Organic solvent extracted EmrE solubilized in dodecyl maltoside is monomeric and binds drug ligand

Winstone, Tara L.; Jidenko, Marie; Maire, Marc le; Ebel, Christine; Duncalf, Karen A; Turner, Raymond J.

doi:10.1016/j.bbrc.2004.11.164

Cited by 30 publications

(25 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3A2 and B2) reflects the good quality of the fit versus the experimental values. It should be mentioned that the analysis of analytical centrifugation data recorded in the presence of detergent is complicated by the fact that the amount of detergent bound to the studied protein is generally unknown, and thus several hypotheses must be considered (30,80). In the presence of 0.1% DM, C HCV 169* migrates as a homogeneous species with a sedimentation coefficient of 3.1 Ϯ 0.1 S (Fig.…”

Section: Resultsmentioning

confidence: 99%

Hepatitis C Virus Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein Features

Boulant

Vanbelle

Ebel

et al. 2005

J Virol

Self Cite

130

156

View full text Add to dashboard Cite

The building block of hepatitis C virus (HCV) nucleocapsid, the core protein, together with viral RNA, is composed of different domains involved in RNA binding and homo-oligomerization. The HCV core protein 1-169 (C HCV 169) and its N-terminal region from positions 1 to 117 (C HCV 117) were expressed in Escherichia coli and purified to homogeneity suitable for biochemical and biophysical characterizations. The overall conformation and the oligomeric properties of the resulting proteins C HCV 169 and C HCV 117 were investigated by using analytical centrifugation, circular dichroism, intrinsic fluorescence measurements, and limited proteolysis. Altogether, our results show that core protein (C HCV 169) behaves as a membranous protein and forms heterogeneous soluble micelle-like aggregates of high molecular weight in the absence of detergent. In contrast, it behaves, in the presence of mild detergent, as a soluble, well-folded, noncovalent dimer. Similar to findings observed for core proteins of HCV-related flaviviruses, the HCV core protein is essentially composed of ␣-helices (50%). In contrast, C HCV 117 is soluble and monodispersed in the absence of detergent but is unfolded. It appears that the folding of the highly basic domain from positions 2 to 117 (2-117 domain) depends on the presence of the 117-169 hydrophobic domain, which contains the structural determinants ensuring the binding of core with cellular membranes. Finally, our findings provide valuable information for further investigations on isolated core protein, as well as for attempts to reconstitute nucleocapsid particles in vitro.Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It is estimated that 1% of the general population in Western Europe and North America and 380 million people globally are infected with HCV worldwide (19). A protective vaccine does not exist to date, and therapeutic options are still limited. HCV has been classified in the Hepacivirus genus within the Flaviviridae family of viruses. It contains a 9.6-kb plus-strand RNA genome (10, 59, 70) composed of a 5Ј noncoding region (5Ј NCR), a long open reading frame (ORF) encoding a polyprotein precursor of about 3,000 amino acids (aa), and a 3Ј NCR. The HCV polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural (NS) proteins. The structural proteins include the core protein, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. They are separated from the NS proteins by the viroporin p7. The NS proteins include the NS2-3 autoprotease and the NS3 serine protease, an NTPase/RNA helicase located in the Cterminal two-thirds of NS3, the NS4A cofactor of NS3, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase (for reviews, see references 55 and 58). Interestingly, alternative reading frames (ARF) were recently identified in the HCV core region that has the potential to encode proteins des...

show abstract

Section: Resultsmentioning

confidence: 99%

Hepatitis C Virus Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein Features

Boulant

Vanbelle

Ebel

et al. 2005

J Virol

Self Cite

130

156

View full text Add to dashboard Cite

show abstract

“…In the case of the multidrug transporters in general and SMR proteins in particular, good assays provide a challenge due to the nature of the substrates. Many studies have used binding assays performed on detergent solubilized protein [3][4][5]. These are not ideal since a protein may bind a substrate without completing its reaction cycle.…”

Section: Functional Studies: a Linked Emre-br (Bacteriorhodopsin) Assaymentioning

confidence: 99%

NMR and fluorescence spectroscopy approaches to secondary and primary active multidrug efflux pumps

Lorch

Lehner

Siarheyeva

et al. 2005

Biochemical Society Transactions

View full text Add to dashboard Cite

Multidrug efflux pumps are found in all major transporter families. Along with a lack of three-dimensional structure information, the mechanism of drug recognition, energy coupling with drug translocation and the catalytic cycle are so far not understood. In the present study, we present first data of a fluorescence-based assay to study the pH-gradient-mediated activity of the multidrug antiporter EmrE, by co-reconstitution with the light-driven proton pump bacteriorhodopsin. In addition to biochemical approaches, the emerging technique, solid-state NMR, can be used for the investigation of these transporters. A number of experiments based on MAS (magic angle sample spinning) NMR are available to provide data on protein structure and dynamics, drug binding and protein-lipid interactions. However, these experiments dictate a number of constraints with respect to sample preparation that will be discussed for proteins from the SMR (small multidrug resistance transporter) family. In addition, 2H-NMR is used to probe protein mobility of Lactococcus lactis ABC transporter, LmrA.

show abstract

“…Several proteins are extracted and utilized in organic solvents [20][21][22][23]. However, studies on the conformation of proteins in organic solvents are rare.…”

Section: Introductionmentioning

confidence: 99%

Effect of organic solvents on the conformation and interaction of catalase and anticatalase antibodies

Rehan

Younus

2006

International Journal of Biological Macromolecules

View full text Add to dashboard Cite

Organic solvent extracted EmrE solubilized in dodecyl maltoside is monomeric and binds drug ligand

Cited by 30 publications

References 40 publications

Hepatitis C Virus Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein Features

Hepatitis C Virus Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein Features

NMR and fluorescence spectroscopy approaches to secondary and primary active multidrug efflux pumps

Effect of organic solvents on the conformation and interaction of catalase and anticatalase antibodies

Contact Info

Product

Resources

About