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Current Opinion in Structural Biology Aug 1997Recent advances in protein engineering have facilitated the development of alternative approaches to determine helix packing in polytopic membrane proteins. Using the... (Review)
Review
Recent advances in protein engineering have facilitated the development of alternative approaches to determine helix packing in polytopic membrane proteins. Using the lac permease as a paradigm, several site-directed biophysical and biochemical techniques are described which should be generally applicable.
Topics: Amino Acid Sequence; Escherichia coli; Escherichia coli Proteins; Membrane Proteins; Membrane Transport Proteins; Models, Molecular; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Protein Structure, Secondary; Protein Structure, Tertiary; Symporters
PubMed: 9266176
DOI: 10.1016/s0959-440x(97)80119-4 -
Biochimica Et Biophysica Acta Aug 2002Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We...
Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We present a quantitative analysis of the stability of the Escherichia coli lactose (lac) permease and a series of lac permease fusion proteins containing an insertion of cytochrome(b562), T4 lysozyme or beta-lactamase in the central hydrophilic loop of the permease. The stability of the proteins was evaluated under a variety of storage conditions by both a qualitative SDS-PAGE assay and by a quantitative hplc assay. Long-chain maltoside detergents were more effective at maintaining purified protein in solution than detergents with smaller head groups and/or shorter alkyl tails. A full factorial experiment established that the proteins were insensitive to sodium chloride concentrations, but greatly stabilized by glycerol, low temperature and the combination of glycerol and low temperature. The accurate quantitation of the protein by absorbance spectroscopy required exclusion of all contact with clarified polypropylene or polyvinyl chloride (PVC) materials. Although some of the fusion proteins were more prone to aggregation than the wild-type permease, the stability of a fusion protein containing a cytochrome(b562) insertion was indistinguishable from that of native lac permease.
Topics: Chromatography, High Pressure Liquid; Cytochrome b Group; Detergents; Drug Stability; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Glycerol; Membrane Transport Proteins; Monosaccharide Transport Proteins; Recombinant Fusion Proteins; Solutions; Spectrophotometry, Ultraviolet; Symporters; Temperature
PubMed: 12100995
DOI: 10.1016/s0005-2736(02)00397-8 -
Trends in Neurosciences Jun 2004During the past several decades, lac permease has assumed almost tutelary proportions as a model for cotransporters. This archetypical membrane protein now exerts its... (Review)
Review
During the past several decades, lac permease has assumed almost tutelary proportions as a model for cotransporters. This archetypical membrane protein now exerts its influence by the most dramatic means possible: its structure is solved. This article describes the configuration and implied transport mechanism of the bacterial lactose transporter and compares its structure and function with those of other transporters and those of ion channels. This juxtaposition of transporters and channels is likely to be helpful because LacY is the first cotransporter with known structure and exclusive carrier properties, and it shares topology with neurotransmitter transporters with unknown structure and channel properties.
Topics: Animals; Binding Sites; Biological Transport; Cell Membrane; Escherichia coli Proteins; Humans; Ion Channels; Membrane Proteins; Membrane Transport Proteins; Models, Biological; Models, Molecular; Protein Structure, Secondary; Protein Structure, Tertiary
PubMed: 15165740
DOI: 10.1016/j.tins.2004.04.007 -
Eukaryotic Cell Feb 2008The conserved AmtB/Mep/Rh family of proteins mediate the transport of ammonium across cellular membranes in a wide range of organisms. Certain fungal members of this...
The conserved AmtB/Mep/Rh family of proteins mediate the transport of ammonium across cellular membranes in a wide range of organisms. Certain fungal members of this group are required to initiate filamentous growth. We have investigated the functions of two members of the AmtB/Mep/Rh family from the pathogenic basidiomycete Cryptococcus neoformans. Amt1 and Amt2 are low- and high-affinity ammonium permeases, respectively, and a mutant lacking both permeases is unable to grow under ammonium-limiting conditions. AMT2 is transcriptionally induced in response to nitrogen limitation, whereas AMT1 is constitutively expressed. Single and double amt mutants exhibit wild-type virulence in two models of cryptococcosis. Consistent with this, the formation of two C. neoformans virulence factors, cell wall melanin and the extracellular polysaccharide capsule, is not impaired in cells lacking either or both of the Amt1 and Amt2 permeases. Amt2 is, however, required for the initiation of invasive growth of haploid cells under low-nitrogen conditions and for the mating of wild-type cells under the same conditions. We propose that Amt2 may be a new fungal ammonium sensor and an element of the signaling cascades that govern the mating of C. neoformans in response to environmental nutritional cues.
