Single-molecule FRET reveals sugar-induced conformational dynamics in LacY (2007)
Nir, Eyal, Kong, Xiangxu, Weiss, Shimon, Kasho, Vladimir, Smirnova, Irina, Kaback, H. Ronald, ...
The N- and C-terminal six-helix bundles of lactose permease (LacY) form a large internal cavity open on the cytoplasmic side and closed on the periplasmic side with a single sugar-binding site at the...
Nie, Y L, Smirnova, I, Kasho, V, Kaback, H. Ronald
Isothermal titration calorimetry has been applied to characterize the thermodynamics of ligand binding to wild-type lactose permease ( LacY) and a mutant (C154G) that strongly favors an inward facing...
Vadyvaloo, V, Smirnova, I N, Kasho, V N, Kaback, H. Ronald
Building a three-dimensional model of the sucrose permease of Escherichia coli (CscB) with the X-ray crystal structure lactose permease (LacY) as template reveals a similar overall fold for CscB....
Access the most recent version at doi: 10.1110/ps.04657704 References (2004)
Eyal Vardy, Isaiah T. Arkin, Kay E. Gottschalk, H. Ronald Kaback, Shimon Schuldiner, Email Alerting
Structural conservation in the major facilitator superfamily as
Vardy, Eyal, Arkin, Isaiah T., Gottschalk, Kay E., Kaback, H. Ronald, Schuldiner, Shimon
The structures of membrane transporters are still mostly unsolved. Only recently, the first two high-resolution structures of transporters of the major facilitator superfamily (MFS) were published....
Iwaarden, Pierre R. Van, Driessen, Arnold J.M., Lolkema, Juke S., Kaback, H. Ronald, Konings, Wil N.
In this study, wild-type lac permease and lac permease mutated at each of the eight cysteinyl residues in the molecule were solubilized from the membrane, purified, and reconstituted into...
Iwaarden, Pierre R. Van, Driessen, Arnold J.M., Menick, Donald R., Kaback, H. Ronald, Konings, Wilhelmus N.
lac permease mutated at each of the 8 cysteinyl residues in the molecule was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The transport activity of proteoliposomes...
Kaback, H. Ronald, Carrasco, Nancy, Lolkema, Julius S.
Lactose exchange catalyzed by purified lac permease reconstituted into proteoliposomes was analyzed with unequal concentrations of lactose on either side of the membrane and at low pH so as to...
Carrasco, Nancy, Püttner, Irene B., Antes, Lisa M., Lee, Jonathan A., Larigan, J. Douglas, Lolkema, Julius S., ...
lac permease with Ala in place of Glu325 was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The reconstituted molecule is completely unable to catalyze lactose/H+...
Püttner, Irene B., Sarkar, Hemanta K., Padan, Etana, Lolkema, Julius S., Kaback, H. Ronald
Wild-type lac permease from Escherichia coli and two site-directed mutant permeases containing Arg in place of His35 and His39 or His322 were purified and reconstituted into proteoliposomes. H35-39R...
Site-Directed Mutagenesis of Pro327 in the lac Permease of Escherichia coli (1988)
Lolkema, Julius S., Püttner, Irene B., Kaback, H. Ronald
By use of oligonucleotide-directed, site-specific mutagenesis, Pro327 in the lac permease of Escherichia coli has been replaced with Ala, Gly, or Leu. Permease with Ala at position 327 catalyzes...
Weinglass, Adam B., Kaback, H. Ronald
Deletion of 5 residues (Δ5) from the central cytoplasmic loop of the lactose permease of Escherichia coli has no significant effect on expression or activity, whereas Δ12 leads to increased rates...
Conformational flexibility at the substrate binding site in the lactose permease of Escherichia coli
Weinglass, Adam B., Kaback, H. Ronald
Glu-126 (helix IV) and Arg-144 (helix V) are charge paired and play a critical role in substrate binding in the lactose permease of Escherichia coli. When Glu-126 is replaced with Asp, the permease...
