Assembly of α-Hemolysin: a Proteinaceous Pore with Potential Applications in Materials Synthesis

1992 ◽  
Vol 292 ◽  
Author(s):  
Hagan Bayley ◽  
Musti Krishnasastry ◽  
Barbara Walker ◽  
John Kasianowicz
Keyword(s):  
Lipid Bilayers ◽  
Three Dimensional ◽  
Structural Data ◽  
Membrane Receptors ◽  
Internal Diameter ◽  
Amino Acid Residues ◽  
Materials Synthesis ◽  
Assembly Mechanism ◽  
Potential Applications ◽  
Membrane Bound

Abstractα-Hemolysin (αHL) is secreted by the bacteriumStaphylococcus aureusas a watersoluble polypeptide of 293 amino acid residues. When presented with lipid bilayers or the detergent deoxycholate (DOC), aHL assembles into hexameric cylindrical pores that each contain one channel ∼ 1 to 2 nm in Internal diameter. A long-term goal of this laboratory is to use wild-type or re-engineered αHL pores as components of nanoscale materials: for example, to confer novel permeability properties upon thin films. The implementation of this concept would be facilitated by a better understanding of the mechanism by which the pore assembles. Reviewed here are findings that have given us insight Into the assembly mechanism, including the results of recent mutagenesis experiments. A critical summary is given of knowledge about the conformation of the monomer In solution, the hexamerIc pore and two proposed intermediates in assembly (a membrane-bound monomer and an oligomeric pore precursor). Future directions are outlined Including the prospects of obtaining three-dimensional structural data on the αHL pore or its precursors, methods for obtaining better monolayer sheets and new experiments on the topography of the pore and its precursors. The role of membrane receptors in facilitating the assembly of αHL is also discussed. Finally, it is demonstrated that despite our rather rudimentary knowledge of the assembly process, the Information gained so far still allows the design of mutant (αHL polypeptides with useful properties. For example, αHL mutants whose pore-forming ability is activated by proteases have been made.

α-Hemolysin: A Self-Assembling Protein Pore With Potential Applications In The Synthesis of New Materials

1991 ◽  
Vol 255 ◽  
Author(s):  
Hagan Bayley ◽  
Musti Krishnasastry ◽  
Barbara Walker ◽  
John Kasianowicz
Keyword(s):  
Genetic Manipulation ◽  
Internal Diameter ◽  
New Materials ◽  
Membrane Pores ◽  
Self Assembling ◽  
Assembly Mechanism ◽  
Potential Applications ◽  
Membrane Bound ◽  
Insight Into ◽  
Selection Of

AbstractA selection of nanoscale membrane pores is being constructed by genetic manipulation of α-hemolysin (αHL), a 33.2 kDa polypeptide secreted by the bacterium Staphylococcus aureus, which can self-assemble into hexameric cylindrical channels -1 to 2 nm In Internal diameter. Ultimately, the new pores will be used to confer novel permeability properties upon materials such as thin films utilizing, for example, monolayer sheets of the hexamer. Recombinant αHL (r-αHL) has now been obtained in multimilligram amounts and purified to homogeneity after overexpression of the αHL gene in Escherichia coli. The properties of r-αHL are closely similar to those of αHL purified from S. aureus. Recent deletion mutagenesis experiments have given us new insight into the assembly mechanism of the pore. Three intermediates have been identified: a membrane-bound monomer; an oligomeric pore precursor; and the hexameric pore itself. Currently, point mutogenesis combined with chemical modification is being used to produce new pores of different internal diameter, with selectivity for the passage of molecules and Ions, and with gating properties (the ability to open and close in response to a physical stimulus, e.g. an electric field or light).

An Update on Recent Advances in Cubosome: A Novel Drug Delivery System

2021 ◽  
Vol 21 ◽  
Author(s):  
Madhukar Garg ◽  
Anju Goyal ◽  
Sapna Kumari
Keyword(s):  
Drug Delivery ◽  
Drug Delivery System ◽  
Lipid Bilayers ◽  
Delivery System ◽  
Liquid Crystalline ◽  
Three Dimensional ◽  
Biologically Active ◽  
Potential Applications ◽  
Health Related ◽  
Novel Drug

: Cubosomes are highly stable nanostructured liquid crystalline dosage delivery form derived from amphiphilic lipids and polymer-based stabilizers converting it in a form of effective biocompatible carrier for the drug delivery. The delivery form comprised of bicontinuous lipid bilayers arranged in three dimensional honeycombs like structure provided with two internal aqueous channels for incorporation of number of biologically active ingredients. In contrast liposomes they provide large surface area for incorporation of different types of ingredients. Due to the distinct advantages of biocompatibility and thermodynamic stability, cubosomes have remained the first preference as method of choice in the sustained release, controlled release and targeted release dosage forms as new drug delivery system for the better release of the drugs. As lot of advancement in the new form of dosage form has bring the novel avenues in drug delivery mechanisms so it was matter of worth to compile the latest updates on the various aspects of mentioned therapeutic delivery system including its structure, routes of applications along with the potential applications to encapsulate variety drugs to serve health related benefits.