Topics: Amino Acid Sequence; Cryptococcosis; Cryptococcus neoformans; Fungal Proteins; Gene Expression Regulation, Fungal; Genes, Mating Type, Fungal; Membrane Transport Proteins; Methylamines; Molecular Sequence Data; Phenotype; Phylogeny; Quaternary Ammonium Compounds; Sequence Homology, Amino Acid; Virulence
PubMed: 18055915
DOI: 10.1128/EC.00079-07 -
Biochemistry Jun 2002The lactose permease of Escherichia coli transports protons and lactose across the plasma membrane and uses a transmembrane ion gradient as the energy source to drive...
The lactose permease of Escherichia coli transports protons and lactose across the plasma membrane and uses a transmembrane ion gradient as the energy source to drive the uphill accumulation of lactose. In this report, the effect of the electrochemical gradient on the permease has been studied. Bacteriorhodopsin was co-reconstituted with the lactose permease to provide a light-triggered electrochemical gradient. Reaction-induced Fourier transform infrared spectra were acquired, and bacteriorhodopsin contributions were subtracted. In previous work, positive bands in the 1765-1730 cm(-1) region of the reaction-induced FT-IR spectrum were attributed to the perturbation of carboxylic acid residues in the permease [Patzlaff, J. S., Brooker, R. J., and Barry, B. A. (2000) J. Biol. Chem. 275, 28695-28700]. In this study, we have globally labeled the permease with (13)C or (15)N. Isotopic labeling demonstrates that features in the reaction-induced FT-IR spectrum arise from permease carboxylic acid, amide I, and amide II vibrational modes. In addition, isotope labeling leads to a tentative assignment of spectral features to lysine, arginine, histidine, glutamine, and/or asparagine in the permease. These results indicate that the electrochemical gradient causes changes in the environment or protonation state of carboxylic acid residues in the permease and suggest an interaction between these carboxylic acid side chains and nitrogen-containing amino acid side chains. Evidence for a change in secondary structure, corresponding to an interconversion of secondary structural elements, a change in the hydrogen-bonding strength, or coupling of peptide vibrational modes, is also presented. These experiments demonstrate the usefulness of reaction-induced spectroscopy in the study of transmembrane transport.
Topics: Bacteriorhodopsins; Carbon Isotopes; Electrochemistry; Escherichia coli Proteins; Isotope Labeling; Membrane Transport Proteins; Monosaccharide Transport Proteins; Nitrogen Isotopes; Photolysis; Spectroscopy, Fourier Transform Infrared; Symporters
PubMed: 12044169
DOI: 10.1021/bi025555g -
Research in Microbiology 1990
Review
Mechanism of sugar transport and phosphorylation via permeases of the bacterial phosphotransferase system: catalytic residues in the beta-glucoside-specific permease as defined by site-specific mutagenesis.
Topics: Bacterial Proteins; Binding Sites; Biological Transport, Active; Carbohydrate Metabolism; Escherichia coli; Genetic Complementation Test; Membrane Transport Proteins; Mutagenesis, Site-Directed; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation
PubMed: 2281195
DOI: 10.1016/0923-2508(90)90014-h -
FEBS Letters Oct 2020The monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic side of lactose permease (LacY) of Escherichia coli and inhibits H /lactose symport and...
The monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic side of lactose permease (LacY) of Escherichia coli and inhibits H /lactose symport and lactose efflux under nonenergized conditions. At the same time, ligand binding and translocation reactions that do not involve net H translocation remain unaffected by 4B1. In this study, surface-enhanced infrared absorption spectroscopy applied to the immobilized LacY was used to study the pH-dependent changes in LacY and to access in situ the effect of the 4B1 antibody on the pK of Glu325, the primary functional H -binding site in LacY. A small shift of the pK value from 10.5 to 9.5 was identified that can be corroborated with the inactivation of LacY upon 4B1 binding.
Topics: Antibodies, Monoclonal; Biological Transport; Escherichia coli; Escherichia coli Proteins; Glutamic Acid; Hydrogen-Ion Concentration; Lactose; Membrane Transport Proteins; Models, Molecular; Monosaccharide Transport Proteins; Spectrophotometry, Infrared; Symporters
PubMed: 32780424
DOI: 10.1002/1873-3468.13907 -
Current Genetics Jul 1996Glutamine uptake in S. cerevisiae is mediated by at least three transporters: high- and low-affinity glutamine permeases and the general amino-acid permease. We have...
