scholarly journals Fluorinated and hemifluorinated surfactants as alternatives to detergents for membrane protein cell-free synthesis

2007 ◽  
Vol 403 (1) ◽  
pp. 183-187 ◽  
Author(s):  
Kyu-Ho Park ◽  
Catherine Berrier ◽  
Florence Lebaupain ◽  
Bernard Pucci ◽  
Jean-Luc Popot ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Lipid Vesicles ◽  
Mechanosensitive Channel ◽  
In Vitro Synthesis ◽  
Fluorinated Surfactants ◽  
Electrophysiological Activity ◽  
Protein Cell ◽  
Suitable Environment

Hemifluorinated and fluorinated surfactants are lipophobic and, as such, non-detergent. Although they do not solubilize biological membranes, they can, after conventional solubilization, substitute for detergents to keep membrane proteins soluble, which generally improves their stability [Breyton, Chabaud, Chaudier, Pucci and Popot (2004) FEBS Lett. 564, 312–318]. In the present study, we show that (hemi)fluorinated surfactants can be used for in vitro synthesis of membrane proteins: they do not interfere with protein synthesis, and they provide a suitable environment for MscL, a pentameric mechanosensitive channel, to fold and oligomerize to its native functional state. Following synthesis, both types of surfactants can be used to deliver MscL directly to pre-formed lipid vesicles. The electrophysiological activity of MscL synthesized in vitro in the presence of either hemi- or per-fluorinated surfactant is similar to that of the protein expressed in vivo.

scholarly journals DNA-Encircled Lipid Bilayers

2018 ◽  
Author(s):  
Katarina Iric ◽  
Madhumalar Subramanian ◽  
Jana Oertel ◽  
Nayan P. Agarwal ◽  
Michael Matthies ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Protein Function ◽  
Dna Nanotechnology ◽  
Lipid Vesicles ◽  
Associated Proteins

ABSTRACTLipid bilayers and lipid-associated proteins play a crucial role in biology. As in vivo studies and manipulation are inherently difficult, several membrane-mimetic systems have been developed to enable investigation of lipidic phases, lipid-protein interactions, membrane protein function and membrane structure in vitro. Controlling the size and shape, or site-specific functionalization is, however, difficult to achieve with established membrane mimetics based on membrane scaffolding proteins, polymers or peptides. In this work, we describe a route to leverage the unique programmability of DNA nanotechnology and create DNA-encircled bilayers (DEBs), which are made of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded minicircle. To stabilize the hydrophobic rim of the lipid bilayer, and to prevent formation of lipid vesicles, we introduced up to 2 alkyl chains per helical that point to the inside of the toroidal DNA ring and interact with the hydrophobic side chains of the encapsulated lipid bilayer. The DEB approach described herein provides unprecedented control of size, and allows the orthogonal functionalizations and arrangement of engineered membrane nanoparticles and will become a valuable tool for biophysical investigation of lipid phases and lipid-associated proteins and complexes including structure determination of membrane proteins and pharmacological screenings of membrane proteins.

scholarly journals Supervised Learning Model Predicts Protein Adsorption to Nanotubes

2021 ◽  
Author(s):  
Rebecca L Pinals ◽  
Nicholas Ouassil ◽  
Jackson Travis Del Bonis-O'Donnell ◽  
Jeffrey W Wang ◽  
Markita P Landry
Keyword(s):  
Amino Acids ◽  
Protein Adsorption ◽  
Biological Systems ◽  
Protein Corona ◽  
Engineered Nanoparticles ◽  
Mass Spectrometry Data ◽  
In Vitro Synthesis ◽  
Intractable Problem

Engineered nanoparticles are advantageous for numerous biotechnology applications, including biomolecular sensing and delivery. However, testing the compatibility and function of nanotechnologies in biological systems requires a heuristic approach, where unpredictable biofouling often prevents effective implementation. Such biofouling is the result of spontaneous protein adsorption to the nanoparticle surface, forming the "protein corona" and altering the physicochemical properties, and thus intended function, of the nanotechnology. To better apply engineered nanoparticles in biological systems, herein, we develop a random forest classifier (RFC) trained with proteomic mass spectrometry data that identifies which proteins adsorb to nanoparticles. We model proteins that populate the corona of a single-walled carbon nanotube (SWCNT)-based optical nanosensor. We optimize the classifier and characterize the classifier performance against other models. To evaluate the predictive power of our model, we then apply the classifier to rapidly identify and experimentally validate proteins with high binding affinity to SWCNTs. Using protein properties based solely on amino acid sequence, we further determine protein features associated with increased likelihood of SWCNT binding: proteins with high content of solvent-exposed glycine residues and non-secondary structure-associated amino acids. Furthermore, proteins with high leucine residue content and beta-sheet-associated amino acids are less likely to form the SWCNT protein corona. The classifier presented herein provides an important tool to undertake the otherwise intractable problem of predicting protein-nanoparticle interactions, which is needed for more rapid and effective translation of nanobiotechnologies from in vitro synthesis to in vivo use.

