scholarly journals Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein

2021 ◽  
Vol 119 (1) ◽  
pp. e2109169119
Author(s):  
Kristen A. Gaffney ◽  
Ruiqiong Guo ◽  
Michael D. Bridges ◽  
Shaima Muhammednazaar ◽  
Daoyang Chen ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Limited Proteolysis ◽  
Water Soluble ◽  
Disordered Proteins ◽  
Resonance Spectroscopy ◽  
Denatured State ◽  
E Coli ◽  
State Ensemble

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron–electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli’s lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

scholarly journals Lipid Bilayer Induces Contraction of the Denatured State Ensemble of a Helical-Bundle Membrane Protein

2021 ◽  
Author(s):  
Kristen Gaffney ◽  
Ruiqiong Guo ◽  
Michael D Bridges ◽  
Daoyang Chen ◽  
Shaima Muhammednazaar ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Water Soluble ◽  
Disordered Proteins ◽  
Resonance Spectroscopy ◽  
Denatured State ◽  
Helical Bundle ◽  
Intrinsically Disordered ◽  
State Ensemble

Defining the denatured state ensemble (DSE) and intrinsically disordered proteins is essential to understanding protein folding, chaperone action, degradation, translocation and cell signaling. While a majority of studies have focused on water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we reconstituted the DSE of a helical bundle membrane protein GlpG of Escherichia coli in native lipid bilayers and measured its conformation and compactness. The DSE was obtained using steric trapping, which couples spontaneous denaturation of a doubly biotinylated GlpG to binding of two bulky monovalent streptavidin molecules. Using limited proteolysis and mass spectrometry, we mapped the flexible regions in the DSE. Using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy, we determined the dimensions of the DSE. Finally, we employed our Upside model for molecular dynamics simulations to generate the DSE including the collapsed and fully expanded states in a bilayer. We find that the DSE is highly dynamic involving the topology changes of transmembrane segments and their unfolding. The DSE is expanded relative to the native state, but only to 55-90% of the fully expanded condition. The degree of expansion depends on the chemical potential with regards to local packing and the lipid composition. Our result suggests that the native lipid bilayer promotes the association of helices in the DSE of membrane proteins and, probably in general, facilitating interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Interaction between lipid bilayers and a new class of antineoplastic agents derived from arylchloroethylurea: a 2H solid-state NMR study

1998 ◽  
Vol 76 (2-3) ◽  
pp. 465-471 ◽  
Author(s):  
Audrey Saint-Laurent ◽  
Nadine Boudreau ◽  
René C.-Gaudreault ◽  
Patrick Poyet ◽  
Michèle Auger
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Antineoplastic Agents ◽  
Acyl Chain ◽  
Resonance Spectroscopy ◽  
Interfacial Region ◽  
Molar Ratios ◽  
New Class ◽  
Nmr Study ◽  
Acyl Chains

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and model membrane of dimyristoylphosphatidylcholine by deuterium nuclear magnetic resonance spectroscopy. The results indicate that the drug incorporates in the bilayer and causes an increase of the lipid acyl chain order, this effect being greater close to the interfacial region of the lipid bilayer. The increase in ordering is dependent on the nature (degree of ramification, length of the alkyl chain, and presence of a sulfur atom) as well as on the position of the R substituent and is correlated with the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky ramified substituent and those for which the ordering effect on the lipid bilayer is the smallest. On the other hand, the ordering effect is greater and seen all along the lipid acyl chains for the long-chain CEUs, such as n-hexadecyl CEU, which have been shown to have very weak cytotoxic activity. Finally, the results obtained as a function of the drug concentration indicate that the ordering effect is seen for lipid to drug molar ratios as low as 20:1.Key words: deuterium, NMR, membrane, arylchloroethylurea, liposome.

