Interresidual Distance Determination by Four-Pulse Double Electron-Electron Resonance in an Integral Membrane Protein: The Na+/Proline Transporter PutP of Escherichia coli
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Abstract
Proximity relationships within three doubly spin-labeled variants of the Na+/proline
transporter PutP of Escherichia coli were studied by means of four-pulse double electron-electron
resonance spectroscopy. The large value of 4.8 nm for the interspin distance determined
between positions 107 in loop 4 and 223 in loop 7 strongly supports the idea of these
positions being located on opposite sides of the membrane. Significant smaller values
of between 1.8 and 2.5 nm were found for the average interspin distances between spin
labels attached to the cytoplasmic loops 2 and 4 (position 37 and 107) and loops 2
and 6 (position 37 and 187). The large distance distribution widths visible in the
pair correlation functions reveal a high flexibility of the studied loop regions.
An increase of the distance between positions 37 and 187 upon Na+ binding suggests
ligand-induced structural alterations of PutP. The results demonstrate that four-pulse
double electron-electron resonance spectroscopy is a powerful means to investigate
the structure and conformational changes of integral membrane proteins reconstituted
in proteoliposomes.