scholarly journals AN ANALYSIS OF COLLAGEN SECRETION BY ESTABLISHED MOUSE FIBROBLAST LINES

1964 ◽  
Vol 22 (1) ◽  
pp. 227-258 ◽  
Author(s):  
Burton Goldberg ◽  
Howard Green
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Mouse Fibroblast ◽  
In Vivo Studies ◽  
In Vitro Synthesis ◽  
Collagen Secretion ◽  
Golgi System ◽  
Alternative Theories

In vitro synthesis of collagen by established mouse fibroblast lines has been examined by electron microscopy. During rapid growth (log phase), when collagen could not be detected in the cultures, the cells lacked a well developed granular ergastoplasm and Golgi system. Upon cessation of growth (stationary phase), collagen accumulated in the cultures and the cells demonstrated highly developed granular and smooth ergastoplasm. Collagen appeared to be synthesized in the rough-surfaced endoplasmic reticulum and to be transported as a soluble protein to the cell surface by vesicular elements of the agranular ergastoplasm. Fusion of the limiting membranes of these vesicles with the cell membrane permitted the discharge of the soluble collagen into the extracellular space, where fibrils of two diameter distributions formed. The secretion of collagen is concluded to be of the merocrine type. Alternative theories of collagen secretion are discussed and the data for established lines compared with the results of other in vitro and in vivo studies of collagen fibrillogenesis.

scholarly journals Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins.

1991 ◽  
Vol 174 (5) ◽  
pp. 1167-1177 ◽  
Author(s):  
J Vuopio-Varkila ◽  
G K Schoolnik
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
De Novo ◽  
Outer Membrane Proteins ◽  
Temporal Correlation ◽  
Enteropathogenic Escherichia Coli ◽  
E Coli

Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine. A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells. The LA phenotype was studied using B171, an O111:NM enteropathogenic E. coli (EPEC) strain, and HEp-2 cell monolayers. LA could be detected 30-60 min after exposure of HEp-2 cells to B171. However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype. Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide. A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC. Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E. coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo. This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.

scholarly journals Dissection of the Golgi complex. II. Density separation of specific Golgi functions in virally infected cells treated with monensin.

1983 ◽  
Vol 96 (3) ◽  
pp. 851-856 ◽  
Author(s):  
P Quinn ◽  
G Griffiths ◽  
G Warren
Keyword(s):  
Membrane Proteins ◽  
Intracellular Transport ◽  
Semliki Forest Virus ◽  
Viral Membrane ◽  
Golgi Stack ◽  
Galactosyl Transferase ◽  
Infected Cells ◽  
Cis And Trans

In the accompanying paper (Griffiths, G., P. Quinn, and G. Warren, 1983, J. Cell Biol., 96:835-850), we suggested that the Golgi stack could be divided into functionally distinct cis, medial, and trans compartments, each comprising one or two adjacent cisternae. These compartments were identified using Baby hamster kidney (BHK) cells infected with Semliki Forest virus (SFV) and treated with monensin. This drug blocked intracellular transport but not synthesis of the viral membrane proteins that were shown to accumulate in the medial cisternae. In consequence, these cisternae bound nucleocapsids. Here we show that this binding markedly increased the density of the medial cisternae and allowed us to separate them from cis and trans Golgi cisternae. A number of criteria were used to show that the intracellular capsid-binding membranes (ICBMs) observed in vivo were the same as those membranes sedimenting to a higher density in sucrose gradients in vitro, and this separation of cisternae was then used to investigate the distribution, within the Golgi stack, of some specific Golgi functions. After labeling for 2.5 min with [3H]palmitate, most of the fatty acid attached to viral membrane proteins was found in the ICBM fraction. Because the viral membrane proteins appear to move from cis to trans, this suggests that fatty acylation occurs in the cis or medial Golgi cisternae. In contrast, the distribution of alpha 1-2-mannosidase, an enzyme involved in trimming high-mannose oligosaccharides, and of galactosyl transferase, which is involved in the construction of complex oligosaccharides, was not affected by monensin treatment. Together with data in the accompanying paper, this would restrict these two Golgi functions to the trans cisternae. Our data strongly support the view that Golgi functions have specific and discrete locations within the Golgi stack.

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