Conformations of the rhodopsin third cytoplasmatic loop grafted onto bacteriorhodopsin. (2000)
Heymann, B., Pfeiffer, M., Hildebrandt, V., Kaback, H. R., Fotiadis, D., De Groot, B. L., ...
Whitelegge, J. P., Le Coutre, J., Lee, J. C., Engel, C. K., Privé, G. G., Faull, K. F., ...
Genes encoding membrane proteins comprise a substantial proportion of genomes sequenced to date, but ability to perform structural studies on this portion of the proteome is limited. Electrospray...
Wu, J, Voss, J, Hubbell, W L, Kaback, H R
Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII. To test this conclusion further, permease containing a biotin-acceptor domain...
Voss, J, Salwiński, L, Kaback, H R, Hubbell, W L
The use of molecular genetics to introduce both a metal ion binding site and a nitroxide spin label into the same protein opens the use of paramagnetic metalnitroxyl interactions to estimate...
Voss, J, Hubbell, W L, Kaback, H R
As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To...
Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage.
Wu, J, Perrin, D M, Sigman, D S, Kaback, H R
Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity...
Sahin-Tóth, M, Lawrence, M C, Kaback, H R
An engineered fusion protein containing two tandem lactose permease molecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance. Introduction...
Properties and purification of an active biotinylated lactose permease from Escherichia coli.
Consler, T G, Persson, B L, Jung, H, Zen, K H, Jung, K, Privé, G G, ...
A simplified approach for purification of functional lactose permease from Escherichia coli is described that is based on the construction of chimeras between the permease and a 100-amino acid...
Functional expression of mouse mdr1 in Escherichia coli.
We describe functional expression of the mouse multidrug-resistance protein (P-glycoprotein; P-gp) in an Escherichia coli mutant defective in the outer membrane protease ompT. Heterologously...
Functional complementation of internal deletion mutants in the lactose permease of Escherichia coli.
Using the lactose permease of Escherichia coli, a well-characterized membrane protein with 12 transmembrane domains, we demonstrated that certain paired in-frame deletion constructs complement each...
Bibi, E, Stearns, S M, Kaback, H R
When the lactose (lac) permease of Escherichia coli is expressed from the lac promoter at relatively low rates, deletion of amino acid residues 2-8 (delta 7) or 2-9 (delta 8) from the hydrophilic N...
Sahin-Tóth, M, Dunten, R L, Gonzalez, A, Kaback, H R
Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys. Individual replacements for Asp-237, Asp-240,...
Insertional mutagenesis of hydrophilic domains in the lactose permease of Escherichia coli.
McKenna, E, Hardy, D, Kaback, H R
The lactose permease of Escherichia coli is a membrane transport protein postulated to contain a hydrophilic N terminus (hydrophilic domain 1), 12 hydrophobic transmembrane alpha-helices that...
McKenna, E, Hardy, D, Pastore, J C, Kaback, H R
Previous experiments are consistent with the notion that residues 396-401 (... SVFTLS ...) at the carboxyl terminus of the last putative transmembrane helix of the lactose (lac) permease of...
Bibi, E, Verner, G, Chang, C Y, Kaback, H R
The overall topology of polytopic membrane proteins is thought to result from either the oriented insertion of the N-terminal alpha-helical domain followed by passive insertion of subsequent helices...
Histidine-94 is the only important histidine residue in the melibiose permease of Escherichia coli.
Pourcher, T, Sarkar, H K, Bassilana, M, Kaback, H R, Leblanc, G
Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the melB gene of Escherichia coli such that each of the seven His residues in the melibiose permease has been replaced...
Design of a membrane transport protein for fluorescence spectroscopy.
Menezes, M E, Roepe, P D, Kaback, H R
To modify the lac permease of Escherichia coli for fluorescence spectroscopy, six tryptophan residues at positions 10, 33, 78, 151, 171, and 223 were first replaced individually with phenylalanine by...
In vivo expression of the lacY gene in two segments leads to functional lac permease.
The lacY gene of Escherichia coli was cut into two approximately equal-size fragments with Afl II and subcloned individually or together under separate lac operator/promoters in plasmid pT7-5. Under...
Mates, S M, Patel, L, Kaback, H R, Miller, M H
The electrical potential (delta psi) across the cytoplasmic membranes of Staphylococcus aureus cells growing under aerobic and anaerobic conditions was determined by measuring the equilibrium...
Eisenberg, E S, Mandel, L J, Kaback, H R, Miller, M H
The relationship between the magnitude of the transmembrane electrical potential and the uptake of [14C]gentamicin was examined in wild-type Staphylococcus aureus in the logarithmic phase of growth....
