scholarly journals Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain

2000 ◽  
Vol 116 (4) ◽  
pp. 587-598 ◽  
Author(s):  
Paul K. Kienker ◽  
Karen S. Jakes ◽  
Alan Finkelstein
Keyword(s):  
Protein Translocation ◽  
Bilayer Membrane ◽  
Water Soluble ◽  
Planar Bilayers ◽  
Amino Terminal ◽  
Water Soluble Protein ◽  
Voltage Gated Channels ◽  
Membrane Spanning ◽  
Carboxy Terminal ◽  
Colicin Ia

Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an ∼68-residue segment translocated across the membrane. Various experimental interventions could cause a longer or shorter segment within the C domain to be translocated, making us wonder why translocation normally stops where it does, near the amino-terminal end of the C domain (approximately residue 450). We hypothesized that regions upstream from the C domain prevent its amino-terminal end from moving into and across the membrane. To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand. The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane. Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane. All of the colicin Ia carboxy-terminal fragments that we examined form channels that pass from a state of relatively normal conductance to a low-conductance state; we interpret this passage as a transition from a channel with four membrane-spanning segments to one with only three.

scholarly journals Sizing the Protein Translocation Pathway of Colicin Ia Channels

2003 ◽  
Vol 122 (2) ◽  
pp. 161-176 ◽  
Author(s):  
Paul K. Kienker ◽  
Karen S. Jakes ◽  
Robert O. Blaustein ◽  
Christopher Miller ◽  
Alan Finkelstein
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Bacterial Toxin ◽  
Upstream Region ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Translocation Pathway ◽  
Membrane Spanning ◽  
Carboxy Terminal ◽  
Colicin Ia

The bacterial toxin colicin Ia forms voltage-gated channels in planar lipid bilayers. The toxin consists of three domains, with the carboxy-terminal domain (C-domain) responsible for channel formation. The C-domain contributes four membrane-spanning segments and a 68-residue translocated segment to the open channel, whereas the upstream domains and the amino-terminal end of the C-domain stay on the cis side of the membrane. The isolated C-domain, lacking the two upstream domains, also forms channels; however, the amino terminus and one of the normally membrane-spanning segments can move across the membrane. (This can be observed as a drop in single-channel conductance.) In longer carboxy-terminal fragments of colicin Ia that include ≤169 residues upstream from the C-domain, the entire upstream region is translocated. Presumably, a portion of the C-domain creates a pathway for the polar upstream region to move through the membrane. To determine the size of this translocation pathway, we have attached “molecular stoppers,” small disulfide-bonded polypeptides, to the amino terminus of the C-domain, and determined whether they could be translocated. We have found that the translocation rate is strongly voltage dependent, and that at voltages ≥90 mV, even a 26-Å stopper is translocated. Upon reduction of their disulfide bonds, all of the stoppers are easily translocated, indicating that it is the folded structure, rather than some aspect of the primary sequence, that slows translocation of the stoppers. Thus, the pathway for translocation is ≥26 Å in diameter, or can stretch to this value. This is large enough for an α-helical hairpin to fit through.

scholarly journals Topography of Diphtheria Toxin's T Domain in the Open Channel State

2000 ◽  
Vol 115 (4) ◽  
pp. 421-434 ◽  
Author(s):  
Lisa Senzel ◽  
Michael Gordon ◽  
Robert O. Blaustein ◽  
K. Joon Oh ◽  
R. John Collier ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Open Channel ◽  
Catalytic Domain ◽  
Water Soluble ◽  
Soluble Form ◽  
Crystallographic Structure ◽  
Channel State ◽  
Amino Terminal ◽  
T Domain ◽  
Membrane Spanning

When diphtheria toxin encounters a low pH environment, the channel-forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a “double dagger” model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed “daggers,” TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of ∼70 residues, is translocated across the membrane to the trans side.

scholarly journals The ybxI Gene of Bacillus subtilis 168 Encodes a Class D β-Lactamase of Low Activity

