Biochemistry 2000 Apr 25;39(16):4831-4837
Spin Labeling Analysis of Structure and Dynamics of the
Na(+)/Proline Transporter of Escherichia coli.

Wegener C, Tebbe S, Steinhoff HJ, Jung H

Universitat Osnabruck, Fachbereich Biologie/Chemie, Arbeitsgruppe Mikrobiologie,
Barbarastrasse 11, D-49069 Osnabruck, Germany, and Lehrstuhl fur Biophysik,
Ruhr-Universitat Bochum, D-44780 Bochum, Germany.

With respect to the functional importance attributed to the N-terminal part of the
Na(+)/proline transporter of Escherichia coli (PutP), we report here on the structural
arrangement and functional dynamics of transmembrane domains (TMs) II and III and
the adjoining loop regions. Information on membrane topography was obtained by
 analyzing the residual mobility of site-specifically-attached nitroxide spin label and by
determination of collision frequencies of the nitroxide with oxygen and a polar metal ion
complex using electron paramagnetic resonance (EPR) spectroscopy. The studies
suggest that amino acids Phe45, Ser50, Ser54, Trp59, and Met62 are part of TM II
while Gly39 and Arg40 are located at a membrane-water interface probably forming
 the cytoplasmic cap of the TM. Also Ala67 and Glu75 are at a membrane-water
interface, suggesting a location close to the periplasmic ends of TMs II and III,
respectively. Ser71 between these residues is clearly in a water-exposed loop
(periplasmic loop 3). Spin labels attached to positions 80, 86, and 91 show EPR
properties typical for a TM location (TM III). Leu97 may be part of a structured loop
region while Ala107 is clearly located in a water-exposed loop (cytoplasmic loop 4).
Finally, spin labels attached to the positions of Asp33 and Leu37 are clearly on the
 surface of the transporter and are directed into an apolar environment. These findings
strongly support the recently proposed 13-helix model of PutP [Jung, H., Rubenhagen,
R., Tebbe, S., Leifker, K., Tholema, N., Quick, M., and Schmid, R. (1998) J. Biol.
 Chem. 273, 26400-26407] and suggest that TMs II and III of the transporter are
formed by amino acids Ser41 to Gly66 and Ser76 to Gly95, respectively. In addition to
the topology analysis, it is shown that binding of Na(+) and/or proline to the transporter
alters the mobility of the nitroxide group at the positions of Leu37 and Phe45. From
 these findings, it is concluded that binding of the ligands induces conformational
 alterations of PutP that involve at least parts of TM II and the preceding cytoplasmic