REGULATION OF SUGAR TRANSPORT IN ISOLATED BACTERIAL MEMBRANE PREPARATIONS FROM Escherichia coli
The experiments presented in this paper demonstrate that glucose transport via the phosphoenolpyruvate-phosphotransferase system as studied in isolated E. coli membrane preparations is subject to...
Boonstra, J, Gutnick, D L, Kaback, H R
Transport properties of membrane vesicles isolated from two adenosine triphosphatase-deficient mutants of Escherichia coli, NR70 and DL54, were compared with those of vesicles prepared from the...
Vinylglycolate resistance in Escherichia coli.
Shaw, L, Grau, F, Kaback, H R, Hong, J S, Walsh, C
Escherichia coli K-12 vinylglycolate-resistant mutants have been isolated and characterized. Two of the mutants, JSH 150 and JSH 151, have been determined to be double mutants, lacking both...
lac permease of Escherichia coli containing a single histidine residue is fully functional.
Arg-302, His-322, and Glu-325, neighboring residues in putative helices IX and X of the lac permease (lacY gene product) of Escherichia coli, play an important role in lactose/H+ symport, possibly as...
Expression of the human erythrocyte glucose transporter in Escherichia coli.
Sarkar, H K, Thorens, B, Lodish, H F, Kaback, H R
The gene encoding the human erythrocyte glucose transporter, cloned from HepG2 hepatoma cells, was expressed in Escherichia coli by introducing a prokaryote-type ribosome binding site, subcloning the...
Phospholipase D (cabbage) inhibits the vectorial phosphorylation of α-methylglucoside by isolated membrane preparations from Escherichia coli ML 308-225 without increasing the efflux of...
The conversion of D-lactate to pyruvate in isolated membrane preparations of E. coli ML 308-225 markedly stimulates the transport of proline, glutamic acid, aspartic acid, aspargine, tryptophan,...
Roepe, P D, Zbar, R I, Sarkar, H K, Kaback, H R
The lac permease (lacY gene product) of Escherichia coli contains 417 amino acid residues and is predicted to have a short hydrophilic amino terminus on the inner surface of the cytoplasmic membrane,...
Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.
Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane...
Lac permease, a polytopic membrane protein from Escherichia coli, has been purified in soluble form by overexpressing the lacY gene by means of the T7 RNA polymerase system. Soluble permease is...
Relationship between the Na+/H+ antiporter and Na+/substrate symport in Bacillus alcalophilus.
Guffanti, A A, Cohn, D E, Kaback, H R, Krulwich, T A
The Na+/H+ antiporter of the obligate alkalophile Bacillus alcalophilus facilitates growth at alkaline pH and precludes growth below pH 8.5. Thus, nonalkalophilic mutant strains do not exhibit Na+/H+...
ATP-driven active transport in right-side-out bacterial membrane vesicles.
Hugenholtz, J, Hong, J S, Kaback, H R
Membrane vesicles from Salmonella typhimurium induced for phosphoglycerate transport, were loaded with pyruvate kinase and ADP by lysing spheroplasts under appropriate conditions. Vesicles so...
Barnes, Eugene M., Kaback, H. R.
The transport of β-galactosides by isolated membrane preparations from Escherichia coli strains containing a functional y gene is markedly stimulated by the conversion of D-lactate to pyruvate. The...
Schuldiner, S, Weil, R, Kaback, H R
High specific activity 6'-N-[3H]dansyl)aminohexyl 1-thio-beta-D-galactopyranoside (Dns6-Gal) has been synthesized, and its binding to Escherichia coli membrane vesicles measured directly by flow...
Goldkorn, T, Rimon, G, Kempner, E S, Kaback, H R
Cytoplasmic membrane vesicles prepared from Escherichia coli containing multiple copies of the lac y gene were frozen in liquid nitrogen before or after generation of a proton electrochemical...
Purified reconstituted lac carrier protein from Escherichia coli is fully functional.
Viitanen, P, Garcia, M L, Kaback, H R
Proteoliposomes reconstituted with lac carrier protein purified from the plasma membrane of Escherichia coli catalyze each of the translocation reactions typical of the beta-galactoside transport...
Membrane potential and gentamicin uptake in Staphylococcus aureus.
Mates, S M, Eisenberg, E S, Mandel, L J, Patel, L, Kaback, H R, Miller, M H
At pH 5.0, the electrical potential (delta psi, interior negative) across the plasma membrane of Staphylococcus aureus exhibits a minimum of -85 to -90 mV; the pH gradient (delta pH, interior...
Carrasco, N, Tahara, S M, Patel, L, Goldkorn, T, Kaback, H R
Monoclonal antibodies directed against the lac carrier protein purified from the membrane of Escherichia coli were prepared by somatic cell fusion of mouse myeloma cells with splenocytes from an...
