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Biochemistry Jan 1998The magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances within proteins [Voss, J., Salwinski, L., Kaback, H....
The magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances within proteins [Voss, J., Salwinski, L., Kaback, H. R., and Hubbell, W. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12295-12299; Voss, J., Hubbell, W. L., and Kaback, H. R. (1995) Proc. Natl. Acad. Sci. U.S. A. 92, 12300-12303], and the approach is utilized here to measure helix proximities in the lactose permease of Escherichia coli. A high-affinity divalent metal binding site was created by replacing Arg302 (helix IX) and Glu325 (helix X) with His residues in permease mutants containing single Cys residues in helices II, V, or VII and a biotin acceptor domain to facilitate purification. Mutant proteins were purified by avidin affinity chromatography, labeled specifically with a nitroxide free radical and investigated by electron paramagnetic resonance spectroscopy in the absence or presence of Cu(II). Spectral broadening due to bound Cu(II) was used to estimate distances between the metal center and the spin-labeled side chains. For each of the transmembrane domains probed, the variation in interspin distance with sequence position is consistent with an alpha-helical structure. The measured distances were also used to construct a model that is in good agreement with packing data obtained from other approaches.
Topics: Bacterial Proteins; Cations, Divalent; Copper; Electron Spin Resonance Spectroscopy; Escherichia coli; Escherichia coli Proteins; Membrane Transport Proteins; Models, Molecular; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Nitrogen Oxides; Protein Structure, Secondary; Spin Labels; Symporters
PubMed: 9425041
DOI: 10.1021/bi972152l -
Biochemistry Jun 1995A substrate for lactose permease of Escherichia coli was synthesized that binds to the protein with a relatively high affinity, but is not transported to any detectable...
A substrate for lactose permease of Escherichia coli was synthesized that binds to the protein with a relatively high affinity, but is not transported to any detectable extent. This substrate, 6'-[(N-phenylalanylphenylalanyl)amino]hexyl 1-thio-beta-D-galactoside, is a peptide galactoside composed of a bulky aromatic dipeptide that is linked to galactose via an aminohexyl spacer. Binding of the peptide galactoside to lactose permease in cytoplasmic membranes was determined in a competition assay yielding a dissociation constant of 150 microM. Transport was measured by a counterflow assay using lipid vesicles with reconstituted lactose permease. An upper limit for the rate constant of transport was obtained as 0.02 s-1, 3 orders of magnitude smaller than the value for lactose.
Topics: Biological Transport; Dipeptides; Escherichia coli; Escherichia coli Proteins; Galactosides; Membrane Transport Proteins; Models, Chemical; Monosaccharide Transport Proteins; Proteolipids; Symporters; Thiogalactosides
PubMed: 7794892
DOI: 10.1021/bi00024a005 -
The Journal of Biological Chemistry Apr 1996The fructose permease of Escherichia coli, the fructose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), contains a duplicated...
