Binding of p-nitrophenyl-.alpha.-D-galactopyranoside to lac permease of Escherichia coli

Lolkema, Julius S.; Walz, D.

doi:10.1021/bi00503a005

Cited by 15 publications

(23 citation statements)

References 25 publications

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“…Scatchard analysis of TDG enhancement of MIANS-labeled W33C permease fluorescence reveals a single binding site with an apparent K O of 71 NM, which coincides with the high-affinity site described previously (Lolkema & Walz, 1990;Lolkema et al, 1991;van Iwaarden et al, 1993;Wu et al, 1995b). Therefore, it appears that occupancy of a high-affinity site in lactose permease causes position 33 to move into a more hydrophobic environment that is less accessible to MIANS, and positions 28 and 3 1 to move into a more hydrophilic environment that is more accessible to the probe.…”

Section: Discussionsupporting

confidence: 83%

See 1 more Smart Citation

Fluorescence of native single‐Trp mutants in the lactose permease from Escherichia coli: Structural properties and evidence for a substrate‐induced conformational change

1995

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Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Nut1 Acud Sci USA 87: 1638-1642), and fluorescent properties were studied with respect to solvent accessi- Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257-8263) that ligand binding induces a conformational change at the C-terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.

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Section: Discussionsupporting

confidence: 83%

“…The fluorescence of MIANS-labeled W33C permease (0.75 pM) was measured as described above, and bound TDG was calculated from the fluorescence increase measured at given TDG concentrations. It was assumed that at saturation one mol of TDG is bound per mol of permease (Lolkema & Walz, 1990). …”

Section: Ligand Bindingmentioning

confidence: 99%

Fluorescence of native single‐Trp mutants in the lactose permease from Escherichia coli: Structural properties and evidence for a substrate‐induced conformational change

1995

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“…Kinetic measurements, binding and site-directed labeling studies indicate that lac permease has two binding sites (13,14,(27)(28)(29). The site-directed fluorescence studies reported here demonstrate that mutants E126A͞V331C, R144A͞V331C, or R144K͞V331C are defective in both sites.…”

Section: Figmentioning

confidence: 49%

The substrate-binding site in the lactose permease of Escherichia coli

Venkatesan

Kaback

1998

Proc. Natl. Acad. Sci. U.S.A.

120

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Site-directed N-ethylmaleimide labeling was studied with Glu-126 and͞or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site. Replacement of either Glu-126 or Arg-144 with Ala markedly decreases Cys-148 reactivity, whereas interchanging the residues, double-Ala replacement, or replacement of Arg-144 with Lys or His does not alter reactivity, indicating that Glu-126 and Arg-144 are charge-paired. Importantly, although alkylation of Cys-148 is blocked by ligand in wild-type permease, no protection whatsoever is observed with any of the Glu-126 or Arg-144 mutants. Site-directed f luorescence with 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS) in mutant Val-331 3 Cys was also studied. In marked contrast to Val-331 3 Cys permease, ligand does not alter MIANS reactivity in mutant Glu-126 3 Ala͞Val-331 3 Cys, Arg-144 3 Ala͞Val-331 3 Cys, or Arg-144 3 Lys͞Val-331 3 Cys and does not cause either quenching or a shift in the emission maximum of the MIANS-labeled mutants. However, mutation Glu-126 3 Ala or Arg-144 3 Ala and, to a lesser extent, Arg-144 3 Lys cause a red-shift in the emission spectrum and render the f luorophore more accessible to I ؊ . The results demonstrate that Glu-126 and Arg-144 are irreplaceable for substrate binding and suggest a model for the substrate-binding site in the permease. In addition, the findings are consistent with the notion that alterations in the substrate translocation pathway at the interface between helices IV and V are transmitted conformationally to the H ؉ translocation pathway at the interface between helices IX and X.The lactose permease (lac permease) of Escherichia coli is representative of secondary active transport proteins that convert free energy stored in electrochemical ion gradients into work in the form of a concentration gradient (reviewed in ref. 1). This hydrophobic, polytopic, cytoplasmic membrane protein catalyzes the coupled stoichiometric translocation of ␤-galactosides and H ϩ . The lacY gene that encodes the permease has been cloned and sequenced, and the product of the lacY gene has been solubilized, purified, reconstituted into proteoliposomes, and shown to be solely responsible for ␤-galactoside transport as a monomer (see ref.2). All available evidence indicates that the permease consists of 12 hydrophobic, membrane-spanning, ␣-helical domains connected by hydrophilic loops with both the N and C termini on the cytoplasmic face of the membrane (Fig. 1) (reviewed in refs. 3 and 4).Site-directed mutagenesis of wild-type permease and Cysscanning mutagenesis of a functional mutant devoid of Cys residues (C-less permease) reveals that 4 of 417 residues in the protein are irreplaceable with respect to coupling between lactose and H ϩ translocation-Glu-269 (helix VIII), Arg-302 (helix IX), His-322 (helix X), and Glu-325 (helix X) (reviewed in refs. 3-5). Although the permease has not been crystallized, application of a battery of site-directed biochemical and biophysical te...

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“…Binding studies and nonequilibrium exchange measurements have revealed two distinct binding sites per lac permease molecule (monomer) for NPG and lactose (Lolkema & Walz, 1990;Lolkema et al, 1991). The findings that two kinetic phases are observed with respect to external lactose during exchange but only one phase with respect to internal lactose suggest that one site is located on the periplasmic aspect of the permease.…”

Section: Discussionmentioning

confidence: 99%

Exchange, efflux and substrate binding by cysteine mutants of the lactose permease of Escherichia coli

Iwaarden

Driessen²,

Lolkema³

et al. 1993

Biochemistry

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Binding of p-nitrophenyl-.alpha.-D-galactopyranoside to lac permease of Escherichia coli

Abstract: Registry No. d(AC)4-d(GT)4, 81609-64-5.

Cited by 15 publications

References 25 publications

Fluorescence of native single‐Trp mutants in the lactose permease from Escherichia coli: Structural properties and evidence for a substrate‐induced conformational change

Fluorescence of native single‐Trp mutants in the lactose permease from Escherichia coli: Structural properties and evidence for a substrate‐induced conformational change

The substrate-binding site in the lactose permease of Escherichia coli

Exchange, efflux and substrate binding by cysteine mutants of the lactose permease of Escherichia coli

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