0 part is mostly buried in the lipid bilayer. The gamma subunit provides a domain that extends into the central cavity of the F1 part. The gamma and epsilon 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 gamma 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.

Binding Sites↗