scholarly journals One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces

2015 ◽  
Vol 113 (3) ◽  
pp. 608-613 ◽  
Author(s):  
Peter J. Yunker ◽  
Haruichi Asahara ◽  
Kuo-Chan Hung ◽  
Corey Landry ◽  
Laura R. Arriaga ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Membrane Proteins ◽  
Self Assembly ◽  
Hydrophobic Interactions ◽  
Cell Communication ◽  
Transmembrane Helices ◽  
One Pot ◽  
Surface Receptors ◽  
Coupling Protein

Single-span membrane proteins (ssMPs) represent approximately one-half of all membrane proteins and play important roles in cellular communications. However, like all membrane proteins, ssMPs are prone to misfolding and aggregation because of the hydrophobicity of transmembrane helices, making them difficult to study using common aqueous solution-based approaches. Detergents and membrane mimetics can solubilize membrane proteins but do not always result in proper folding and functionality. Here, we use cell-free protein synthesis in the presence of oil drops to create a one-pot system for the synthesis, assembly, and display of functional ssMPs. Our studies suggest that oil drops prevent aggregation of some in vitro-synthesized ssMPs by allowing these ssMPs to localize on oil surfaces. We speculate that oil drops may provide a hydrophobic interior for cotranslational insertion of the transmembrane helices and a fluidic surface for proper assembly and display of the ectodomains. These functionalized oil drop surfaces could mimic cell surfaces and allow ssMPs to interact with cell surface receptors under an environment closest to cell–cell communication. Using this approach, we showed that apoptosis-inducing human transmembrane proteins, FasL and TRAIL, synthesized and displayed on oil drops induce apoptosis of cultured tumor cells. In addition, we take advantage of hydrophobic interactions of transmembrane helices to manipulate the assembly of ssMPs and create artificial clusters on oil drop surfaces. Thus, by coupling protein synthesis with self-assembly at the water–oil interface, we create a platform that can use recombinant ssMPs to communicate with cells.

scholarly journals Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation

2021 ◽  
Author(s):  
Sudeep Banjade ◽  
Lu Zhu ◽  
Jeffrey Jorgensen ◽  
Sho Suzuki ◽  
Scott D. Emr
Keyword(s):  
Membrane Proteins ◽  
Self Assembly ◽  
Cell Biology ◽  
Substrate Recognition ◽  
Initial Step ◽  
Binding Interactions ◽  
Vacuole Membrane ◽  
Multivalent Interactions ◽  
Cargo Sorting

AbstractThe general mechanisms by which ESCRTs are specifically recruited to various membranes, and how ESCRT subunits are spatially organized remain central questions in cell biology. At the endosome and lysosomes, ubiquitination of membrane proteins triggers ESCRT-mediated substrate recognition and degradation. Using the yeast lysosome/vacuole, we define the principles by which substrate engagement by ESCRTs occurs at this organelle. We find that multivalent interactions between ESCRT-0 and polyubiquitin is critical for substrate recognition at yeast vacuoles, with a lower-valency requirement for cargo engagement at endosomes. Direct recruitment of ESCRT-0 induces dynamic foci on the vacuole membrane, and forms fluid condensates in vitro with polyubiquitin. We propose that self-assembly of early ESCRTs induces condensation, an initial step in ESCRT-assembly/nucleation at membranes. This property can be tuned specifically at various organelles by modulating the number of binding interactions.One-Sentence SummaryCondensation of multivalent ESCRT-0/polyubiquitin assemblies organizes cargo sorting reactions at lysosomes

Norbornene-Functionalised Chitosan Hydrogels and Microgels via an Unprecedented Photo-Initiated Self-Assembly for Potential Biomedical Applications

2020 ◽  
Author(s):  
Sarah michel ◽  
Alice Kilner ◽  
Jean-Charles Eloi ◽  
Sarah E rogers ◽  
Wuge H. Briscoe ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Self Assembly ◽  
Biomedical Applications ◽  
Hydrophobic Interactions ◽  
Gelation Mechanism ◽  
Swelling Behaviour ◽  
Assembly Mechanism ◽  
Significant Toxicity ◽  
Chitosan Hydrogels

<p><br></p><p> Access to biocompatible self-assembled gels and microgels is of great interests for a variety of biological applications from tissue engineering to drug delivery. Here, the facile synthesis of supramolecular hydrogels of norbornene (nb)-functionalised chitosan (CS-nb) via UV-triggered self-assembly in the presence of Irgacure 2959 (IRG) is reported. The <i>in vitro </i>stable hydrogels are injectable and showed pH-responsive swelling behaviour, while their structure and mechanical properties could be tuned by tailoring the stereochemistry of the norbornene derivative (e.g. <i>endo</i>- or -<i>exo</i>). Interestingly, unlike other nb-type hydrogels, the gels possess nanopores within their structure, which might lead to potential drug delivery applications. A gelation mechanism was proposed based on hydrophobic interactions following the combination of IRG on norbornene, as supported by 1H NMR. This self-assembly mechanism was used to access microgels of size 100-150 nm which could be further functionalised and showed no significant toxicity to human dermofibroblast cells. </p>

scholarly journals Norbornene-Functionalised Chitosan Hydrogels and Microgels via an Unprecedented Photo-Initiated Self-Assembly for Potential Biomedical Applications

