oxalates and nitroxyl

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., Rübenhagen, 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 loop.

Topics: Amino Acid Sequence; Amino Acid Transport Systems, Neutral; Cysteine; Electron Spin Resonance Spectroscopy; Escherichia coli; Ligands; Membrane Proteins; Membrane Transport Proteins; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitrogen Oxides; Oxalates; Oxygen; Proline; Protein Conformation; Proteolipids; Sodium; Spin Labels

Colicin E1 is an E. coli plasmid-encoded water-soluble protein that spontaneously inserts into lipid membranes to form a voltage-gated ion channel. We have employed a novel approach in which site-directed mutagenesis is used to provide highly specific attachment points for nitroxide spin labels. A series of colicin mutants, differing only by the position of a single cysteine residue, were prepared and selectively labeled at that cysteine. A hydrophilic sequence (398-406) within the C-terminal domain of the water-soluble form of the protein was investigated and exhibited an electron paramagnetic resonance (EPR) spectral periodicity strongly suggesting an amphiphilic alpha-helix. After removal of the N-terminus of the protein with trypsin, the spectra for this sequence indicate increased label mobility and a more flexible structure.

Topics: Amino Acid Sequence; Colicins; Cysteine; Electron Spin Resonance Spectroscopy; Escherichia coli; Molecular Sequence Data; Mutation; Nitrogen Oxides; Oxalates; Oxalic Acid; Protein Conformation; Spin Labels; Trypsin