Infection with the hepatitis C virus (HCV) has a huge impact on global health putting more than 170 million people at risk of developing severe liver disease. The HCV encoded p7 ion channel is essential for the production of infectious viruses. Despite a growing body of functional data, little is known about the 3-dimensional (3D) structure of the channel. Here, we present the 3D structure of a full-length viroporin, the detergent-solubilized hexameric 42 kDa form of the HCV p7 ion channel, as determined by single-particle electron microscopy using the random conical tilting approach. The reconstruction of such a small protein complex was made possible by a combination of high-contrast staining, the symmetry, and the distinct structural features of the channel. The orientation of the p7 monomers within the density was established using immunolabeling with N and C termini specific F ab fragments. The density map at a resolution of Ϸ16 Å reveals a flower-shaped protein architecture with protruding petals oriented toward the ER lumen. This broadest part of the channel presents a comparatively large surface area providing potential interaction sites for cellular and virally encoded ER resident proteins. membrane protein ͉ viroporin ͉ single particle analysis ͉ random conical tilt reconstruction T he hepatitis C virus (HCV) poses a major global health problem. It puts more than 170 million people worldwide at risk of developing liver cirrhosis and hepatocellular carcinoma. HCV comprises 6 different genotypes and is one of the fastest mutating viruses known to man. There is no vaccine available, and treatment options are genotype-specific, prone to viral escape mutations, and inadequate.The HCV p7 ion channel is a more recent addition to the growing list of potential drug targets encoded by HCV, reflecting the urgent need for a therapeutic approach. p7 is critical for the release of infectious virions in vitro (1, 2) and in vivo (3). It is not involved in HCV RNA replication (4, 5), but is required for late steps of viral particle assembly (2) and potentially cell entry (6). However, the prerequisite incorporation of p7 into budding virions has not been demonstrated. p7 belongs to the viroporins, small virally encoded proteins with at least 1 membrane-spanning helix that oligomerize to form channels or pores that modify the permeability of the cell membrane to ions and other small molecules (7). In planar lipid bilayers, p7 monomers oligomerize to form cation-selective ion channels that can be specifically inhibited by long alkylchain iminosugars, amiloride, and amantadine derivatives, with varying reported efficacies (6,(8)(9)(10)(11)(12)(13)(14)(15). Each HCV p7 monomer consists of 63 aa, most of which are hydrophobic and possibly contain endoplasmic reticulum (ER) retention signals (16-18). Computational secondary structure predictions suggest that the monomers contain 2 transmembrane spanning helices connected by a short basic loop (19,20). The loop is assumed to face the cytoplasm, with the N and C termini facing...

The adamantanes are a class of compounds that have found use in the treatment of influenza A and Parkinson's disease, among others. The mode of action for influenza A is based on the adamantanes' interaction with the transmembrane M2 channel, whereas the treatment of Parkinson's disease is thought to relate to a channel block of N-methyl-D-aspartate receptors. An understanding of how these compounds interact with the lipid bilayer is thus of great interest. We used molecular-dynamics simulations to calculate the potential of mean force of adamantanes in a lipid bilayer. Our results demonstrate a preference for the interfacial region of the lipid bilayer for both protonated and deprotonated species, with the protonated species proving significantly more favorable. However, the protonated species have a large free-energy barrier in the center of the membrane. In contrast, there is no barrier (compared with aqueous solution) at the center of the bilayer for deprotonated species, suggesting that the permeant species is indeed the neutral form, as commonly assumed. We discuss the results with respect to proposed mechanisms of action and implications for drug-delivery in general.

The p7 protein from hepatitis C virus is critical for the assembly and secretion of infectious virus, making it an attractive drug target. It is thought to be a viroporin with a demonstrated ion channel activity when reconstituted into planar lipid bilayers. Electron microscopy experiments suggest that p7 oligomers coexist as hexamers and heptamers. Proposed models of p7 oligomers assume the N-terminal helix to be the pore lining helix. Here, we demonstrate, via electrophysiology, that Cu(2+) has an inhibitory effect on the p7 ion channel and that the amino acid responsible for this inhibition is one histidine in each monomer. This information coupled with the p7 sequence data suggests that the N-terminal helix of p7 does indeed form the transmembrane pore and that this histidine is pore-lining. The information will aid in the construction of oligomeric pore-models and the interpretation of electron microscopy data.

In many neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, proteinaceous aggregates are observed in damaged neuronal regions. The relationship of neuronal inclusions to disease has been intensively studied and provided strong support for the importance of protein aggregation for neurodegeneration. Accumulating evidence, however, suggests that it is not the insoluble aggregates identified by light microscopy, but rather soluble oligomers that are the most neurotoxic species. Despite their importance for neurodegeneration and for development of therapeutic treatments, little is known about the structure of soluble oligomers and their structure-toxicity relationship. Soluble oligomers are potent toxins in many neurodegenerative diseases, but little is known about the structure of soluble oligomers and their structure-toxicity relationship. Here, we showed that amyloid fibrils formed by the protein alphasynuclein (aS), one of the key players in Parkinson's disease, are rapidly dissociated in supercooled water at À15 C, conditions in which many globular proteins remain folded. NMR studies indicate that the weakening of hydrophobic and electrostatic interactions contribute to the cold-induced destabilization of the amyloid fibrils. Taking advantage of the vulnerability of aS fibrils in supercooled solution, we prepared on-pathway oligomers of the 140-residue protein aS, at concentrations and order of magnitude higher than previously possible. The oligomers form ion channels with well-defined conductance states in a variety of membranes and their b-structure differs from that of amyloid fibrils of aS. The ability to prepare soluble oligomers of aS at high concentrations is essential not only for understanding the structural basis of oligomers toxicity, but also for the development of therapeutic treatments and imaging agents for monitoring aS oligomerization in vivo.

One important feature that differentiates membrane proteins from soluble ones is topology. This is the specific entanglement between the membrane protein and the lipid bilayer. Probing this specific orientation of the protein has been best achieved using oriented solid-state NMR experiments such as PISEMA, SAMPI4, and HIMSELF. These experiments correlate an anisotropic 15 N (or 13