It is now clear that the understanding of halophilic adaptation at a molecular level requires a strategy of complementary experiments, combining molecular biology, biochemistry, and cellular approaches with physical chemistry and thermodynamics. In this review, after a discussion of the definition and composition of halophilic enzymes, the effects of salt on their activity, solubility, and stability are reviewed. We then describe how thermodynamic observations, such as parameters pertaining to solvent-protein interactions or enzyme-unfolding kinetics, depend strongly on solvent composition and reveal the important role played by water and ion binding to halophilic proteins. The three high-resolution crystal structures now available for halophilic proteins are analyzed in terms of haloadaptation, and finally cellular response to salt stress is discussed briefly.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

The membrane protein bacteriorhodopsin (BR) can be kept soluble in its native state for months in the absence of detergent by amphipol (APol) A8-35, an amphiphilic polymer. After an actinic flash, A8-35-complexed BR undergoes a complete photocycle, with kinetics intermediate between that in detergent solution and that in its native membrane. BR/APol complexes form well defined, globular particles comprising a monomer of BR, a complete set of purple membrane lipids, and, in a peripheral distribution, approximately 2 g APol/g BR, arranged in a compact layer. In the absence of free APol, BR/APol particles can autoassociate into small or large ordered fibrils.

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly ␣-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1 H and 13 C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal ␣-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism. Hepatitis C virus (HCV)8 infection is a major cause of human chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1). About 170 million individuals worldwide are chronically infected with HCV, and current therapy based on a combination of pegylated interferon and ribavirin is poorly tolerated and ineffective in 50% of patients. In this context, the ongoing search for new drugs and targets is very active, and the structural and functional characterization of the HCV viroporin p7 is essential for the molecular understanding of its role in HCV replication and for antiviral drug development.HCV is a highly variable enveloped positive-stranded RNA virus, and patient isolates are classified into seven genotypes and numerous subtypes (2, 3) within the genus Hepacivirus of the family Flaviviridae (4). The HCV genome encodes a polyprotein precursor,

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.

Amphipols are short amphilic polymers designed for applications in membrane biochemistry and biophysics and used, in particular, to stabilize membrane proteins in aqueous solutions. Amphipol A8-35 was obtained by modification of a short-chain parent polymer (poly(acrylic acid); PAA) with octyl- and isopropylamine, to yield an amphiphilic product with an average molar mass of 9-10 kg x mol(-1) (sodium salt form) and a polydispersity index of 2.0 to 3.1, depending on the source of PAA. The behavior of A8-35 in aqueous buffers was studied by size exclusion chromatography, static and dynamic light scattering, equilibrium and sedimentation velocity analytical ultracentrifugation, and small angle neutron scattering. Despite the variable length of the chains and the random distribution of hydrophobic groups along them, A8-35 self-organizes into well-defined assemblies. The data are best compatible with most of the polymer forming compact assemblies (particles) with a molar mass of approximately 40 kg x mol(-1), a radius of gyration of approximately 2.4 nm, and a Stokes radius of approximately 3.15 nm. Each particle contains, on average, four A8-35 macromolecules and 75-80 octyl chains. Neutron scattering reveals a sharp interface between the particles and water. A minor (approximately 0.1%) mass fraction of the material forms much larger aggregates, whose proportion may increase under certain conditions of preparation or handling, such as low pH. They can be removed by gel filtration.

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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...