Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants

2014 ◽  
Vol 32 (3) ◽  
pp. 564-574 ◽  
Author(s):  
Xuan Zheng ◽  
Shuangshuang Dong ◽  
Jie Zheng ◽  
Duanhua Li ◽  
Feng Li ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Membrane Proteins ◽  
Self Assembly

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.

Simulation studies of the interactions between membrane proteins and detergents

2005 ◽  
Vol 33 (5) ◽  
pp. 910-912 ◽  
Author(s):  
P.J. Bond ◽  
J. Cuthbertson ◽  
M.S.P. Sansom
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Self Assembly ◽  
Kinetic Mechanism ◽  
Coarse Grained ◽  
Simulation Studies ◽  
High Resolution Data ◽  
Biologically Relevant ◽  
Structure And Dynamics ◽  
Detergent Interactions

Interactions between membrane proteins and detergents are important in biophysical and structural studies and are also biologically relevant in the context of folding and transport. Despite a paucity of high-resolution data on protein–detergent interactions, novel methods and increased computational power enable simulations to provide a means of understanding such interactions in detail. Simulations have been used to compare the effect of lipid or detergent on the structure and dynamics of membrane proteins. Moreover, some of the longest and most complex simulations to date have been used to observe the spontaneous formation of membrane protein–detergent micelles. Common mechanistic steps in the micelle self-assembly process were identified for both α-helical and β-barrel membrane proteins, and a simple kinetic mechanism was proposed. Recently, simplified (i.e. coarse-grained) models have been utilized to follow long timescale transitions in membrane protein–detergent assemblies.

Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins

2011 ◽  
Vol 28 (3) ◽  
pp. 262-271 ◽  
Author(s):  
Friederike Junge ◽  
Stefan Haberstock ◽  
Christian Roos ◽  
Susanne Stefer ◽  
Davide Proverbio ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Structural Analysis ◽  
Membrane Proteins ◽  
Free Protein ◽  
Cell Free Protein Synthesis

scholarly journals A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties v1

2020 ◽  
Author(s):  
Anne Zemella ◽  
Theresa Richter ◽  
Lena Thoring ◽  
Stefan Kubick
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Trna Synthetase ◽  
Fluorescent Labeling ◽  
Laser Scanning Microscopy ◽  
Free System ◽  
Free Protein ◽  
Canonical Amino Acid ◽  
Cell Free Protein Synthesis

Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In this protocol the first part describes the fluorescent labeling by using a precharged tRNA, loaded with a fluorescent amino acid. The second part describes the preparation of a modified aminoacyl-tRNA-synthetase and a suppressor tRNA that are applied to the CHO cell-free system to enable the incorporation of a non-canonical amino acid. The reactive group of the non-canonical amino acid is further coupled to a fluorescent dye. Both methods utilize the amber stop codon suppression technology. The successful fluorescent labeling of the model G protein-coupled receptor adenosine A2A (Adora2a) is analyzed by in-gel-fluorescence, a reporter protein assay, and confocal laser scanning microscopy (CLSM). Moreover, a ligand-dependent conformational change of the fluorescently labeled Adora2a was analyzed by bioluminescence resonance energy transfer (BRET).

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

scholarly journals The Bacterial Cytoskeleton based on Bacterial Translation Elongation Factor EF-Tu: Novel Insights

2022 ◽  
Vol 10 (2) ◽  
pp. 01-06
Author(s):  
Frank Mayer
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Self Assembly ◽  
Elongation Factor ◽  
Short Review ◽  
Cell Integrity ◽  
Bacterial Cytoskeleton ◽  
Sequence Of Events ◽  
Independent Functions ◽  
Ribosomal Protein Synthesis