Kaczorowski, G J, LeBlanc, G, Kaback, H R
4-Nitrophenyl-alpha-D-galactopyranoside (NPG) was used as a photoaffinity reagent to specifically inactivate the beta-galactoside transport system in Escherichia coli ML 308-225 membrane vesicles....
Factors influencing the accumulation of tetraphenylphosphonium cation in HeLa cells.
Hiller, R, Schaefer, A, Zibirre, R, Kaback, H R, Koch, G
Exposure of HeLa cells to tetraphenylphosphonium cation (TPP+) results in a rapid accumulation intracellularly, and a steady-state level is reached within 10 min. Accumulation of [3H]TPP+ in HeLa...
Mechanism of monensin-induced hyperpolarization of neuroblastoma-glioma hybrid NG108-15.
Lichtshtein, D, Dunlop, K, Kaback, H R, Blume, A J
Addition of the ionophore monensin to mouse neuroblastoma-rat glioma hybrid NG108-15 cells leads to a 20 to 30-mV increase in the electrical potential across the plasma membrane as shown by direct...
Matsushita, K, Patel, L, Gennis, R B, Kaback, H R
Most active transport across the bacterial cell membrane is driven by a proton electrochemical gradient (delta-muH+, interior negative and alkaline) generated via electron transfer through a...
Hong, Jen-Shiang, Kaback, H. R.
Two classes of mutants isolated from E. coli and Salmonella typhimurium are altered in respiration-coupled active transport, as studied in whole cells and/or isolated membrane vesicles. Mutant cells...
Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.
Padan, E, Sarkar, H K, Viitanen, P V, Poonian, M S, Kaback, H R
The lacY gene of Escherichia coli, which encodes the lac permease, has been modified by oligonucleotide-directed, site-specific mutagenesis such that each of the four histidine residues in the...
Carrasco, N, Herzlinger, D, Mitchell, R, DeChiara, S, Danho, W, Gabriel, T F, ...
A dodecapeptide corresponding to the carboxyl terminus of the lac carrier of Escherichia coli was synthesized, coupled to thyroglobulin, and the conjugate was used to generate site-directed...
Macrophage membrane potential changes associated with gamma 2b/gamma 1 Fc receptor-ligand binding.
Young, J D, Unkeless, J C, Kaback, H R, Cohn, Z A
We have studied the effects of specific ligands of the receptor for the IgG Fc fragment (FcR) on the membrane potential (delta psi) of the macrophage cell line J774 by the [3H]tetraphenylphosphonium...
Young, J D, Unkeless, J C, Kaback, H R, Cohn, Z A
We tested the effect of specific ligands to the mouse macrophage IgG gamma 2b/gamma 1 Fc fragment receptor (FcR) on ion permeability of plasma membrane vesicles prepared from J774 macrophages by...
Topology of the lac carrier protein in the membrane of Escherichia coli.
Goldkorn, T, Rimon, G, Kaback, H R
Proteolysis of topologically sealed right-side-out and inside-out membrane vesicles from Escherichia coli with chymotrypsin, trypsin, or papain inactivates lac carrier function in a symmetrical...
Hamamoto, T, Carrasco, N, Matsushita, K, Kaback, H R, Montal, M
Turnover of o-type cytochrome oxidase purified from Escherichia coli and reconstituted into proteoliposomes leads to the generation of a transmembrane electrical potential (interior negative) by...
Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles.
Padan, E, Patel, L, Kaback, H R
Exposure of Escherichia coli ML 308-225 membrane vesicles to the histidine-specific reagent diethylpyrocarbonate (DEPC) led to concentration- and time-dependent inactivation of active lactose...
Gordon, Adrienne S., Lombardi, F. J., Kaback, H. R.
A protein-containing fraction has been solubilized from E. coli ML 308-225 membrane vesicles that has many of the properties of the amino acid “carrier proteins” of the D-lactate...
Dansyl-Galactoside, a Fluorescent Probe of Active Transport in Bacterial Membrane Vesicles*
Reeves, John P., Shechter, Emanuel, Weil, Rudolf, Kaback, H. R.
A fluorescent galactoside, 2-(N-dansyl)-aminoethyl β-D-thiogalactoside (dansyl-galactoside), competitively inhibits lactose transport by membrane vesicles of Escherichia coli, but is not actively...
Ramos, S, Schuldiner, S, Kaback, H R
Membrane vesicles isolated from E. coli generate a trans-membrane proton gradient of 2 pH units under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to...
Microenvironment of the binding site in the lac carrier protein.
