Structure and functional dynamics of the KdpFABC P-type ATPase from Escherichia coli

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Titel: Structure and functional dynamics of the KdpFABC P-type ATPase from Escherichia coli
Autor(en): Heitkamp, Thomas
Erstgutachter: Prof. Dr. Karlheinz Altendorf
Zweitgutachter: Prof. Dr. Robert Tampé
Zusammenfassung: The KdpFABC complex from E. coli functions as a high affinity K uptake system and belongs to the superfamily of P-type ATPases. So far, no information is available about the orientation of the subunits within the complex as well as its oligomeric state. By chemical crosslinking, gel filtration, electron transmission microscopy and single particle FRET analysis this study shows that the KdpFABC complex occurs as a homodimer with a dissociation constant between 30 to 50 nM. Furthermore, by means of single particle analysis of transmission electron micrographs, the solution structure of the complex at 1.9 nm resolution could be solved, thus providing the first structural analysis resolving all subunits of the holoenzyme. Based on crystal structures, it is generally assumed that P-type ATPases undergo large domain movements during catalysis. However, these conformational changes have never been shown directly. By use of single molecule FRET with alternating laser excitation, distance changes could be measured directly within KdpB during ATP hydrolysis. With this technique, distances and dwell times were determined for three conformational states in the working enzyme as well as in the orthovanadate- and the OCS-inhibited state.
URL: https://repositorium.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-2009042019
Schlagworte: KdpFABC; P-type ATPase; potassium transport; single particle analysis; electron microscopy; ALEX-FRET
Erscheinungsdatum: 17-Apr-2009
Enthalten in den Sammlungen:FB05 - E-Dissertationen

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