A mechanism for the coupled translocation of substrate and H+ by the lactose permease of Escherichia coli is proposed, based on a variety of experimental observations. The permease is composed of 12...
The substrate-binding site in the lactose permease of Escherichia coli
Venkatesan, Pushpa, Kaback, H. Ronald
Site-directed N-ethylmaleimide labeling was studied with Glu-126 and/or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site....
Sondej, Melissa, Sun, Jianzhong, Seok, Yeong-Jae, Kaback, H. Ronald, Peterkofsky, Alan
Mediated by the protein IIAGlc, the phosphoenolpyruvate:sugar phosphotransferase system plays a role in the regulation of activity of other sugar transport systems in Escherichia coli. By using a...
The lipid bilayer determines helical tilt angle and function in lactose permease of Escherichia coli
Le Coutre, Johannes, Narasimhan, L. R., Patel, C. Kumar N., Kaback, H. Ronald
The structure of lactose permease from Escherichia coli in its lipid environment was studied by attenuated total reflection Fourier transform infrared spectroscopy. The protein exhibits an α-helical...
Unraveling the mechanism of the lactose permease of Escherichia coli
Sahin-Tóth, Miklós, Karlin, Arthur, Kaback, H. Ronald
We studied the effect of pH on ligand binding in wild-type lactose permease or mutants in the four residues—Glu-269, Arg-302, His-322, and Glu-325—that are the key participants in H+...
Le Coutre, Johannes, Kaback, H. Ronald, Patel, C. Kumar N., Heginbotham, Lise, Miller, Christopher
The structure of the tetrameric K+ channel from Streptomyces lividans in a lipid bilayer environment was studied by polarized attenuated total reflection Fourier transform infrared spectroscopy. The...
Topology of allosteric regulation of lactose permease
Seok, Yeong-Jae, Sun, Jianzhong, Kaback, H. Ronald, Peterkofsky, Alan
Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar...
Sahin-Tóth, Miklós, Kaback, H. Ronald
A mechanistic model for lactose/H+ symport via the lactose permease of Escherichia coli proposed recently indicates that the permease must be protonated to bind ligand with high affinity. Moreover,...
Guan, Lan, Murphy, Franklin D., Kaback, H. Ronald
Intermolecular thiol cross-linking was used to determine surface-exposed positions in 250 lactose permease mutants containing single-Cys replacements in each transmembrane helix. Significant...
Vázquez-Ibar, José Luis, Weinglass, Adam B., Kaback, H. Ronald
Luminescence resonance energy transfer with a lanthanide like Tb3+ as donor is a useful technique for estimating intra- and intermolecular distances in macromolecules. However, the technique usually...
Changing the lactose permease of Escherichia coli into a galactose-specific symporter
Guan, Lan, Sahin-Tóth, Miklós, Kaback, H. Ronald
N-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and...
An approach to membrane protein structure without crystals
Sorgen, Paul L., Hu, Yonglin, Guan, Lan, Kaback, H. Ronald, Girvin, Mark E.
The lactose permease of Escherichia coli catalyzes coupled translocation of galactosides and H+ across the cell membrane. It is the best-characterized member of the Major Facilitator Superfamily, a...
Elucidation of substrate binding interactions in a membrane transport protein by mass spectrometry
Weinglass, Adam B., Whitelegge, Julian P., Hu, Yonglin, Verner, Gillian E., Faull, Kym F., Kaback, H.Ronald
Integration of biochemical and biophysical data on the lactose permease of Escherichia coli has culminated in a molecular model that predicts substrate–protein proximities which include interaction...
Intermolecular thiol cross-linking via loops in the lactose permease of Escherichia coli
Ermolova, Natalia, Guan, Lan, Kaback, H. Ronald
Previous experiments using intermolecular thiol cross-linking to determine surface-exposed positions in the transmembrane helices of the lactose permease suggest that only positions accessible from...