Bacteria possess an EF-Tu-based cytoskeleton.This article presents a short review. A number of questions which are not discussed in the former publications can be asked, such as: all bacteria possess a ribosomal protein synthesis system and, hence, also EF-Tu. EF-Tu is produced in an amount that is higher than the need for a function as translation elogation factor in ribsomal protein synthesis. This article tries to answer the question regarding the surplus of EF-Tu: formation of a "cell-wide web" by self-assembly as a feafure that stabilizes cell integrity. An additional question can be asked: what is the origin of this bacterial cytoskeleton? This article contains a speculation on this topic. A third question regards the'ntteructjon of ribosomes in the process of protemsynthesis: does the EF-Tu protein move to the ribosome, or does the ribosome move to the EF-Tu intergated in a fibril of the bacterial cytoskeleton? The former publication depicts electron micrographs which show colocalizatton of botth entities. EF-Tu is an example for aprotein with two independent functions: participation in the ribosomal protein synthesis as a kanslation elongation factor, and component of a bacterial cytoskeleton. This situation can open up a discussion ofthe sequence of events and states of early cells during evolution.

scholarly journals Self-assembly of polymer-encased lipid nanodiscs and membrane protein reconstitution

2018 ◽  
Author(s):  
Bikash R. Sahoo ◽  
Takuya Genjo ◽  
Kanhu C. Moharana ◽  
Ayyalusamy Ramamoorthy
Keyword(s):  
Membrane Proteins ◽  
Self Assembly ◽  
Md Simulation ◽  
Mechanistic Model ◽  
Coarse Graining ◽  
Spatial Arrangement ◽  
Molar Ratio ◽  
Dynamics And Function ◽  
And Function ◽  
Lipid Nanodiscs

AbstractThe absence of detergent and curvature makes nanodiscs to be excellent membrane mimetics. The lack of structural and mechanistic model of polymer-encapsulated lipid-nanodiscs limits their use to study the structure, dynamics and function of membrane proteins. In this study, we parametrized and optimized the coarse-graining (CG) bead-mapping for two differently charged and functionalized copolymers, namely styrene-maleic acid (SMAEA) and polymethacrylate (PMAQA), for the Martini force-field framework and showed nanodisc formation (< 8 nm diameter) on a time scale of tens of microseconds using molecular dynamics (MD) simulation. Structural models of ~ 2.0 or 4.8 kDa PMAQA and ~2.2 kDa SMAEA polymer based lipid-nanodiscs highlights the importance of polymer chemical structure, size and polymer:lipid molar ratio in the optimization of nanodisc structure. The ideal spatial arrangement of polymers in nanodisc, nanodisc size and thermal stability obtained from our MD simulation correlates well with the experimental observations. The polymer-nanodisc were tested for the reconstitution of single-pass or multi-pass transmembrane proteins. We expect this study to be useful in the development of novel polymer based lipid-nanodiscs and for the structural studies of membrane proteins.TOC GRAPHICS

scholarly journals Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins.

1982 ◽  
Vol 94 (1) ◽  
pp. 179-186 ◽  
Author(s):  
G Tarone ◽  
G Galetto ◽  
M Prat ◽  
P M Comoglio
Keyword(s):  
Protein Synthesis ◽  
Cell Adhesion ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Rabbit Antiserum ◽  
Proteinase K ◽  
Proteolytic Digestion ◽  
Adhesive Properties ◽  
Surface Molecules ◽  
Protein Nature

Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to restore the adhesive properties of pronase-treated cells, showing the protein nature of the molecules involved in adhesion to fibronectin. A peculiar feature of these proteins was their resistance to cleavage by trypsin. After prolonged trypsin treatment (1 mg/ml for 20 min at 37 degrees C), cells adhered and spread on fibronectin-coated dishes, even when protein synthesis was inhibited by 4 microM cycloheximide. Under these conditions only three glycoproteins (gp) of molecular weight 130,000, 120,000, and 80,000 were left on the cell surface. These were precipitated by a rabbit antiserum against BHK cells that also inhibited adhesion of trypsin-treated cells. gp120 and gp80 were left at the cell surface after mild pronase digestion (0.2 mg/ml for 20 min at 37 degrees C), under conditions not affecting adhesion. These data suggest that these glycoproteins may be involved in fibronectin-mediated cell adhesion in some yet unknown way.

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