REMOVED: Study on Separation Properties of Aquaporin-Based Proteoliposomes and Synthesizing of High Performance Aquaporin Based Biomimetic Membrane

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Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel

The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target

The Integrin Receptor in Biologically Relevant Bilayers: Insights from Molecular Dynamics Simulations

Integrins are heterodimeric (αβ) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2–F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2–F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes

Two different conformations in hepatitis C virus p7 protein account for proton transport and dye release

The p7 protein from the hepatitis C virus (HCV) is a 63 amino acid long polypeptide that is essential for replication, and is involved in protein trafficking and proton transport. Therefore, p7 is a possible target for antivirals. The consensus model for the channel formed by p7 protein is a hexameric or heptameric oligomer of α-helical hairpin monomers, each having two transmembrane domains, TM1 and TM2, where the N-terminal TM1 would face the lumen of this channel. A reported high-throughput functional assay to search for p7 channel inhibitors is based on carboxyfluorescein (CF) release from liposomes after p7 addition. However, the rationale for the dual ability of p7 to serve as an ion or proton channel in the infected cell, and to permeabilize membranes to large molecules like CF is not clear. We have recreated both activities in vitro, examining the conformation present in these assays using infrared spectroscopy. Our results indicate that an α-helical form of p7, which can transport protons, is not able to elicit CF release. In contrast, membrane permeabilization to CF is observed when p7 contains a high percentage of β-structure, or when using a C-terminal fragment of p7, encompassing TM2. We propose that the reported inhibitory effect of some small compounds, e.g., rimantadine, on both CF release and proton transport can be explained via binding to the membrane-inserted C-terminal half of p7, increasing its rigidity, in a similar way to the influenza A M2-rimantadine interaction.Published versio

Incorporation of p7 into lipid bilayers.

<p>(A) Schematic representation of the flotation system, where fractions with increasing density are found: (LIP), fraction with expected liposome fraction and (PEL), the most dense fraction; (B) SDS-PAGE gel corresponding to samples in the addition experiment: (BF) sample after addition and before liposome flotation, (PEL) sample at the bottom of the tube (see A), (LIP) sample associated to liposomes (see A). Sample loading was nominally 4 µg (1×) except in samples PEL and LIP, as indicated. Arrows and dots are shown for reference to indicate significant bands; (C) same as (B), but for the extruded sample where detergent was removed before extrusion; (D) ATR-IR spectra of the LIP fractions for the ‘addition’ sample (red) and the ‘extrusion’ sample (blue). Spectra for other p7 containing fractions were similar and are not shown; (E) same as (D), after a mild deconvolution (FWHH = 25 cm⁻¹ and k = 1.5). The main maxima in the amide I region are indicated.</p

Secondary structure of p7 protein in lipid bilayers.

<p>(A) amide I region corresponding to recombinant p7 protein reconstituted into hydrated DMPC bilayers by the dialysis method, after having been solubilized and dried from methanol (M, blue) or HFIP (H, red); (B) Same for a sample reconstituted by the direct method after drying from methanol (1), after freeze-thawing (2), after extrusion (3), and supernatant after 5 min centrifugation of the proteoliposomes at 2,000 g (4); (C) Same for sample obtained by the addition method, where p7 was dissolved at ∼10 mg/ml in the indicated solvents: MeOH, HFIP or TFE; (D), Amide I region of dry recombinant p7 protein obtained after lyophilizing a solution of p7 dissolved in either methanol (M) or HFIP (H).</p

Expression and purification of coronavirus envelope proteins using a modified β-barrel construct

Coronavirus envelope (E) proteins are short (∼100 residues) polypeptides that contain at least one transmembrane (TM) domain and a cluster of 2–3 juxtamembrane cysteines. These proteins are involved in viral morphogenesis and tropism, and their absence leads in some cases to aberrant virions, or to viral attenuation. In common to other viroporins, coronavirus envelope proteins increase membrane permeability to ions. Although an NMR-based model for the TM domain of the E protein in the severe acute respiratory syndrome virus (SARS-CoV E) has been reported, structural data and biophysical studies of full length E proteins are not available because efficient expression and purification methods for these proteins are lacking. Herein we have used a novel fusion protein consisting of a modified β-barrel to purify both wild type and cysteine-less mutants of two representatives of coronavirus E proteins: the shortest (76 residues), from SARS-CoV E, and one of the longest (109 residues), from the infectious bronchitis virus (IBV E). The fusion construct was subsequently cleaved with cyanogen bromide and all polypeptides were obtained with high purity. This is an approach that can be used in other difficult hydrophobic peptides

Secondary structure of N- and C-terminal fragments of p7.

<p>Infrared amide I region of (A) fragment p7(1-26), and its Fourier self deconvolved spectrum (B); C and D, same for fragment p7(27-63). Samples pre-solubilized and dried from methanol and HFIP are indicated as dotted and solid lines, respectively.</p

Proton transport of p7 and its fragments.

<p>Comparison of liposomal proton transport mediated by p7 full length, p7-TM1 fragment and p7-TM2 fragment solubilized in HFIP prior mixing with lipids (protein to lipid molar ratio 1∶125); LIP, negative control without peptide.</p