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Interresidual Distance Determination by Four-Pulse Double Electron-Electron Resonance in an Integral Membrane Protein: The Na⁺/Proline Transporter PutP of Escherichia coli

Gunnar Jeschke ^*, Christoph Wegener , Monika Nietschke , Heinrich Jung  and Heinz-Jürgen Steinhoff 

^* Max-Planck-Institut für Polymerforschung, 55128 Mainz, Germany; Universität Osnabrück, Fachbereich Physik, D-49069 Osnabrück, Germany; and Universität Osnabrück, Fachbereich Biologie/Chemie, D-49069 Osnabrück, Germany

Correspondence: Address reprint requests to Heinz-Jürgen Steinhoff, Fachbereich Physik Universität Osnabrück, Barbarastrasse 7, 49069 Osnabrück, Germany. Tel.: 49-541-9692675; E-mail: hsteinho{at}uos.de.

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.

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