2004 ◽  
Vol 48 (2) ◽  
pp. 484-490 ◽  
Author(s):  
Maria-Luigi Colombo ◽  
Sophie Hanique ◽  
Stéphane L. Baurin ◽  
Cédric Bauvois ◽  
Kris De Vriendt ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Amino Acid Residues ◽  
Putative Signal Peptide ◽  
Bacillus Subtilis 168 ◽  
Class D ◽  
Water Soluble Protein ◽  
Carboxy Terminal

ABSTRACT The ybxI gene of Bacillus subtilis 168 encodes a preprotein of 267 amino acid residues, including a putative signal peptide of 23 residues. The YbxI primary structure exhibits high similarity scores with two members of the superfamily of the serine penicillin-recognizing enzymes: the class D β-lactamases and the hydrophilic carboxy-terminal domains of the BlaR and MecR penicillin receptors. To determine the function and the activity of this putative penicillin-recognizing enzyme, we have subcloned the ybxI gene in the pET-26b expression vector. Transformation of Escherichia coli BL21(DE3) by the recombinant plasmid pCIP51 resulted in the export of the mature YbxI in the periplasm as a water-soluble protein. The recombinant protein was purified to 95% homogeneity. YbxI interacts with several β-lactam antibiotics and can hydrolyze some of them. YbxI is not inactivated by clavulanic acid. The YbxI function and its enzymatic activity in B. subtilis remain unknown. The acyl-enzyme obtained after incubation of YbxI with a fluorescent derivative of ampicillin can be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that YbxI can be acylated by β-lactam antibiotics. YbxI does not hydrolyze some of the standard substrates of d-alanyl-d-alanine peptidases, the targets of penicillin. YbxI belongs to the penicillin-recognizing enzyme family but has an activity intermediate between those of a penicillin-binding protein and a β-lactamase.

scholarly journals A specific transmembrane domain of a coronavirus E1 glycoprotein is required for its retention in the Golgi region.

1987 ◽  
Vol 105 (3) ◽  
pp. 1205-1214 ◽  
Author(s):  
C E Machamer ◽  
J K Rose
Keyword(s):  
Transmembrane Domain ◽  
Signal Sequence ◽  
Mutant Protein ◽  
Specific Domain ◽  
Amino Terminal ◽  
Golgi Region ◽  
Membrane Spanning ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Membrane Spanning Domains

The E1 glycoprotein of the avian coronavirus infectious bronchitis virus contains a short, glycosylated amino-terminal domain, three membrane-spanning domains, and a long carboxy-terminal cytoplasmic domain. We show that E1 expressed from cDNA is targeted to the Golgi region, as it is in infected cells. E1 proteins with precise deletions of the first and second or the second and third membrane-spanning domains were glycosylated, thus suggesting that either the first or third transmembrane domain can function as an internal signal sequence. The mutant protein with only the first transmembrane domain accumulated intracellularly like the wild-type protein, but the mutant protein with only the third transmembrane domain was transported to the cell surface. This result suggests that information specifying accumulation in the Golgi region resides in the first transmembrane domain, and provides the first example of an intracellular membrane protein that is transported to the plasma membrane after deletion of a specific domain.

scholarly journals Identification of Channel-lining Amino Acid Residues in the Hydrophobic Segment of Colicin Ia

2008 ◽  
Vol 132 (6) ◽  
pp. 693-707 ◽  
Author(s):  
Paul K. Kienker ◽  
Karen S. Jakes ◽  
Alan Finkelstein
Keyword(s):  
Crystal Structure ◽  
Amino Acid ◽  
Lipid Bilayer ◽  
Open Channel ◽  
Structural Model ◽  
Water Soluble ◽  
Amino Acid Residues ◽  
Lipid Bilayer Membranes ◽  
Planar Bilayers ◽  
Colicin Ia