Glutamine uptake in S. cerevisiae is mediated by at least three transporters: high- and low-affinity glutamine permeases and the general amino-acid permease. We have isolated the gene encoding the high-affinity glutamine permease and named it GNP1. The amino-acid sequence of GNP1, and its hydropathy profile of 12 transmembrane domains, closely resemble those of known amino-acid permeases. The Km of GNP1 for glutamine uptake was determined to be 0.59 mM. Cells lacking GNP1 exhibit reduced levels of glutamine transport, and are resistant to a toxic analog of glutamine, L-glutamic acid gamma-monohydroxamate. Unlike other amino-acid permeases, whose expression is nitrogen-source limited, GNP1 is expressed on both rich and poor nitrogen sources.
Topics: Amino Acid Sequence; Amino Acid Transport Systems, Basic; Amino Acid Transport Systems, Neutral; Glutamine; Membrane Transport Proteins; Molecular Sequence Data; Nitrogen; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid
PubMed: 8660458
DOI: 10.1007/s002940050108 -
Membrane Biochemistry 1993In the present study lactose permease mutants were isolated which recognize the monosaccharide, L-arabinose. Although the wild-type permease exhibits a poor recognition...
In the present study lactose permease mutants were isolated which recognize the monosaccharide, L-arabinose. Although the wild-type permease exhibits a poor recognition for L-arabinose, seven independent mutants were identified by their ability to grow on L-arabinose minimal plates. When subjected to DNA sequencing, it was found that all seven of these mutants were single-site mutations in which alanine 177 was changed to valine. The wild type and valine 177 mutant were then analyzed with regard to their abilities to recognize and transport monosaccharides and disaccharides. Free L-arabinose was shown to competitively inhibit [14C]-lactose transport yielding a Ki value of 121 mM for the Val177 mutant and a much higher value of 320 mM for the wild-type. Among several monosaccharides, D-glucose as well as L-arabinose inhibited lactose transport in the Val177 mutant to a significantly greater extent, while D-arabinose and D-xylose only caused a slight inhibition. On the other hand, kinetic studies with sugars which are normally recognized by the wild-type permease such as [14C]-galactose and [14C]-lactose revealed that the Val177 mutant and wild-type strains had similar transport characteristics for these two sugars. Overall, these results are consistent with the notion that the Val177 substitution causes an enhanced recognition for particular sugars (i.e. L-arabinose) but does not universally affect the recognition and unidirectional transport for all sugars. This idea is further supported by the observation that site-directed mutants containing isoleucine, leucine, phenylalanine, or proline at position 177 also were found to possess an enhanced recognition for L-arabinose.
Topics: Arabinose; Biological Transport; Carbohydrate Sequence; DNA; Escherichia coli; Escherichia coli Proteins; Galactose; Lactose; Membrane Transport Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Substrate Specificity; Symporters
PubMed: 8510563
DOI: 10.3109/09687689309150253 -
Biochimica Et Biophysica Acta Jun 1998The PHO84 and PHO89 genes of Saccharomyces cerevisiae encode two high-affinity phosphate cotransporters of the plasma membrane. Hydropathy analysis suggests a secondary... (Review)
Review
The PHO84 and PHO89 genes of Saccharomyces cerevisiae encode two high-affinity phosphate cotransporters of the plasma membrane. Hydropathy analysis suggests a secondary structure arrangements of the proteins in 12 transmembrane domains. The derepressible Pho84 and Pho89 transporters appear to have characteristic similarities with the phosphate transporters of Neurospora crassa. The Pho84 protein catalyzes a proton-coupled phosphate transport at acidic pH, while the Pho89 protein catalyzes a sodium-dependent phosphate uptake at alkaline pH. The Pho84 transporter can be stably overproduced in the cytoplasmic membrane of Escherichia coli, purified and reconstituted in a functional state into proteoliposomes.
Topics: Amino Acid Sequence; Animals; Biological Transport, Active; Carrier Proteins; Fungal Proteins; Humans; Membrane Transport Proteins; Molecular Sequence Data; Organophosphates; Phosphate Transport Proteins; Protein Structure, Secondary; Proton-Phosphate Symporters; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Sodium-Phosphate Cotransporter Proteins; Sodium-Phosphate Cotransporter Proteins, Type III; Symporters
PubMed: 9693717
DOI: 10.1016/s0005-2728(98)00037-1