Vázquez-Ibar, José Luis, Guan, Lan, Svrakic, Maja, Kaback, H. Ronald
The crystal structure of the Escherichia coli lactose permease at 3.5 Å with a bound substrate has been reported recently. The structure reveals the sugar–protein contacts, which include...
Membrane potential changes during mitogenic stimulation of mouse spleen lymphocytes
Kiefer, Hansruedi, Blume, Arthur J., Kaback, H. Ronald
By monitoring differences in accumulation of the lipophilic cation [3H]tetraphenylphosphonium in media containing low or high potassium concentrations [Lichtshtein, D., Kaback, H. R. & Blume, A. J....
Lichtshtein, David, Kaback, H. Ronald, Blume, Arthur Joel
Neuroblastoma-glioma hybrid cells (NG108-15) in suspension accumulate the permeant lipophilic cation [3H]tetraphenylphosphonium (TPP+) against a concentration gradient. The steady-state level of TPP+...
Short, Steven A., Kaback, H. Ronald, Kohn, Leonard D.
When membrane vesicles prepared from a D-lactate dehydrogenase mutant of E. coli ML 308-225 are treated with a homogeneous preparation of D-lactate dehydrogenase, the enzyme binds to the vesicles and...
Molecular structure of membrane vesicles from Escherichia coli
Owen, Peter, Kaback, H. Ronald
The molecular architecture of membrane vesicles prepared from Escherichia coli ML 308-225 has been studied by using crossed immunoelectrophoresis, and a reference pattern of 52 discrete...
Ramos, Sofia, Grollman, Evelyn F., Lazo, Pedro S., Dyer, Sherry A., Habig, William H., Hardegree, M. Carolyn, ...
Accumulation of the permeant lipophilic cation [3H]tetraphenylphosphonium (TPP+) by synaptosome preparations from guinea pig brain cerebral cortex is inhibited 1:10 by medium containing 193 mM K+ and...
Anaerobic Transport in Escherichia coli Membrane Vesicles*
Konings, Wilhelmus N., Kaback, H. Ronald
Anaerobic β-galactoside transport in whole cells and membrane vesicles from E. coli ML 308-225 is coupled to the oxidation of α-glycerol-P or D-lactate with fumarate as an electron acceptor....
Grollman, Evelyn F., Lee, George, Ambesi-Impiombato, F. S., Meldolesi, Maria F., Aloj, Salvatore M., Coon, Hayden G., ...
Cultured thyroid cells accumulate the lipophilic cation triphenylmethylphosphonium, indicating that there is an electrical potential (interior negative) across the plasma membrane. Thyrotropin...
Reeves, John P., Hong, Jen-Shiang, Kaback, H. Ronald
Membrane-bound, flavin-linked D-lactate dehydrogenase in membrane vesicles of E. coli ML 308-225 is solubilized by extraction with guanidine HCl. When membrane vesicles prepared from a D-lactate...
Binding affinity of lactose permease is not altered by the H+ electrochemical gradient
The x-ray structure of lactose permease of Escherichia coli (LacY) exhibits a single sugar-binding site at the apex of a hydrophilic cavity open to the cytoplasm, and it has been postulated that the...
Manipulating phospholipids for crystallization of a membrane transport protein
Guan, Lan, Smirnova, Irina N., Verner, Gill, Nagamori, Shushi, Kaback, H. Ronald
Crystallization is a major bottleneck to obtaining x-ray structures of membrane proteins. By applying an established crystallization protocol for the lactose permease (LacY) of Escherichia coli, a...
Weinglass, Adam B., Kaback, H. Ronald
Deletion of 5 residues (Δ5) from the central cytoplasmic loop of the lactose permease of Escherichia coli has no significant effect on expression or activity, whereas Δ12 leads to increased rates...
Conformational flexibility at the substrate binding site in the lactose permease of Escherichia coli
Weinglass, Adam B., Kaback, H. Ronald
Glu-126 (helix IV) and Arg-144 (helix V) are charge paired and play a critical role in substrate binding in the lactose permease of Escherichia coli. When Glu-126 is replaced with Asp, the permease...