scholarly journals Discovery of novel membrane binding structures and functions

2014 ◽  
Vol 92 (6) ◽  
pp. 555-563 ◽  
Author(s):  
Irina Kufareva ◽  
Marc Lenoir ◽  
Felician Dancea ◽  
Pooja Sridhar ◽  
Eugene Raush ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Lipid Bilayers ◽  
Protein Interactions ◽  
Membrane Binding ◽  
Three Dimensional ◽  
Intrinsic Activity ◽  
Resonance Spectroscopy ◽  
Pleckstrin Homology ◽  
Lysobisphosphatidic Acid ◽  
Membrane Interactive

The function of a protein is determined by its intrinsic activity in the context of its subcellular distribution. Membranes localize proteins within cellular compartments and govern their specific activities. Discovering such membrane-protein interactions is important for understanding biological mechanisms and could uncover novel sites for therapeutic intervention. We present a method for detecting membrane interactive proteins and their exposed residues that insert into lipid bilayers. Although the development process involved analysis of how C1b, C2, ENTH, FYVE, Gla, pleckstrin homology (PH), and PX domains bind membranes, the resulting membrane optimal docking area (MODA) method yields predictions for a given protein of known three-dimensional structures without referring to canonical membrane-targeting modules. This approach was tested on the Arf1 GTPase, ATF2 acetyltransferase, von Willebrand factor A3 domain, and Neisseria gonorrhoeae MsrB protein and further refined with membrane interactive and non-interactive FAPP1 and PKD1 pleckstrin homology domains, respectively. Furthermore we demonstrate how this tool can be used to discover unprecedented membrane binding functions as illustrated by the Bro1 domain of Alix, which was revealed to recognize lysobisphosphatidic acid (LBPA). Validation of novel membrane-protein interactions relies on other techniques such as nuclear magnetic resonance spectroscopy (NMR), which was used here to map the sites of micelle interaction. Together this indicates that genome-wide identification of known and novel membrane interactive proteins and sites is now feasible and provides a new tool for functional annotation of the proteome.

Characterizing the Locus of a Peripheral Membrane Protein–Lipid Bilayer Interaction Underlying Protein Export Activity in E. coli

Langmuir ◽  
2020 ◽  
Vol 36 (8) ◽  
pp. 2143-2152 ◽  
Author(s):  
Tina R. Matin ◽  
Milica Utjesanovic ◽  
Krishna P. Sigdel ◽  
Virginia F. Smith ◽  
Ioan Kosztin ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Lipid Bilayer ◽  
Protein Export ◽  
E Coli ◽  
Export Activity ◽  
Peripheral Membrane Protein

Structure of a shear-thickening polysaccharide extracted from the New Zealand black tree fern, Cyathea medullaris

2020 ◽  
Author(s):  
M Wee ◽  
M Mastrangelo ◽  
Susan Carnachan ◽  
Ian Sims ◽  
K Goh
Keyword(s):  
New Zealand ◽  
Uronic Acid ◽  
Average Molecular Weight ◽  
Shear Thickening ◽  
Water Soluble ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Tree Fern ◽  
13C Nuclear Magnetic Resonance ◽  
Exclusion Chromatography

A shear-thickening water-soluble polysaccharide was purified from mucilage extracted from the fronds of the New Zealand black tree fern (Cyathea medullaris or 'mamaku' in Māori) and its structure characterised. Constituent sugar analysis by three complementary methods, combined with linkage analysis (of carboxyl reduced samples) and 1H and 13C nuclear magnetic resonance spectroscopy (NMR) revealed a glucuronomannan comprising a backbone of 4-linked methylesterified glucopyranosyl uronic acid and 2-linked mannopyranosyl residues, branched at O-3 of 45% and at both O-3 and O-4 of 53% of the mannopyranosyl residues with side chains likely comprising terminal xylopyranosyl, terminal galactopyranosyl, non-methylesterified terminal glucopyranosyl uronic acid and 3-linked glucopyranosyl uronic acid residues. The weight-average molecular weight of the purified polysaccharide was ~1.9×106Da as determined by size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS). The distinctive rheological properties of this polysaccharide are discussed in relation to its structure. © 2014 Elsevier B.V.