The fructose permease of Escherichia coli, the fructose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), contains a duplicated IIB domain. The protein therefore consists of three distinct domains, B', B, and C (N-terminal to C-terminal), joined by flexible linkers and is thus designated FruB'BC. The N-terminal B' domain was removed using molecular genetic techniques, and the truncated Enzyme II (FruBC) was characterized relative to the wild-type enzyme both in vivo and in vitro. In vivo, FruBC exhibited depressed fermentation characteristics at low fructose concentrations. [14C]Fructose uptake measurements revealed reduced rates only when the permease was rate-limiting for transport. In vitro, FruBC exhibited a 10-fold lower affinity for its phosphoryl donating protein, the IIA-FPr diphosphoryl transfer protein (DTP), than was observed with the wild-type enzyme, and the maximal velocity of fructose phosphorylation was 7-fold depressed. Because the fructose-1-phosphate:[14C]fructose transphosphorylation reaction appeared normal, we conclude that the loss of the B' domain primarily affected phosphoryl transfer between the IIA and IIB domains of the permease. A mutant FruBC derivative with cysteine 112 replaced by serine (C112S FruBC) was inactive as a phosphoryl carrier and a sugar transport protein. Expression of the plasmid-encoded mutant protein inhibited the in vivo activity of the chromosomally encoded wild-type fructose permease, but it did not observably affect the activities of the mannitol or glucitol PTS permeases or of non-PTS sugar permeases. Further, the presence of the detergent extracted mutant protein inhibited the activity of the detergent solubilized wild-type or FruBC enzyme. In contrast, the wild-type FruB BC permease was apparently epistatic over the truncated FruBC permease in vivo. The experiments reported 1) show that the B' domain of the fructose permease functions to facilitate phosphoryl transfer between DTP and the permease, 2) establish the essentiality of cysteine 112 in the B domain of the permease, 3) provide evidence that a functional fructose permease consists of an oligomer in which both IIB domains must be active for the enzyme to catalyze normal rates of phosphoryl transfer and transport, 4) suggest that a single B' domain in the oligomeric Enzyme II is sufficient to allow high efficiency phosphoryl transfer between the IIA domain of DTP and the IIB domain of the permease, and 5) show that the B' domain is not important for oligomerization.
Topics: Bacterial Proteins; Base Sequence; Binding, Competitive; Biological Transport; DNA Primers; Escherichia coli; Fermentation; Fructose; Fructosephosphates; Genes, Dominant; Kinetics; Membrane Transport Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation; Protein Conformation; Structure-Activity Relationship
PubMed: 8626640
DOI: 10.1074/jbc.271.17.9997 -
Gene May 2013One of the oldest known gene clusters that are involved in biological oxidation processes is the sox operon. This operon is present in different microbial species. In...
One of the oldest known gene clusters that are involved in biological oxidation processes is the sox operon. This operon is present in different microbial species. In the present study an attempt has been made to analyze the probable structural role of SoxT protein from Pseudaminobacter salicylatoxidans. This protein has been predicted to be a permease-like protein. A comparative model of the protein has been made and analyzed. The possible membrane spanning region of the protein has been detected by structural bioinformatics approach. The inducer of the sulfur oxidation process has been predicted. And thereby the plausible mechanism of the transport of the sulfur anion inside the bacterial cell has been elucidated. Since this is the first study regarding the structural aspect of the protein this study may shed light on the theory of the yet unknown molecular mechanism of the sulfur oxidation process by sox operon.
Topics: Bacterial Proteins; Catalytic Domain; Cell Membrane; Membrane Transport Proteins; Models, Molecular; Operon; Phyllobacteriaceae; Protein Conformation; Sulfur
PubMed: 23500599
DOI: 10.1016/j.gene.2013.02.038 -
Scientific Reports Oct 2017Lipids play key roles in Biology. Mechanical properties of the lipid bilayer influence their neighbouring membrane proteins, however it is unknown whether different...
Lipids play key roles in Biology. Mechanical properties of the lipid bilayer influence their neighbouring membrane proteins, however it is unknown whether different membrane protein properties have the same dependence on membrane mechanics, or whether mechanics are tuned to specific protein processes of the protein. We study the influence of lipid lateral pressure and electrostatic effects on the in vitro reconstitution, folding, stability and function of a representative of the ubiquitous major facilitator transporter superfamily, lactose permease. Increasing the outward chain lateral pressure in the bilayer, through addition of lamellar phosphatidylethanolamine lipids, lowers lactose permease folding and reconstitution yields but stabilises the folded state. The presence of phosphatidylethanolamine is however required for correct folding and function. An increase in headgroup negative charge through the addition of phosphatidylglycerol lipids favours protein reconstitution but is detrimental to topology and function. Overall the in vitro folding, reconstitution, topology, stability and function of lactose permease are found to have different dependences on bilayer composition. A regime of lipid composition is found where all properties are favoured, even if suboptimal. This lays ground rules for rational control of membrane proteins in nanotechnology and synthetic biology by manipulating global bilayer properties to tune membrane protein behaviour.