A mechanism for the coupled translocation of substrate and H+ by the lactose permease of Escherichia coli is proposed, based on a variety of experimental observations. The permease is composed of 12...
The substrate-binding site in the lactose permease of Escherichia coli
Venkatesan, Pushpa, Kaback, H. Ronald
Site-directed N-ethylmaleimide labeling was studied with Glu-126 and/or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site....
Sondej, Melissa, Sun, Jianzhong, Seok, Yeong-Jae, Kaback, H. Ronald, Peterkofsky, Alan
Mediated by the protein IIAGlc, the phosphoenolpyruvate:sugar phosphotransferase system plays a role in the regulation of activity of other sugar transport systems in Escherichia coli. By using a...
The lipid bilayer determines helical tilt angle and function in lactose permease of Escherichia coli
Le Coutre, Johannes, Narasimhan, L. R., Patel, C. Kumar N., Kaback, H. Ronald
The structure of lactose permease from Escherichia coli in its lipid environment was studied by attenuated total reflection Fourier transform infrared spectroscopy. The protein exhibits an α-helical...
Unraveling the mechanism of the lactose permease of Escherichia coli
Sahin-Tóth, Miklós, Karlin, Arthur, Kaback, H. Ronald
We studied the effect of pH on ligand binding in wild-type lactose permease or mutants in the four residues—Glu-269, Arg-302, His-322, and Glu-325—that are the key participants in H+...
Le Coutre, Johannes, Kaback, H. Ronald, Patel, C. Kumar N., Heginbotham, Lise, Miller, Christopher
The structure of the tetrameric K+ channel from Streptomyces lividans in a lipid bilayer environment was studied by polarized attenuated total reflection Fourier transform infrared spectroscopy. The...
Topology of allosteric regulation of lactose permease
Seok, Yeong-Jae, Sun, Jianzhong, Kaback, H. Ronald, Peterkofsky, Alan
Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar...
Sahin-Tóth, Miklós, Kaback, H. Ronald
A mechanistic model for lactose/H+ symport via the lactose permease of Escherichia coli proposed recently indicates that the permease must be protonated to bind ligand with high affinity. Moreover,...
Guan, Lan, Murphy, Franklin D., Kaback, H. Ronald
Intermolecular thiol cross-linking was used to determine surface-exposed positions in 250 lactose permease mutants containing single-Cys replacements in each transmembrane helix. Significant...
Vázquez-Ibar, José Luis, Weinglass, Adam B., Kaback, H. Ronald
Luminescence resonance energy transfer with a lanthanide like Tb3+ as donor is a useful technique for estimating intra- and intermolecular distances in macromolecules. However, the technique usually...
Changing the lactose permease of Escherichia coli into a galactose-specific symporter
Guan, Lan, Sahin-Tóth, Miklós, Kaback, H. Ronald
N-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and...
An approach to membrane protein structure without crystals
Sorgen, Paul L., Hu, Yonglin, Guan, Lan, Kaback, H. Ronald, Girvin, Mark E.
The lactose permease of Escherichia coli catalyzes coupled translocation of galactosides and H+ across the cell membrane. It is the best-characterized member of the Major Facilitator Superfamily, a...
Elucidation of substrate binding interactions in a membrane transport protein by mass spectrometry
Weinglass, Adam B., Whitelegge, Julian P., Hu, Yonglin, Verner, Gillian E., Faull, Kym F., Kaback, H.Ronald
Integration of biochemical and biophysical data on the lactose permease of Escherichia coli has culminated in a molecular model that predicts substrate–protein proximities which include interaction...
Intermolecular thiol cross-linking via loops in the lactose permease of Escherichia coli
Ermolova, Natalia, Guan, Lan, Kaback, H. Ronald
Previous experiments using intermolecular thiol cross-linking to determine surface-exposed positions in the transmembrane helices of the lactose permease suggest that only positions accessible from...