2020 ◽  
Author(s):  
Sarah michel ◽  
Alice Kilner ◽  
Jean-Charles Eloi ◽  
Sarah E rogers ◽  
Wuge H. Briscoe ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Self Assembly ◽  
Biomedical Applications ◽  
Hydrophobic Interactions ◽  
Gelation Mechanism ◽  
Swelling Behaviour ◽  
Assembly Mechanism ◽  
Significant Toxicity ◽  
Chitosan Hydrogels

<p><br></p><p> Access to biocompatible self-assembled gels and microgels is of great interests for a variety of biological applications from tissue engineering to drug delivery. Here, the facile synthesis of supramolecular hydrogels of norbornene (nb)-functionalised chitosan (CS-nb) via UV-triggered self-assembly in the presence of Irgacure 2959 (IRG) is reported. The <i>in vitro </i>stable hydrogels are injectable and showed pH-responsive swelling behaviour, while their structure and mechanical properties could be tuned by tailoring the stereochemistry of the norbornene derivative (e.g. <i>endo</i>- or -<i>exo</i>). Interestingly, unlike other nb-type hydrogels, the gels possess nanopores within their structure, which might lead to potential drug delivery applications. A gelation mechanism was proposed based on hydrophobic interactions following the combination of IRG on norbornene, as supported by 1H NMR. This self-assembly mechanism was used to access microgels of size 100-150 nm which could be further functionalised and showed no significant toxicity to human dermofibroblast cells. </p>

scholarly journals Polymeric Composites with Embedded Nanocrystalline Cellulose for the Removal of Iron(II) from Contaminated Water

Polymers ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 1377 ◽  
Author(s):  
Anayet Kabir ◽  
Matthew Dunlop ◽  
Bishnu Acharya ◽  
Rabin Bissessur ◽  
Marya Ahmed
Keyword(s):  
Self Assembly ◽  
Three Dimensional ◽  
Polymeric Composites ◽  
Industrial Applications ◽  
Nanocrystalline Cellulose ◽  
Soluble Form ◽  
Contaminated Water ◽  
One Pot ◽  
Treated Water

The exponential increase in heavy metal usage for industrial applications has led to the limited supply of clean water for human needs. Iron is one of the examples of heavy metals, which is responsible for an unpleasant taste of water and its discoloration, and is also associated with elevated health risks if it persists in drinking water for a prolonged period of time. The adsorption of a soluble form of iron (Fe2+) from water resources is generally accomplished in the presence of natural or synthetic polymers or nanoparticles, followed by their filtration from treated water. The self-assembly of these colloidal carriers into macroarchitectures can help in achieving the facile removal of metal-chelated materials from treated water and hence can reduce the cost and improve the efficiency of the water purification process. In this study, we aim to develop a facile one-pot strategy for the synthesis of polymeric composites with embedded nanocrystalline cellulose (NCC) for the chelation of iron(II) from contaminated water. The synthesis of the polymeric composites with embedded nanoparticles was achieved by the facile coating of ionic monomers on the surface of NCC, followed by their polymerization, crosslinking, and self-assembly in the form of three-dimensional architectures at room temperature. The composites prepared were analyzed for their physiochemical properties, antifouling properties, and for their iron(II)-chelation efficacies in vitro. The results indicate that the embedded-NCC polymeric composites have antifouling properties and exhibit superior iron(II)-chelation properties at both acidic and basic conditions.

scholarly journals Probing Membrane Protein Assembly into Nanodiscs by In Situ Dynamic Light Scattering: A2A Receptor as a Case Study

Biology ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 400 ◽  
Author(s):  
Rosana I. Reis ◽  
Isabel Moraes
Keyword(s):  
Light Scattering ◽  
Membrane Proteins ◽  
Dynamic Light Scattering ◽  
Membrane Protein ◽  
Cell Communication ◽  
Cell Physiology ◽  
Functional Characterisation

Membrane proteins play a crucial role in cell physiology by participating in a variety of essential processes such as transport, signal transduction and cell communication. Hence, understanding their structure–function relationship is vital for the improvement of therapeutic treatments. Over the last decade, based on the development of detergents, amphipoles and styrene maleic-acid lipid particles (SMALPs), remarkable accomplishments have been made in the field of membrane protein structural biology. Nevertheless, there are still many drawbacks associated with protein–detergent complexes, depending on the protein in study or experimental application. Recently, newly developed membrane mimetic systems have become very popular for allowing a structural and functional characterisation of membrane proteins in vitro. The nanodisc technology is one such valuable tool, which provides a more native-like membrane environment than detergent micelles or liposomes. In addition, it is also compatible with many biophysical and biochemical methods. Here we describe the use of in situ dynamic light scattering to accurately and rapidly probe membrane proteins’ reconstitution into nanodiscs. The adenosine type 2A receptor (A2AR) was used as a case study.