Topics: Lipid Bilayers; Membrane Transport Proteins; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Folding; Protein Stability
PubMed: 29026149
DOI: 10.1038/s41598-017-13290-7 -
Biochemistry Jul 2006Combining structure determinations from nuclear magnetic resonance (NMR) data and molecular dynamics simulations (MD) under the same environmental conditions revealed a... (Comparative Study)
Comparative Study
Combining structure determinations from nuclear magnetic resonance (NMR) data and molecular dynamics simulations (MD) under the same environmental conditions revealed a startling asymmetry in the intrinsic conformational stability of secondary structure in the transmembrane domain of lactose permease (LacY). Eleven fragments, corresponding to transmembrane segments (TMs) of LacY, were synthesized, and their secondary structure in solution was determined by NMR. Eight of the TMs contained significant regions of helical structure. MD simulations, both in DMSO and in a DMPC bilayer, showed sites of local stability of helical structure in these TMs, punctuated by regions of conformational instability, in substantial agreement with the NMR data. Mapping the stable regions onto the crystal structure of LacY reveals a marked asymmetry, contrasting with the pseudosymmetry in the static structure: the secondary structure in the C-terminal half is more stable than in the N-terminal half. The relative stability of secondary structure is likely exploited in the transport mechanism of LacY. Residues supporting proton conduction are in more stable regions of secondary structure, while residues key to substrate binding are found in considerably unstable regions of secondary structure.
Topics: Amino Acid Sequence; Magnetic Resonance Spectroscopy; Membrane Transport Proteins; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Protein Structure, Secondary
PubMed: 16800633
DOI: 10.1021/bi060355g -
European Journal of Biochemistry Aug 1996Ser57 in the Na+/proline permease of Escherichia coli has been replaced with alanine, cysteine, glycine, or threonine, and properties of the corresponding putP mutants...
Ser57 in the Na+/proline permease of Escherichia coli has been replaced with alanine, cysteine, glycine, or threonine, and properties of the corresponding putP mutants have been analyzed. Although Ser57 is not essential for activity, the amino acid side chain at this position is critical for proline uptake. Thus, alanine, cysteine, glycine, or threonine in place of Ser57 reduces the initial rate of proline transport under standard conditions to less than 10% of the wild-type value. In addition, substitution of Ser57 in the Na+/proline permease reduces the sensitivity of E. coli cells to the toxic proline analogs L-azetidine-2-carboxylate and 3.4-dehydro-D.L-proline. Replacement of Ser57 with alanine or cysteine results in apparent affinities for proline that are reduced by more than two orders of magnitude, and permeases with threonine and glycine in place of Ser57 yield apparent affinities reduced by a factor of 60 and 18 respectively, relative to wild-type. In contrast, all of the Ser57 replacements analyzed cause only small changes in Vmax values. All permease molecules containing Ser57 substitutions are inserted into the membrane in amounts comparable to the wild-type protein as shown by immunoblot analysis. These results indicate that alterations of proline transport and sensitivity to toxic proline analogs have to be attributed primarily to defects in substrate binding. It is suggested that the serine residue at position 57 of the permease is located within the substrate-binding domain of the protein.
Topics: Amino Acid Sequence; Amino Acid Transport Systems, Neutral; Binding Sites; Biological Transport; Cell Compartmentation; Escherichia coli; Genetic Complementation Test; Kinetics; Membrane Transport Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Proline; Sequence Analysis, DNA; Serine
PubMed: 8774720
DOI: 10.1111/j.1432-1033.1996.0732u.x -
Proceedings of the National Academy of... Aug 1989Lac 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...
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 dissociated from membranes with urea or other chaotropes and appears after the membrane is saturated with newly synthesized permease. Remarkably, this form of the permease appears to remain soluble in phosphate buffer at neutral pH after removal of urea, although it aggregates in a time- and concentration-dependent manner. Importantly, soluble permease behaves as a monomer during size-exclusion chromatography with or without urea, contains less than 3 mol of organic phosphate per mol of protein, and is largely helical. Soluble permease binds p-nitrophenyl alpha-D-galactopyranoside approximately 40% as well as permease in the native environment of the membrane and can be reconstituted into phospholipid vesicles that catalyze lactose counterflow or active transport in response to a membrane potential (interior negative). The results suggest that lac permease can assume a nondenatured conformation in aqueous solution.
