Relating Surfactant Properties to Activity and Solubilization of the Human Adenosine A3 Receptor

Berger, Bryan W.; García, Roxana Y.; Lenhoff, Abraham M.; Kaler, Eric W.; Robinson, Clifford R.

doi:10.1529/biophysj.104.051417

Cited by 30 publications

(29 citation statements)

References 43 publications

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“…Among them, DDM, LDAO, and CHAPS extracted the protein more efficiently than the other detergents (data not shown). DDM is a mild and nondenaturing nonionic detergent with a critical micelle concentration (CMC) of 0.17 mM (0.009% [wt/vol]); many membrane proteins were successfully solubilized in DDM and retained their protein functions (30,31). Our optimized protocol used 1% DDM (wt/vol) to extract NS4B from the cellular membrane, followed by reconstituting the protein in 0.03% DDM (wt/vol).…”

Section: Resultsmentioning

confidence: 99%

Dimerization of Flavivirus NS4B Protein

Zou

Xie

Lee

et al. 2014

J Virol

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Flavivirus replication is mediated by a complex machinery that consists of viral enzymes, nonenzymatic viral proteins, and host factors. Many of the nonenzymatic viral proteins, such as NS4B, are associated with the endoplasmic reticulum membrane. How these membrane proteins function in viral replication is poorly understood. Here we report a robust method to express and purify dengue virus (DENV) and West Nile virus NS4B proteins. The NS4B proteins were expressed in Escherichia coli, reconstituted in dodecyl maltoside (DDM) detergent micelles, and purified to >95% homogeneity. The recombinant NS4B proteins dimerized in vitro, as evidenced by gel filtration, chemical cross-linking, and multiangle light scattering experiments. The dimeric form of NS4B was also detected when the protein was expressed alone in cells as well as in cells infected with DENV type 2 (DENV-2). Mutagenesis analysis showed that the cytosolic loop (amino acids 129 to 165) and the C-terminal region (amino acids 166 to 248) are responsible for NS4B dimerization. trans-Complementation experiments showed that (i) two genome-length RNAs containing distinct NS4B lethal mutations could not trans-complement each other, (ii) the replication defect of NS4B mutant RNA could be restored in cells containing DENV-2 replicons, and (iii) expression of wild-type NS4B protein alone was not sufficient to restore the replication of the NS4B mutant RNA. Collectively, the results indicate that trans-complementation of a lethal NS4B mutant RNA requires wild-type NS4B presented from a replication complex. IMPORTANCEThe reported expression and purification system has made it possible to study the biochemistry and structure of flavivirus NS4B proteins. The finding of flavivirus NS4B dimerization and the mapping of regions important for NS4B dimerization provide the possibility to inhibit viral replication through blocking NS4B dimerization. The requirement of NS4B in the context of the replication complex for successful trans-complementation enhances our understanding of NS4B in flavivirus replication. Many flaviviruses are significant human pathogens, including dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV). The four serotypes of DENV represent the most prevalent mosquito-borne viral pathogen for humans. DENV causes about 390 million human infections annually, leading to 96 million cases with manifest symptoms (1). No clinically approved vaccine or antiviral is currently available for DENV. A pilot study of the most advanced DENV vaccine candidate reported an overall efficacy of only 30.2% in a recent clinical trial (2). More efforts are needed to understand the biology of DENV and other flaviviruses to enable vaccine development.The genomes of flaviviruses are single-stranded, positive-sense RNAs of 11 kb in length. The genomic RNA harbors a long open reading frame (ORF) flanked by the 5=-and 3=-untranslated regions (UTRs). The ORF encodes a polyprotein which...

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Section: Resultsmentioning

confidence: 99%

Dimerization of Flavivirus NS4B Protein

Zou

Xie

Lee

et al. 2014

J Virol

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“…It is known that detergents with a narrow range of hydrophile-lipophile balance (HLB) are effective for membrane protein study. [16] The initial versions of fluorine-containing amphiphiles were fully fluorinated surfactants (FSs). [6] These agents, however, were often suboptimal for membrane protein study, presumably because they are too lipophobic to form strong interactions with the hydrophobic part of the protein.…”

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confidence: 99%

Hemifluorinated Maltose‐Neopentyl Glycol (HF‐MNG) Amphiphiles for Membrane Protein Stabilisation

Cho¹,

Byrne²,

Chae³

2013

ChemBioChem

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“…Previous experiments have shown that this technique works well for analyzing activity of GPCRs, and mimics proper solution conditions for ligand binding (Berger et al, 2005). After the second 50 mM imidazole wash during batch purification, approximately 50 μL of settled resin was separated from the mixture, centrifuged at 3,220g, and the supernatant was decanted.…”

Section: Ligand Bindingmentioning

confidence: 99%

“…Initially, solubilization on a small scale was attempted using digitonin. Though digitonin has been successfully used for the functional purification of other adenosine receptors (Berger et al, 2005;Nakata, 1989), its heterogeneity compromises spectroscopic characterization, and it tends to recrystallize over time. The surfactant n-dodecyl-β-D-maltoside (DDM) was substituted for digitonin, since it has been extensively cited as a useful surfactant for maintaining proper structure of membrane proteins, and many solved membrane protein structures have been solubilized in DDM (Protein Data Bank).…”

Section: Purification Of a 2 Ar From Batch Culturementioning

confidence: 99%

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

O’Malley

Lazarova

Britton

et al. 2007

Journal of Structural Biology

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The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (~4 mg/L of culture) of functional human adenosine A 2 a receptor fused to a green fluorescent protein (A 2 aR-GFP) from Saccharomyces cerevisiae. In this work we re-engineered A 2 aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A 2 aR and A 2 aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A 2 aR-GFP-His 10 . One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-β-D-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A 2 aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A 2 a-His 10 receptor was purified in quantities of 6 +/− 2 mg/L of culture, with ligand-binding yields of 1 mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A 2 aR yet achieved from any heterologous expression system.

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Relating Surfactant Properties to Activity and Solubilization of the Human Adenosine A3 Receptor

Cited by 30 publications

References 43 publications

Dimerization of Flavivirus NS4B Protein

Dimerization of Flavivirus NS4B Protein

Hemifluorinated Maltose‐Neopentyl Glycol (HF‐MNG) Amphiphiles for Membrane Protein Stabilisation

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

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