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K+-translocating KdpFABC P-type ATPase from Escherichia coli acts as a functional and structural dimer.

Biochemistry (2008-02-27)
Thomas Heitkamp, René Kalinowski, Bettina Böttcher, Michael Börsch, Karlheinz Altendorf, Jörg-Christian Greie
ABSTRACT

The membrane-embedded K (+)-translocating KdpFABC complex from Escherichia coli belongs to the superfamily of P-type ATPases, which share common structural features as well as a well-studied catalytic mechanism. However, little is known about the oligomeric state of this class of enzymes. For many P-type ATPases, such as the Na (+)/K (+)-ATPase, Ca (2+)-ATPase, or H (+)-ATPase, an oligomeric state has been shown or is at least discussed but has not yet been characterized in detail. In the KdpFABC complex, kinetic analyses already indicated the presence of two cooperative ATP-binding sides within the functional enzyme and, thus, also point in the direction of a functional oligomer. However, the nature of this oligomeric state has not yet been fully elucidated. In the present work, a close vicinity of two KdpB subunits within the functional KdpFABC complex could be demonstrated by chemical cross-linking of native cysteine residues using copper 1,10-phenanthroline. The cysteines responsible for cross-link formation were identified by mutagenesis. Cross-linked and non-cross-linked KdpFABC complexes eluted with the same apparent molecular weight during gel filtration, which corresponded to the molecular weight of a homodimer, thereby clearly indicating that the KdpFABC complex was purified as a dimer. Isolated KdpFABC complexes were analyzed by transmission electron microscopy and exhibited an approximately 1:1 distribution of mono- and dimeric particles. Finally, reconstituted functional KdpFABC complexes were site-directedly labeled with flourescent dyes, and intermolecular single-molecule FRET analysis was carried out, from which a dissociation constant for a monomer/dimer equilibrium between 30 and 50 nM could be derived.

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Atto 488 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
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Atto 488-Biotin, BioReagent, suitable for fluorescence, ~75% (HPCE)
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Atto 488 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
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Atto 488 amine
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Atto 488 azide
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Atto 488 iodoacetamide

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