Micelles, Bicelles, Amphipols, Nanodiscs, Liposomes, or Intact Cells: The Hitchhiker’s Guide to the Study of Membrane Proteins by NMR

2014 ◽  
pp. 315-345 ◽  
Author(s):  
Laurent J. Catoire ◽  
Xavier L. Warnet ◽  
Dror E. Warschawski
Keyword(s):  
Membrane Proteins ◽  
Intact Cells

scholarly journals Visualizing conformational dynamics of proteins in solution and at the cell membrane

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sharona E Gordon ◽  
Mika Munari ◽  
William N Zagotta
Keyword(s):  
Membrane Proteins ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Computational Method ◽  
Intact Cells ◽  
Distance Distributions ◽  
Amber Codon Suppression ◽  
Noncanonical Amino Acid

Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

scholarly journals Phosphorylation of chlamydomonas reinhardi chloroplast membrane proteins in vivo and in vitro.

1982 ◽  
Vol 93 (3) ◽  
pp. 712-718 ◽  
Author(s):  
G C Owens ◽  
I Ohad
Keyword(s):  
Membrane Proteins ◽  
Photosystem Ii ◽  
Binding Site ◽  
Reaction Center ◽  
Thylakoid Membrane ◽  
Red Light ◽  
Intact Cells ◽  
Turnover Process

Phosphorylation of thylakoid membrane proteins in the chloroplast of wild-type and mutant strains of Chlamydomonas reinhardi has been studied in vivo and in vitro. Intact cells or purified membranes were labeled with [32P]orthophosphate or [gamma-32P]ATP, respectively, and the presence of phosphorylated polypeptides was detected by autoradiography after membrane fractionation by SDS PAGE. The 32P was esterified to serine and threonine residues. At least six polypeptides were phosphorylated in vitro and in vivo, and corresponded to components of the photosystem II complex contributing to the formation of the light-harvesting-chlorophyll (LHC) a,b-protein complex, the DCMU binding site (32-35 kdaltons), and the reaction center (26 kdaltons). In agreement with previous reports (Alfonzo, et al., 1979, Plant Physiol., 65:730-734; and Bennett, 1979, FEBS (Fed. Eur. Biochem. Soc.) Lett., 103:342-344), the membrane-bound protein kinase was markedly stimulated by light in vitro via a mechanism requiring photosystem II activity. Phosphorylation of thylakoid membrane polypeptides in vivo was, however, completely independent of illumination. Similar amounts of phosphate were incorporated into the photosynthetic membranes of cells incubated in the dark, in white light with or without 3-(3,4-dichlorophenyl-1,1-dimethyl urea (DCMU), or in red or far-red light. Different turnovers of the phosphate were observed in the light and dark, and a phosphoprotein phosphatase involved in this turnover process was also associated with the membrane. Comparison of the amount of esterified phosphate per protein in vivo and the maximum incorporation in isolated membranes revealed that only a small fraction of the available sites could be phosphorylated in vitro. In contrast to the DCMU binding site, the LHC and 26-kdalton polypeptide were not phosphorylated in vivo when the reaction center II polypeptides of 44-54 kdaltons were missing. The finding that all the phosphoproteins appear to be components of the photosystem II complex and are only partially dephosphorylated in vivo suggests strongly that protein phosphorylation might play an important role in the maintenance of the organizational integrity of this complex. The observation that the LHC is not phosphorylated in the absence of the reaction center lends support to this idea.

Express incorporation of membrane proteins from various human cell types into phospholipid bilayer nanodiscs

2017 ◽  
Vol 474 (8) ◽  
pp. 1361-1371 ◽  
Author(s):  
Stefanie Mak ◽  
Ruoyu Sun ◽  
Michael Schmalenberg ◽  
Carsten Peters ◽  
Peter B. Luppa
Keyword(s):  
Membrane Proteins ◽  
Human Cell ◽  
Cell Types ◽  
Phospholipid Bilayer ◽  
Size Exclusion ◽  
Cell Organelles ◽  
Laboratory Equipment ◽  
Intact Cells ◽  
Hek 293 ◽  
Analytical Approaches