Colicin Ia is a bactericidal protein of 626 amino acid residues that kills its target cell by forming a channel in the inner membrane; it can also form voltage-dependent channels in planar lipid bilayer membranes. The channel-forming activity resides in the carboxy-terminal domain of ∼177 residues. In the crystal structure of the water-soluble conformation, this domain consists of a bundle of 10 α-helices, with eight mostly amphipathic helices surrounding a hydrophobic helical hairpin (helices H8-H9). We wish to know how this structure changes to form a channel in a lipid bilayer. Although there is evidence that the open channel has four transmembrane segments (H8, H9, and parts of H1 and H6-H7), their arrangement relative to the pore is largely unknown. Given the lack of a detailed structural model, it is imperative to better characterize the channel-lining protein segments. Here, we focus on a segment of 44 residues (573–616), which in the crystal structure comprises the H8-H9 hairpin and flanking regions. We mutated each of these residues to a unique cysteine, added the mutant colicins to the cis side of planar bilayers to form channels, and determined whether sulfhydryl-specific methanethiosulfonate reagents could alter the conduction of ions through the open channel. We found a pattern of reactivity consistent with parts of H8 and H9 lining the channel as α-helices, albeit rather short ones for spanning a lipid bilayer (12 residues). The effects of the reactions on channel conductance and selectivity tend to be greater for residues near the amino terminus of H8 and the carboxy terminus of H9, with particularly large effects for G577C, T581C, and G609C, suggesting that these residues may occupy a relatively constricted region near the cis end of the channel.

scholarly journals Molecular cloning of integrin-associated protein: an immunoglobulin family member with multiple membrane-spanning domains implicated in alpha v beta 3-dependent ligand binding.

1993 ◽  
Vol 123 (2) ◽  
pp. 485-496 ◽  
Author(s):  
F P Lindberg ◽  
H D Gresham ◽  
E Schwarz ◽  
E J Brown
Keyword(s):  
Ligand Binding ◽  
Cho Cells ◽  
Peptide Sequence ◽  
Human Antibody ◽  
Amino Terminal ◽  
Membrane Spanning ◽  
Highly Hydrophobic ◽  
Leukocyte Response ◽  
Carboxy Terminal ◽  
Alpha V Beta 3

Integrin Associated Protein (IAP) is a 50-kD membrane protein which copurifies with the integrin alpha v beta 3 from placenta and coimmunoprecipitates with beta 3 from platelets. IAP also is functionally associated with signal transduction from the Leukocyte Response Integrin. Using tryptic peptide sequence, human and murine IAP cDNAs have been isolated. The protein has an extracellular amino-terminal immunoglobulin domain that binds all monoclonal anti-IAP antibodies. The carboxy-terminal region is highly hydrophobic with three or five membrane-spanning segments and a short hydrophilic tail. Immunofluorescence microscopy suggests that this hydrophilic tail is located on the inside of the cytoplasmic membrane. Monoclonal anti-IAP antibody inhibits the binding of vitronectin-coated beads to alpha v beta 3 on human erythroleukemia cells, and polyclonal anti-IAP recognizes hamster IAP on CHO cells and inhibits vitronectin bead binding. When CHO cells are transfected with human IAP, monoclonal anti-human antibody completely inhibits vitronectin bead binding. These data suggest a model in which ligand binding by alpha v beta 3 is regulated by IAP.

Recent Advances in Novasome Formulation Technology

2014 ◽  
Vol 7 (2) ◽  
pp. 2407-2411
Author(s):  
J M Shah ◽  
N.H Shah ◽  
Hadiya P D
Keyword(s):  
Drug Delivery ◽  
Effective Means ◽  
Entrapment Efficiency ◽  
Bilayer Membrane ◽  
Water Soluble ◽  
Delivery Methods ◽  
Liposomal Drug ◽  
Pharmaceutical Technology ◽  
Insoluble Drugs ◽  
Novel Drug

Pharmaceutical technology has developed various newer modes of novel drug delivery aspects. Modifications in the previously existing drug delivery methods have led to various newly innovated technologies serving as a safe and effective means of improvement over the existing ones. Novasome technology is one of the new innovations of liposomes which have solved many of the problems related to liposomal drug delivery system. It offers a seven bilayer membrane which has the ability to incorporate both water soluble and insoluble drugs. It has an excellent entrapment efficiency which provides better medication. Formulation of novasomes is achieved in a high shear device. Due to its numerous advantages, novasomes have been used extensively in various fields like cosmetics, chemical, personal care, foods, pharmaceuticals and agrochemicals.