scholarly journals Tentative Peptide‒Lipid Bilayer Models Elucidating Molecular Behaviors and Interactions Driving Passive Cellular Uptake of Collagen-Derived Small Peptides

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 710
Author(s):  
Pathomwat Wongrattanakamon ◽  
Wipawadee Yooin ◽  
Busaban Sirithunyalug ◽  
Piyarat Nimmanpipug ◽  
Supat Jiranusornkul
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Mean Square Displacement ◽  
Dynamics Simulation ◽  
Coordinate Frame ◽  
Mean Square ◽  
Binding Modes ◽  
Small Peptides ◽  
Collagen Peptides ◽  
Electron Density Profiles

Collagen contains hydroxyproline (Hyp), which is a unique amino acid. Three collagen-derived small peptides (Gly-Pro-Hyp, Pro-Hyp, and Gly-Hyp) interacting across a lipid bilayer (POPC model membrane) for cellular uptakes of these collagen-derived small peptides were studied using accelerated molecular dynamics simulation. The ligands were investigated for their binding modes, hydrogen bonds in each coordinate frame, and mean square displacement (MSD) in the Z direction. The lipid bilayers were evaluated for mass and electron density profiles of the lipid molecules, surface area of the head groups, and root mean square deviation (RMSD). The simulation results show that hydrogen bonding between the small collagen peptides and plasma membrane plays a significant role in their internalization. The translocation of the small collagen peptides across the cell membranes was shown. Pro-Hyp laterally condensed the membrane, resulting in an increase in the bilayer thickness and rigidity. Perception regarding molecular behaviors of collagen-derived peptides within the cell membrane, including their interactions, provides the novel design of specific bioactive collagen peptides for their applications.

scholarly journals Anti-EGFR VHH Antibody under Thermal Stress is Better Solubilized with a Lysine than with an Arginine SEP Tag

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 810
Author(s):  
Md. Golam Kibria ◽  
Akari Fukutani ◽  
Yoko Akazawa-Ogawa ◽  
Yoshihisa Hagihara ◽  
Yutaka Kuroda
Keyword(s):  
Thermal Stress ◽  
Phosphate Buffer ◽  
Protein Concentration ◽  
Biochemical Analysis ◽  
Stress Condition ◽  
Binding Activity ◽  
Denatured State ◽  
E Coli ◽  
Structural And Functional Properties ◽  
Model Protein

In this study, we assessed the potential of arginine and lysine solubility-enhancing peptide (SEP) tags to control the solubility of a model protein, anti-EGFR VHH-7D12, in a thermally denatured state at a high temperature. We produced VHH-7D12 antibodies attached with a C-terminal SEP tag made of either five or nine arginines or lysines (7D12-C5R, 7D12-C9R, 7D12-C5K and 7D12-C9K, respectively). The 5-arginine and 5-lysine SEP tags increased the E. coli expression of VHH-7D12 by over 80%. Biophysical and biochemical analysis confirmed the native-like secondary and tertiary structural properties and the monomeric nature of all VHH-7D12 variants. Moreover, all VHH-7D12 variants retained a full binding activity to the EGFR extracellular domain. Finally, thermal stress with 45-minute incubation at 60 and 75 °C, where VHH-7D12 variants are unfolded, showed that the untagged VHH-7D12 formed aggregates in all of the four buffers, and the supernatant protein concentration was reduced by up to 35%. 7D12-C5R and 7D12-C9R did not aggregate in Na-acetate (pH 4.7) and Tris-HCl (pH 8.5) but formed aggregates in phosphate buffer (PB, pH 7.4) and phosphate buffer saline (PBS, pH 7.4). The lysine tags (either C5K or C9K) had the strongest solubilization effect, and both 7D12-C5K and 7D12-C9K remained in the supernatant. Altogether, our results indicate that, under a thermal stress condition, the lysine SEP tags solubilization effect is more potent than that of an arginine SEP tags, and the SEP tags did not affect the structural and functional properties of the protein.

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