Analysis of membrane proteins is still inadequately represented in diagnostics despite their importance as drug targets and biomarkers. One main reason is the difficult handling caused by their insolubility in aqueous buffer solutions. The nanodisc technology was developed to circumvent this challenge and enables analysis of membrane proteins with standard research methods. However, existing nanodisc generation protocols rely on time-consuming membrane isolation and protein purification from overexpression systems. In the present study, we present a novel, simplified procedure for the rapid generation of nanodiscs directly from intact cells. Workflow and duration of the nanodisc preparation were shortened without reducing the reconstitution efficiency, and all the steps were modified for the use of only standard laboratory equipment. This protocol was successfully applied to various human cell types, such as cultivated human embryonic kidney 293 (HEK-293) cells, as well as freshly isolated human red blood cells and platelets. In addition, the reconstitution of membrane proteins from cell organelles was achieved. The use of endogenous lipids ensures a native-like environment, which promotes native protein (re)folding. Nanodisc generation was verified by size exclusion chromatography and EM, whereas incorporation of different membrane proteins was demonstrated by Western blot analysis. Our protocol enabled the rapid incorporation of endogenous membrane proteins from human cells into nanodiscs, which can be applied to analytical approaches.

scholarly journals N-glycosylation and topology of the human SLC26 family of anion transport membrane proteins

2014 ◽  
Vol 306 (10) ◽  
pp. C943-C960 ◽  
Author(s):  
Jing Li ◽  
Fan Xia ◽  
Reinhart A. F. Reithmeier
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Complex Form ◽  
Cell Surface Expression ◽  
Intact Cells ◽  
Hek 293 ◽  
Permeabilized Cells ◽  
Glycosylation Sites ◽  
High Mannose

The human solute carrier ( SLC26) family of anion transporters consists of 10 members ( SLCA1–11, SLCA10 being a pseudogene) that encode membrane proteins containing ∼12 transmembrane (TM) segments with putative N-glycosylation sites (-NXS/T-) in extracellular loops and a COOH-terminal cytosolic STAS domain. All 10 members of the human SLC26 family, FLAG-tagged at the NH2 terminus, were transiently expressed in HEK-293 cells. While most proteins were observed to contain both high-mannose and complex oligosaccharides, SLC26A2 was mainly in the complex form, SLC26A4 in the high-mannose form, and SLC26A8 was not N-glycosylated. Mutation of the putative N-glycosylation sites showed that most members contain multiple N-glycosylation sites in the second extracytosolic (EC) loop, except SLC26A11, which was N-glycosylated in EC loop 4. Immunofluorescence staining of permeabilized cells localized the proteins to the plasma membrane and the endoplasmic reticulum, with SLC26A2 highly localized to the plasma membrane. N-glycosylation was not a necessary requirement for cell surface expression as the localization of nonglycosylated proteins was similar to their wild-type counterparts, although a lower level of cell-surface biotinylation was observed. No immunostaining of intact cells was observed for any SLC26 members, demonstrating that the NH2-terminal FLAG tag was located in the cytosol. Topological models of the SLC26 proteins that contain an even number of transmembrane segments with both the NH2 and COOH termini located in the cytosol and utilized N-glycosylation sites defining the positions of two EC loops are presented.

Effective Transfer of Membrane Proteins from Intact Cells to Liposomes and Preparation of Liposomal Vaccines

1990 ◽  
Vol 613 (1 Enzyme Engine) ◽  
pp. 116-127 ◽  
Author(s):  
JUNZO SUNAMOTO ◽  
KAZUNARI AKIYOSHI ◽  
MITSUAKI GOTO ◽  
TETSUROU NOGUCHI ◽  
TOSHINORI SATO ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Intact Cells ◽  
Effective Transfer

scholarly journals The phosphorylation of coated membrane proteins in intact neurons.

1986 ◽  
Vol 102 (4) ◽  
pp. 1325-1333 ◽  
Author(s):  
J H Keen ◽  
M M Black
Keyword(s):  
Membrane Proteins ◽  
Gel Electrophoresis ◽  
Sympathetic Neurons ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Two Dimensional ◽  
Intact Cells ◽  
Membrane Structure And Function ◽  
Coated Membrane