Interaction Between Mutations in the suppressor of Hairy wing and modifier of mdg4 Genes of Drosophila melunogaster Affecting the Phenotype of gypsy-Induced Mutations

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 425-436 ◽  
Author(s):  
Pavel Georgiev ◽  
Marina Kozycina
Keyword(s):  
Mutagenic Effect ◽  
Induced Mutations ◽  
Binding Region ◽  
Direct Inhibition ◽  
Amino Terminal ◽  
Acidic Domain ◽  
Complete Inactivation ◽  
Insulation Effect ◽  
Gypsy Retrotransposon ◽  
Carboxy Terminal

Abstract The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers located distally from the promoter with respect to the position of the su(Hw)-binding region. Mutations in a second gene, modifier of mdg4, also affect the gypsy-induced phenotype. Two major effects of the mod(mdg4)lul mutation can be distinguished: the interference with insulation by the su(Hw)-binding region and direct inhibition of gene expression that is not dependent on the su(Hw)-binding region position. The mod(mdg4)lul mutation partially suppresses ct6, scD1 and Hw1 mutations, possibly by interfering with the insulation effect of the su(Hw)-binding region. An example of the second effect of mod(mdg4)lul is a complete inactivation of yellow expression in combination with the y  2 allele. Phenotypic analyses of flies with combinations of mod(mdg4)lul and different su(Hw) mutations, or with constructions carrying deletions of the acidic domains of the su(Hw) protein, suggest that the carboxy-terminal acidic domain is important for direct inhibition of yellow transcription in bristles, while the amino-terminal acidic domain is more essential for insulation.

scholarly journals Pore-forming Esx proteins mediate toxin secretion by Mycobacterium tuberculosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Uday Tak ◽  
Terje Dokland ◽  
Michael Niederweis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pore Formation ◽  
Molecular Switches ◽  
Water Soluble ◽  
Host Cells ◽  
Solvent Exposure ◽  
Membrane Channel ◽  
Toxin Secretion ◽  
Outer Membrane Channel ◽  
Membrane Spanning

AbstractMycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) to kill host cells. Here, we show that the WXG100 proteins EsxE and EsxF are essential for TNT secretion. EsxE and EsxF form a water-soluble heterodimer (EsxEF) that assembles into oligomers and long filaments, binds to membranes, and forms stable membrane-spanning channels. Electron microscopy of EsxEF reveals mainly pentameric structures with a central pore. Mutations of both WXG motifs and of a GXW motif do not affect dimerization, but abolish pore formation, membrane deformation and TNT secretion. The WXG/GXW mutants are locked in conformations with altered thermostability and solvent exposure, indicating that the WXG/GXW motifs are molecular switches controlling membrane interaction and pore formation. EsxF is accessible on the bacterial cell surface, suggesting that EsxEF form an outer membrane channel for toxin export. Thus, our study reveals a protein secretion mechanism in bacteria that relies on pore formation by small WXG proteins.

THE SOLUBLE PROTEINS OF THE MUSCLE TISSUE OF THE HADDOCK

1931 ◽  
Vol 6 (1) ◽  
pp. 1-11 ◽  
Author(s):  
J. F. LOGAN
Keyword(s):  
Sodium Chloride ◽  
Aqueous Solutions ◽  
Muscle Tissue ◽  
Soluble Protein ◽  
Potassium Phosphate ◽  
Extraction Methods ◽  
Water Soluble ◽  
Tabular Form ◽  
Water Soluble Protein ◽  
Isoelectric Points

As a contribution to the chemistry of muscle tissue, the solubility of the protein of haddock muscle in aqueous solutions of sodium chloride and neutral potassium phosphate, respectively, was determined. The results are expressed in tabular form and graphically in the form of solubility curves. A water-soluble protein and also a salt-soluble protein were isolated from dialyzed haddock muscle by extraction methods. These proteins were obtained in a comparatively pure condition by precipitation from solution in the region of their isoelectric points.

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