Schuldiner, S, Weil, R, Robertson, D E, Kaback, H R
Studies with a homologous series of (N-dansyl)aminoalkyl-1-thio-beta-D-galactopyranosides containing two to six methylene carbons bridging the galactosyl and dansyl ends of the molecules are...
Patel, L, Schuldiner, S, Kaback, H R
Extraction of E. coli ML 308-225 membrane vesicles with chaotropic agents causes the vesicles to become specifically permeable to protons. As a result, the vesicles no longer generate a membrane...
Addition of ubiquinone-1 to E. coli ML 308-225 membrane vesicles dramatically increases coupling between NADH oxidation and active transport such that initial rates and steady-state levels of lactose...
Short, S. A., Kaback, H. R., Kaczorowski, G., Fisher, J., Walsh, C. T., Silverstein, S. C.
Transport of vinylglycolate (2-hydroxy-3-butenoic acid) via the lactate transport system is the limiting step for covalent labeling of membrane vesicles prepared from E. coli ML 308-225. Thus, the...
Whitelegge, J. P., Le Coutre, J., Lee, J. C., Engel, C. K., Privé, G. G., Faull, K. F., ...
Genes encoding membrane proteins comprise a substantial proportion of genomes sequenced to date, but ability to perform structural studies on this portion of the proteome is limited. Electrospray...
Wu, J, Voss, J, Hubbell, W L, Kaback, H R
Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII. To test this conclusion further, permease containing a biotin-acceptor domain...
Voss, J, Salwiński, L, Kaback, H R, Hubbell, W L
The use of molecular genetics to introduce both a metal ion binding site and a nitroxide spin label into the same protein opens the use of paramagnetic metalnitroxyl interactions to estimate...
Voss, J, Hubbell, W L, Kaback, H R
As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To...
Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage.
Wu, J, Perrin, D M, Sigman, D S, Kaback, H R
Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity...
Sahin-Tóth, M, Lawrence, M C, Kaback, H R
An engineered fusion protein containing two tandem lactose permease molecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance. Introduction...
Properties and purification of an active biotinylated lactose permease from Escherichia coli.
Consler, T G, Persson, B L, Jung, H, Zen, K H, Jung, K, Privé, G G, ...
A simplified approach for purification of functional lactose permease from Escherichia coli is described that is based on the construction of chimeras between the permease and a 100-amino acid...
Functional expression of mouse mdr1 in Escherichia coli.
We describe functional expression of the mouse multidrug-resistance protein (P-glycoprotein; P-gp) in an Escherichia coli mutant defective in the outer membrane protease ompT. Heterologously...
Functional complementation of internal deletion mutants in the lactose permease of Escherichia coli.
Using the lactose permease of Escherichia coli, a well-characterized membrane protein with 12 transmembrane domains, we demonstrated that certain paired in-frame deletion constructs complement each...
Bibi, E, Stearns, S M, Kaback, H R
When the lactose (lac) permease of Escherichia coli is expressed from the lac promoter at relatively low rates, deletion of amino acid residues 2-8 (delta 7) or 2-9 (delta 8) from the hydrophilic N...
Sahin-Tóth, M, Dunten, R L, Gonzalez, A, Kaback, H R
Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys. Individual replacements for Asp-237, Asp-240,...
Insertional mutagenesis of hydrophilic domains in the lactose permease of Escherichia coli.
McKenna, E, Hardy, D, Kaback, H R
The lactose permease of Escherichia coli is a membrane transport protein postulated to contain a hydrophilic N terminus (hydrophilic domain 1), 12 hydrophobic transmembrane alpha-helices that...
McKenna, E, Hardy, D, Pastore, J C, Kaback, H R
Previous experiments are consistent with the notion that residues 396-401 (... SVFTLS ...) at the carboxyl terminus of the last putative transmembrane helix of the lactose (lac) permease of...
Bibi, E, Verner, G, Chang, C Y, Kaback, H R
The overall topology of polytopic membrane proteins is thought to result from either the oriented insertion of the N-terminal alpha-helical domain followed by passive insertion of subsequent helices...
Histidine-94 is the only important histidine residue in the melibiose permease of Escherichia coli.
Pourcher, T, Sarkar, H K, Bassilana, M, Kaback, H R, Leblanc, G
Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the melB gene of Escherichia coli such that each of the seven His residues in the melibiose permease has been replaced...
Design of a membrane transport protein for fluorescence spectroscopy.
Menezes, M E, Roepe, P D, Kaback, H R
To modify the lac permease of Escherichia coli for fluorescence spectroscopy, six tryptophan residues at positions 10, 33, 78, 151, 171, and 223 were first replaced individually with phenylalanine by...