scholarly journals Nanogold as a Specific Marker for Electron Cryotomography

2009 ◽  
Vol 15 (3) ◽  
pp. 183-188 ◽  
Author(s):  
Yongning He ◽  
Grant J. Jensen ◽  
Pamela J. Bjorkman
Keyword(s):  
Lipid Bilayers ◽  
Outer Surface ◽  
Three Dimensional ◽  
Fc Receptor ◽  
Specific Marker ◽  
Neonatal Fc Receptor ◽  
Membrane Bound ◽  
Nanogold Particles ◽  
Intracellular Vesicles ◽  
Electron Cryotomography

AbstractWhile electron cryotomography (ECT) provides “molecular” resolution, three-dimensional images of unique biological specimens, sample crowdedness, and/or resolution limitations can make it difficult to identify specific macromolecular components. Here we used a 1.4 nm Nanogold® cluster specifically attached to the Fc fragment of IgG to monitor its interaction with the neonatal Fc receptor (FcRn), a membrane-bound receptor that transports IgG across cells in acidic intracellular vesicles. ECT was used to image complexes formed by Nanogold-labeled Fc bound to FcRn attached to the outer surface of synthetic liposomes. In the resulting three-dimensional reconstructions, 1.4 nm Nanogold particles were distributed predominantly along the interfaces where 2:1 FcRn-Fc complexes bridged adjacent lipid bilayers. These results demonstrate that the 1.4 nm Nanogold cluster is visible in tomograms of typically thick samples (∼250 nm) recorded with defocuses appropriate for large macromolecules and is thus an effective marker.

Detection of Botulinum Neurotoxin/A Insertion Using an Encapsulated Interface Bilayer

2012 ◽  
Author(s):  
Graham Taylor ◽  
Donald Leo ◽  
Andy Sarles
Keyword(s):  
Nervous System ◽  
Lipid Bilayers ◽  
Motor Neurons ◽  
Botulinum Neurotoxin ◽  
Lipid Membrane ◽  
Membrane Receptors ◽  
High Concentration ◽  
Neural Signals ◽  
Surface Receptors ◽  
Membrane Bound

Many signaling mechanisms in living cells occur at biological boundaries via cell surface receptors and membrane proteins embedded in lipid bilayers. The coordination of actions of sensory and motor neurons in the nervous system represents one example of many that heavily depends on lipid membrane bound receptor mediated signaling. As a result, chemical and biological toxins that disrupt these neural signals can have severe physiological effects, including paralysis and death. Botulinum neurotoxin Type A (BoNT/A) is a proteolytic toxin that inserts through vesicle membranes and cleaves membrane receptors involved with synaptic acetylcholine uptake and nervous system signal conduction. In this work, we investigate the use of a Bioinspired liquid-supported interface bilayer for studying the insertion of BoNT/A toxin molecules into synthetic lipid bilayers. DPhPC lipid bilayers are formed using the regulated attachment method (RAM), as developed by Sarles and Leo, and we perform current measurements on membranes exposed to BoNT/A toxin to characterize activity of toxin interacting with the synthetic bilayer. Control tests without toxin present are also presented. The results of these tests show an increase in the magnitude of current through the bilayer when the toxin is included. We interpret these initial results to mean that incorporation of BoNT/A toxin at a high concentration in an interface bilayer increases the permeability of the membrane as a result of toxin molecules spanning the thickness of the bilayer.

scholarly journals A Membrane-Bound Flavocytochromec-Sulfide Dehydrogenase from the Purple Phototrophic Sulfur Bacterium Ectothiorhodospira vacuolata

2000 ◽  
Vol 182 (11) ◽  
pp. 3097-3103 ◽  
Author(s):  
Vesna Kostanjevecki ◽  
Ann Brigé ◽  
Terrance E. Meyer ◽  
Michael A. Cusanovich ◽  
Yves Guisez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Signal Sequence ◽  
Sulfide Oxidation ◽  
Three Dimensional ◽  
Chromatium Vinosum ◽  
Dimensional Structure ◽  
Northern Hybridization ◽  
Amino Acid Residues ◽  
Membrane Bound

