Probing of the rates of alternating access in LacY with Trp fluorescence.
Sugar/H(+) symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H(+) binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward- and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward- or outward-facing conformers. In the inward-facing conformer, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pK(a) of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is approximately 100 s(-1). On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-140-->Trp/Phe-334-->His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pK(a) = 8), but remarkably, the rate of sugar-induced quenching is only approximately 0.4 s(-1). The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins.
Smirnova
Irina
I
Department of Physiology, University of California, Los Angeles, CA 90095-7327, USA.
Kasho
Vladimir
V
Sugihara
Junichi
J
Kaback
H Ronald
HR
D003593:Cytoplasm / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D015335:Molecular Probes; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013395:Sucrose / Q000378:metabolism; D027981:Symporters / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D015335:Molecular Probes; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D006639:Histidine; D013395:Sucrose; D014364:Tryptophan