Engineering a terbium-binding site into an integral membrane protein for luminescence energy transfer.
Luminescence resonance energy transfer with a lanthanide like Tb(3+) as donor is a useful technique for estimating intra- and intermolecular distances in macromolecules. However, the technique usually requires the use of a bulky chelator with a flexible linker attached to a Cys residue to bind Tb(3+) and, for intramolecular studies, an acceptor fluorophor attached to another Cys residue in the same protein. Here, an engineered EF- hand motif is incorporated into the central cytoplasmic loop of the lactose permease of Escherichia coli generating a high-affinity site for Tb(3+) (K(Tb)(3+) approximately 4.5 microM) or Gd(3+) (K(Gd)(3+) approximately 2.3 microM). By exciting a Trp residue in the coordination sequence, Tb(3+) bound to the EF-hand motif is sensitized specifically, and the efficiency of energy transfer to strategically placed Cys residues labeled with fluorophors is measured. In this study, we use the technique to measure distance from the EF-hand in the central cytoplasmic loop of lactose permease to positions 179 or 169 at the center or periplasmic end of helix VI, respectively. The average calculated distances of approximately 23 A (position 179) and approximately 33 A (position 169) observed with three different fluorophors as acceptors agree well with the geometry of a slightly tilted alpha-helix. The approach should be of general use for studying static and dynamic aspects of polytopic membrane protein structure and function.
Vázquez-Ibar
José Luis
JL
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662, USA.
Weinglass
Adam B
AB
Kaback
H Ronald
HR
D001665:Binding Sites; D003593:Cytoplasm / Q000378:metabolism; D020832:EF Hand Motifs / Q000235:genetics; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D005453:Fluorescence; D008163:Luminescent Measurements; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D027981:Symporters; D013725:Terbium / Q000378:metabolism*
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; D013725:Terbium; C021499:lactose permease