ABSTRACT The amino acid sequence of Ectothiorhodospira vacuolatacytochrome c-552, isolated from membranes withn-butanol, shows that it is a protein of 77 amino acid residues with a molecular mass of 9,041 Da. It is closely related to the cytochrome subunit of Chlorobium limicola f. sp.thiosulfatophilum flavocytochrome c-sulfide dehydrogenase (FCSD), having 49% identity. These data allowed isolation of a 5.5-kb subgenomic clone which contains the cytochrome gene and an adjacent flavoprotein gene as in other species which have an FCSD. The cytochrome subunit has a signal peptide with a normal cleavage site, but the flavoprotein subunit has a signal sequence which suggests that the mature protein has an N-terminal cysteine, characteristic of a diacyl glycerol-modified lipoprotein. The membrane localization of FCSD was confirmed by Western blotting with antibodies raised against Chromatium vinosum FCSD. When aligned according to the three-dimensional structure of ChromatiumFCSD, all but one of the side chains near the flavin are conserved. These include the Cys 42 flavin adenine dinucleotide binding site; the Cys 161-Cys 337 disulfide; Glu 167, which modulates the reactivity with sulfite; and aromatic residues which may function as charge transfer acceptors from the flavin-sulfite adduct (C. vinosumnumbering). The genetic context of FCSD is different from that in other species in that flanking genes are not conserved. The transcript is only large enough to encode the two FCSD subunits. Furthermore, Northern hybridization showed that the production of E. vacuolata FCSD mRNA is regulated by sulfide. All cultures that contained sulfide in the medium had elevated levels of FCSD RNA compared with cells grown on organics (acetate, malate, or succinate) or thiosulfate alone, consistent with the role of FCSD in sulfide oxidation.

scholarly journals A Novel Pseudomonas geniculata AGE Family Epimerase/Isomerase and Its Application in d-Mannose Synthesis

Foods ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1809
Author(s):  
Zhanzhi Liu ◽  
Ying Li ◽  
Jing Wu ◽  
Sheng Chen
Keyword(s):  
3D Structure ◽  
Three Dimensional ◽  
Optimal Temperature ◽  
Reaction Conditions ◽  
Enzymatic Production ◽  
Physiological Properties ◽  
Potential Applications ◽  
Kinetics Of ◽  
Optimal Reaction ◽  
Gene Age

d-mannose has exhibited excellent physiological properties in the food, pharmaceutical, and feed industries. Therefore, emerging attention has been applied to enzymatic production of d-mannose due to its advantage over chemical synthesis. The gene age of N-acetyl-d-glucosamine 2-epimerase family epimerase/isomerase (AGEase) derived from Pseudomonas geniculata was amplified, and the recombinant P. geniculata AGEase was characterized. The optimal temperature and pH of P. geniculata AGEase were 60 °C and 7.5, respectively. The Km, kcat, and kcat/Km of P. geniculata AGEase for d-mannose were 49.2 ± 8.5 mM, 476.3 ± 4.0 s−1, and 9.7 ± 0.5 s−1·mM−1, respectively. The recombinant P. geniculata AGEase was classified into the YihS enzyme subfamily in the AGE enzyme family by analyzing its substrate specificity and active center of the three-dimensional (3D) structure. Further studies on the kinetics of different substrates showed that the P. geniculata AGEase belongs to the d-mannose isomerase of the YihS enzyme. The P. geniculata AGEase catalyzed the synthesis of d-mannose with d-fructose as a substrate, and the conversion rate was as high as 39.3% with the d-mannose yield of 78.6 g·L−1 under optimal reaction conditions of 200 g·L−1d-fructose and 2.5 U·mL−1P. geniculata AGEase. This novel P. geniculata AGEase has potential applications in the industrial production of d-mannose.

scholarly journals Controlled Fabrication of Quality ZnO NWs/CNTs and ZnO NWs/Gr Heterostructures via Direct Two-Step CVD Method

Nanomaterials ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1836
Author(s):  
Nicholas Schaper ◽  
Dheyaa Alameri ◽  
Yoosuk Kim ◽  
Brian Thomas ◽  
Keith McCormack ◽  
...  
Keyword(s):  
Direct Synthesis ◽  
Chemical Vapor ◽  
Single Walled Carbon Nanotubes ◽  
Materials Synthesis ◽  
Oxide Nanowires ◽  
Cvd Method ◽  
Thermal Actuators ◽  
Potential Applications ◽  
Walled Carbon Nanotubes ◽  
Scalable Production

A novel and advanced approach of growing zinc oxide nanowires (ZnO NWs) directly on single-walled carbon nanotubes (SWCNTs) and graphene (Gr) surfaces has been demonstrated through the successful formation of 1D–1D and 1D–2D heterostructure interfaces. The direct two-step chemical vapor deposition (CVD) method was utilized to ensure high-quality materials’ synthesis and scalable production of different architectures. Iron-based universal compound molecular ink was used as a catalyst in both processes (a) to form a monolayer of horizontally defined networks of SWCNTs interfaced with vertically oriented ZnO NWs and (b) to grow densely packed ZnO NWs directly on a graphene surface. We show here that our universal compound molecular ink is efficient and selective in the direct synthesis of ZnO NWs/CNTs and ZnO NWs/Gr heterostructures. Heterostructures were also selectively patterned through different fabrication techniques and grown in predefined locations, demonstrating an ability to control materials’ placement and morphology. Several characterization tools were employed to interrogate the prepared heterostructures. ZnO NWs were shown to grow uniformly over the network of SWCNTs, and much denser packed vertically oriented ZnO NWs were produced on graphene thin films. Such heterostructures can be used widely in many potential applications, such as photocatalysts, supercapacitors, solar cells, piezoelectric or thermal actuators, as well as chemical or biological sensors.

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