To complement studies that have demonstrated the prominent phosphorylation of a 50-kD coated vesicle polypeptide in vitro, we have evaluated the phosphorylation of coated membrane proteins in intact cells. A co-assembly assay has been devised in which extracts of cultured rat sympathetic neurons labeled with [32P]-Pi were combined with unlabeled carrier bovine brain coat proteins and reassembled coat structures were isolated by gradient centrifugation. Two groups of phosphorylated polypeptides, of 100-110 kD (pp100-110) and 155 kD (pp155) apparent molecular mass, were incorporated into reassembled coats. The neuronal pp100-110 are structurally and functionally related to the 100-110-kD component of the bovine brain assembly protein (AP), a protein complex that also contains 50-kD and 16.5-kD components and is characterized by its ability to promote the reassembly of clathrin coat structures under physiological conditions of pH and ionic strength (Zaremba, S. and J. H. Keen, 1983, J. Cell Biol., 97:1337-1348). The neuronal pp155 detected in reassembled coat structures was readily observable in total extracts of [32P]-Pi-labeled neurons dissolved in SDS-containing buffer. A bovine brain counterpart to the neuronal pp155 was also observed when brain coated vesicles were subjected to two-dimensional gel electrophoresis. Phosphoserine was the predominant phosphoaminoacid found in both the pp100 and pp155. A structural and functional counterpart to the 50-kD brain assembly polypeptide (AP50) was also identified in these neurons. Although the brain AP50 is prominently phosphorylated by an endogenous protein kinase in isolated coated vesicle preparations, the neuronal AP50 was not detectably phosphorylated in intact cells as assessed by two-dimensional non-equilibrium pH gradient gel electrophoresis of labeled cells dissolved directly in SDS-containing buffers. These results demonstrate that the bovine brain assembly polypeptides of 50 kD and 100-110 kD that we have previously described, as well as a novel 155-kD polypeptide reported here, have structural and functional counterparts in cultured neurons. They also indicate that phosphorylation of the 100-110-kD AP may be involved in the regulation of coated membrane structure and function. The extent of phosphorylation of the AP50 in intact cells and in isolated coated vesicles is strikingly different: it has been suggested that the latter process reflects an autophosphorylation reaction (Campbell C., J. Squicciarini, M. Shia, P. F. Pilch, and R. E. Fine, 1984, Biochemistry, 23:4420-4426).(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of protein modification reagents on the chemotactic response in Salmonella typhimurium

1979 ◽  
Vol 57 (12) ◽  
pp. 1332-1336 ◽  
Author(s):  
Steven Clarke ◽  
D. E. Koshland Jr.
Keyword(s):  
Membrane Proteins ◽  
Salmonella Typhimurium ◽  
Protein Modification ◽  
Bacterial Chemotaxis ◽  
Chemotactic Response ◽  
Intact Cells ◽  
Flagellar Rotation ◽  
High Concentrations

Several classes of reagents that covalently modify proteins have been shown to inhibit the ability of Salmonella typhimurium to reverse the direction of its flagellar rotation. Such reversal normally allows the bacterium to tumble and reorient its movement in a more favorable direction. These reagents include those that react with amino, guanidino, sulfhydryl, and disulfide groups on proteins. At high concentrations, most of these compounds also cause the paralysis of flagellar rotation.Tumbling in bacterial chemotaxis has been shown to be dependent on the methylation of a class of membrane proteins. The effects of these reagents in an in vitro methylation system have been studied. The results obtained suggest that most of these compounds are probably not acting on intact cells by inhibiting the activities of the cheR methyltransferase or the methyl-accepting proteins.

Inhibition of fusion of skeletal myoblasts by tunicamycin and its reversal by N-acetylglucosamine

1984 ◽  
Vol 62 (1) ◽  
pp. 28-35 ◽  
Author(s):  
G. A. Gates ◽  
H. Kaur ◽  
B. D. Sanwal
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Protease Inhibitors ◽  
Concanavalin A ◽  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Cytoplasmic Membrane ◽  
Intact Cells ◽  
Skeletal Myoblasts ◽  
Insoluble Material

The differentiation of a permanent line of rat skeletal myoblasts is inhibited by a low concentration of tunicamycin in the growth medium. At a level of 0.9 μg/mL, mannose incorporation in trichloroacetic-acid-insoluble material is inhibited to the extent of about 50% by the antibiotic. Blotting of glycoproteins of the cytoplasmic membrane of myoblasts separated by gel electrophoresis by radioiodinated concanavalin A revealed that four major glycoproteins of 230 000, 145 000, 119 000, and 46 000 daltons were present in lower relative amounts in the plasma membrane following tunicamycin treatment. The 119 000 dalton glycoprotein was a major radioiodinated protein in intact cells and was presumably localized on the periphery of the membrane. The effect of tunicamycin on both fusion and glycosylation of membrane proteins could be reversed by N-acetylglucosamine, but not by the protease inhibitors leupeptin and pepstatin.

scholarly journals Visualizing conformational dynamics of proteins in solution and at the cell membrane

2018 ◽  
Author(s):  
Sharona E. Gordon ◽  
Mika Munari ◽  
William N. Zagotta
Keyword(s):  
Membrane Proteins ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Computational Method ◽  
Intact Cells ◽  
Distance Distributions ◽  
Amber Codon Suppression ◽  
Noncanonical Amino Acid

AbstractConformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

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