scholarly journals Self-assembly of large, ordered lamellae from non-bilayer lipids and integral membrane proteins in vitro

2000 ◽  
Vol 97 (4) ◽  
pp. 1473-1476 ◽  
Author(s):  
I. Simidjiev ◽  
S. Stoylova ◽  
H. Amenitsch ◽  
T. Javorfi ◽  
L. Mustardy ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Self Assembly ◽  
Integral Membrane Proteins

scholarly journals Synthesis of Extended, Self-Assembled Biological Membranes containing Membrane Proteins from Gas Phase

2019 ◽  
Author(s):  
Matthias Wilm
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Self Assembly ◽  
Protein Function ◽  
Lipid Membranes ◽  
Protein Complexes ◽  
Biological Membranes ◽  
The Self ◽  
In Vitro Systems

1.AbstractMembrane proteins carry out a wide variety of biological functions. The reproduction of specific properties that have evolved over millions of years of biological membranes in a technically controlled environment is of significant interest. Here a method is presented that allows the self-assembly of a macroscopically large, freely transportable membrane with Outer membrane porin G from Escherichia Coli. The technique does not use protein specific characteristics and therefore, could represent a method for the generation of extended layers of membranes with arbitrary membrane protein content. Such in-vitro systems are relevant in the study of membrane-protein function and structure and the self-assembly of membrane-based protein complexes. They might become important for the incorporation of the lipid-membranes in technological devices.

Peptides Co-Assembling into Hydrangea-Like Microstructures

2020 ◽  
Vol 20 (5) ◽  
pp. 3239-3245
Author(s):  
Zhen Guo ◽  
Zhiwei Shen ◽  
Yujiao Wang ◽  
Tingyuan Tan ◽  
Yi Zhang
Keyword(s):  
Self Assembly ◽  
Hydrophobic Interactions ◽  
Supramolecular Assembly ◽  
Force Microscopy ◽  
Peptide Assembly ◽  
Atomic Force ◽  
Potential Applications ◽  
Cellular Microenvironments ◽  
Functional Biomaterials

Supramolecular assembly in vitro is a simple and effective way to produce multi-level biostructures to mimic the self-assembly of biomolecules in organisms. The study on peptide assembly behaviors would benefit a lot to understand what goes on in life, as well as in the construction of plenty of functional biomaterials that have potential applications in various fields. Since cellular microenvironments are crowded and contain various biomolecules, studying protein and peptide co-assembly is of great interest. Here, we introduced the co-assembly of 5-FAM-ELVFFAE-NH2 (EE-7) and (CY5)-KLVFFAK-NH2 (KK-7), which are sequences derived from the core of the amyloid β (Aβ) peptide, a key protein in Alzheimer’s diseases. Morphologic studies employing atomic force microscopy and scanning electron microscopy indicated that the co-assembled entities had a novel hydrangea-like microstructure, in contrast to micro-sheet structures formed from monocomponent EE-7 or KK-7, respectively. Fluorescence co-localization experiments confirmed that the hydrangealike microstructures were indeed made of both EE-7 and KK-7. We suggest that the formation of the hydrangea-like microstructures is driven by both the electrostatic and hydrophobic interactions between EE-7 and KK-7. A molecular mechanism has been provided to explain the formation of the hydrangea-like microstructures.

Structural comparison of substrate entry gate for rhomboid intramembrane peptidasesThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (2) ◽  
pp. 216-223 ◽  
Author(s):  
Christelle Lazareno-Saez ◽  
Cory L. Brooks ◽  
M. Joanne Lemieux
Keyword(s):  
Membrane Proteins ◽  
Hydrophobic Interactions ◽  
Peer Review Process ◽  
Mobile Element ◽  
Transmembrane Helices ◽  
General Role ◽  
E Coli ◽  
Signalling Molecules ◽  
Health And Disease ◽  
Selection Of

Rhomboids are intramembrane serine peptidases conserved in all kingdoms of life. Their general role is to cleave integral membrane proteins to release signalling molecules. These signals, when disrupted, can contribute to various diseases. Crystal structures of H. influenzae (hiGlpG) and E. coli GlpG (ecGlpG) rhomboids have revealed a structure with six transmembrane helices and a Ser–His catalytic dyad buried within the membrane. One emerging issue was the identification of the mobile element in the protein that allows substrate docking. It has been proposed that the substrate entry gate is composed of helix 5 and loop 5. The present review studies the structures of these two orthologs. In ecGlpG structures, different conformations of loop 5 and helix 5 are observed. Open and closed conformations of ecGlpG structures are compared with each other and with hiGlpG, surveying differences in hydrophobic interactions within loop 5 and helix 5. Furthermore, a comparison of the ecGlpG and hiGlpG structures reveals differences in loop 4. Overall, less variation is observed in loop 4, suggesting this region acts as an anchor for the substrate gate. Functional and regulatory implications of these variations are discussed.

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