Topics: Cell Membrane; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Kinetics; Lactose; Membrane Proteins; Membrane Transport Proteins; Molecular Weight; Monosaccharide Transport Proteins; Solubility; Symporters
PubMed: 2668955
DOI: 10.1073/pnas.86.16.6087 -
Current Genetics 1986The yeast CAN1 gene, thought to encode arginine permease, has found use in genetics as a selectable locus. We have sequenced the cloned CAN1 gene, which contains an open...
The yeast CAN1 gene, thought to encode arginine permease, has found use in genetics as a selectable locus. We have sequenced the cloned CAN1 gene, which contains an open reading frame of 1770 nucleotides, encoding a polypeptide of calculated molecular weight 65,766. Disruption of this open reading frame largely abolishes CAN1 gene expression, while subcloned fragments of the open reading frame hybridize strand-specifically to a 2.3 kb yeast RNA message. The encoded protein has no leader signal sequence, and is highly hydrophobic, with a possible twelve membrane-spanning domains, several of which have the high hydrophobic moments seen in channel-forming or permease proteins. This protein structure is consistent with the CAN1 product being the plasma membrane arginine permease.
Topics: Amino Acid Sequence; Amino Acid Transport Systems; Amino Acid Transport Systems, Basic; Base Sequence; Cloning, Molecular; Genes; Genes, Fungal; Membrane Proteins; Membrane Transport Proteins; Molecular Sequence Data; Protein Conformation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Solubility; Transcription, Genetic
PubMed: 3327612
DOI: 10.1007/BF00418125 -
Proceedings of the National Academy of... Nov 1992Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys. Individual...
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, Glu-269, Arg-302, Lys-319, His-322, Glu-325, or Lys-358 abolish active lactose transport. When Asp-237 and Lys-358 are simultaneously replaced with Cys and/or Ala, however, high activity is observed. Therefore, when either Asp-237 or Lys-358 is replaced with a neutral residue, leaving an unpaired charge, the permease is inactivated, but neutral replacement of both residues yields active permease [King, S. C., Hansen, C. L. & Wilson, T. H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Remarkably, moreover, when Asp-237 is interchanged with Lys-358, high activity is observed. The observations provide a strong indication that Asp-237 and Lys-358 interact to form a salt bridge. In addition, the data demonstrate that neither residue nor the salt bridge plays an important role in the transport mechanism. Thirteen additional double mutants were constructed in which a negative and a positively charged residue were replaced with Cys. Only Asp-240-->Cys/Lys-319-->Cys exhibits significant activity, accumulating lactose to 25-30% of the steady state observed with C-less permease. Replacing either Asp-240 or Lys-319 individually with Ala also inactivates the permease, but double mutants with neutral substitutions (Cys and/or Ala) at both positions exhibit essentially the same activity as Asp-240-->Cys/Lys-319-->Cys. In marked contrast to Asp-237 and Lys-358, interchanging Asp-240 and Lys-319 abolishes active lactose transport. The results demonstrate that Asp-240 and Lys-319, like Asp-237 and Lys-358, interact functionally and may form a salt bridge. However, the interaction between Asp-240 and Lys-319 is clearly more complex than the interaction between Asp-237 and Lys-358. In any event, the findings suggest that putative transmembrane helix VII lies next to helices X and XI in the tertiary structure of lactose permease.
Topics: Amino Acid Sequence; Base Sequence; Biological Transport, Active; Cell Membrane; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Kinetics; Membrane Transport Proteins; Models, Structural; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Plasmids; Protein Structure, Secondary; Symporters
PubMed: 1438245
DOI: 10.1073/pnas.89.21.10547