Vázquez-Ibar, José Luis, Guan, Lan, Svrakic, Maja, Kaback, H. Ronald
The crystal structure of the Escherichia coli lactose permease at 3.5 Å with a bound substrate has been reported recently. The structure reveals the sugar–protein contacts, which include...
Membrane potential changes during mitogenic stimulation of mouse spleen lymphocytes
Kiefer, Hansruedi, Blume, Arthur J., Kaback, H. Ronald
By monitoring differences in accumulation of the lipophilic cation [3H]tetraphenylphosphonium in media containing low or high potassium concentrations [Lichtshtein, D., Kaback, H. R. & Blume, A. J....
Lichtshtein, David, Kaback, H. Ronald, Blume, Arthur Joel
Neuroblastoma-glioma hybrid cells (NG108-15) in suspension accumulate the permeant lipophilic cation [3H]tetraphenylphosphonium (TPP+) against a concentration gradient. The steady-state level of TPP+...
Short, Steven A., Kaback, H. Ronald, Kohn, Leonard D.
When membrane vesicles prepared from a D-lactate dehydrogenase mutant of E. coli ML 308-225 are treated with a homogeneous preparation of D-lactate dehydrogenase, the enzyme binds to the vesicles and...
Molecular structure of membrane vesicles from Escherichia coli
Owen, Peter, Kaback, H. Ronald
The molecular architecture of membrane vesicles prepared from Escherichia coli ML 308-225 has been studied by using crossed immunoelectrophoresis, and a reference pattern of 52 discrete...
Ramos, Sofia, Grollman, Evelyn F., Lazo, Pedro S., Dyer, Sherry A., Habig, William H., Hardegree, M. Carolyn, ...
Accumulation of the permeant lipophilic cation [3H]tetraphenylphosphonium (TPP+) by synaptosome preparations from guinea pig brain cerebral cortex is inhibited 1:10 by medium containing 193 mM K+ and...
Anaerobic Transport in Escherichia coli Membrane Vesicles*
Konings, Wilhelmus N., Kaback, H. Ronald
Anaerobic β-galactoside transport in whole cells and membrane vesicles from E. coli ML 308-225 is coupled to the oxidation of α-glycerol-P or D-lactate with fumarate as an electron acceptor....
Grollman, Evelyn F., Lee, George, Ambesi-Impiombato, F. S., Meldolesi, Maria F., Aloj, Salvatore M., Coon, Hayden G., ...
Cultured thyroid cells accumulate the lipophilic cation triphenylmethylphosphonium, indicating that there is an electrical potential (interior negative) across the plasma membrane. Thyrotropin...
Reeves, John P., Hong, Jen-Shiang, Kaback, H. Ronald
Membrane-bound, flavin-linked D-lactate dehydrogenase in membrane vesicles of E. coli ML 308-225 is solubilized by extraction with guanidine HCl. When membrane vesicles prepared from a D-lactate...
Binding affinity of lactose permease is not altered by the H+ electrochemical gradient
The x-ray structure of lactose permease of Escherichia coli (LacY) exhibits a single sugar-binding site at the apex of a hydrophilic cavity open to the cytoplasm, and it has been postulated that the...
Manipulating phospholipids for crystallization of a membrane transport protein
Guan, Lan, Smirnova, Irina N., Verner, Gill, Nagamori, Shushi, Kaback, H. Ronald
Crystallization is a major bottleneck to obtaining x-ray structures of membrane proteins. By applying an established crystallization protocol for the lactose permease (LacY) of Escherichia coli, a...
Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY
Mirza, Osman, Guan, Lan, Verner, Gill, Iwata, So, Kaback, H Ronald
Cation-coupled active transport is an essential cellular process found ubiquitously in all living organisms. Here, we present two novel ligand-free X-ray structures of the lactose permease (LacY) of...
Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY
Mirza, Osman, Guan, Lan, Verner, Gill, Iwata, So, Kaback, H Ronald
Correction to: The EMBO Journal (2006) 25.6, 1177–1183. doi:10.1038/sj.emboj.7601028; Published online 9 March 2006
Site-directed alkylation and the alternating access model for LacY
Kaback, H. Ronald, Dunten, R., Frillingos, S., Venkatesan, P., Kwaw, I., Zhang, W., ...
In a functional lactose permease mutant from Escherichia coli (LacY) devoid of native Cys residues, almost every residue was replaced individually with Cys and tested for reactivity with the permeant...
SecY alterations that impair membrane protein folding and generate a membrane stress
Shimohata, Nobuyuki, Nagamori, Shushi, Akiyama, Yoshinori, Kaback, H. Ronald, Ito, Koreaki
We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/σE stress response pathways. Similar stress induction was also...
Single-molecule FRET reveals sugar-induced conformational dynamics in LacY
Majumdar, Devdoot S., Smirnova, Irina, Kasho, Vladimir, Nir, Eyal, Kong, Xiangxu, Weiss, Shimon, ...
The N- and C-terminal six-helix bundles of lactose permease (LacY) form a large internal cavity open on the cytoplasmic side and closed on the periplasmic side with a single sugar-binding site at the...
Role of YidC in folding of polytopic membrane proteins
Nagamori, Shushi, Smirnova, Irina N., Kaback, H. Ronald
YidC of Echerichia coli, a member of the conserved Alb3/Oxa1/YidC family, is postulated to be important for biogenesis of membrane proteins. Here, we use as a model the lactose permease (LacY), a...
Structural determination of wild-type lactose permease
Guan, Lan, Mirza, Osman, Verner, Gillian, Iwata, So, Kaback, H. Ronald
Here we describe an x-ray structure of wild-type lactose permease (LacY) from Escherichia coli determined by manipulating phospholipid content during crystallization. The structure exhibits the same...
Structural conservation in the major facilitator superfamily as revealed by comparative modeling
Vardy, Eyal, Arkin, Isaiah T., Gottschalk, Kay E., Kaback, H. Ronald, Schuldiner, Shimon
The structures of membrane transporters are still mostly unsolved. Only recently, the first two high-resolution structures of transporters of the major facilitator superfamily (MFS) were published....
Sugar binding induces an outward facing conformation of LacY
Smirnova, Irina, Kasho, Vladimir, Choe, Jun-Yong, Altenbach, Christian, Hubbell, Wayne L., Kaback, H. Ronald
According to x-ray structure, the lactose permease (LacY) is a monomer organized into N- and C-terminal six-helix bundles that form a deep internal cavity open on the cytoplasmic side with a single...
Dong, Yuxia, Palmer, Sara R., Hasona, Adnan, Nagamori, Shushi, Kaback, H. Ronald, Dalbey, Ross E., ...
Oxa/YidC/Alb family proteins are chaperones involved in membrane protein insertion and assembly. Streptococcus mutans has two YidC paralogs. Elimination of yidC2, but not yidC1, results in stress...
Opening and closing of the periplasmic gate in lactose permease
Zhou, Yonggang, Guan, Lan, Freites, J. Alfredo, Kaback, H. Ronald
X-ray crystal structures of lactose permease (LacY) reveal pseudosymmetrically arranged N- and C-terminal six-transmembrane helix bundles surrounding a deep internal cavity open on the cytoplasmic...
Electrophysiological characterization of LacY
Garcia-Celma, Juan J., Smirnova, Irina N., Kaback, H. Ronald, Fendler, Klaus
Electrogenic events due to the activity of wild-type lactose permease from Escherichia coli (LacY) were investigated with proteoliposomes containing purified LacY adsorbed on a solid-supported...
2007 Annual progress report synopsis of the Center for Structures of Membrane Proteins
Stroud, Robert M., Choe, Senyon, Holton, James, Kaback, H. Ronald, Kwiatkowski, Witek, Minor, Daniel L., ...
A synopsis of the 2007 annual progress report for the Center for Structures of Membrane Proteins, a specialized center of the Protein Structure Initiative.