Structural and functional characterization of the human respiratory syncytial virus small hydrophobic protein

The small hydrophobic (SH) protein is a transmembrane surface glycoprotein encoded by the respiratory syncytial virus (RSV). It is 64 amino acids long with one putative transmembrane domain. Although SH protein is important for viral infectivity, its exact role during viral infection is not clear. In this study, we have examined the structure, oligomerization, and function of SH protein and the transmembrane domain (SH-TM) using biochemical, biophysical and computational approaches.DOCTOR OF PHILOSOPHY (SBS

The transmembrane homotrimer of ADAM 1 in model lipid bilayers

Fertilin is a transmembrane protein heterodimer formed by the two subunits fertilin α and fertilin β that plays an important role in sperm–egg fusion. Fertilin α and β are members of the ADAM family, and contain each one transmembrane α-helix, and are termed ADAM 1 and ADAM 2, respectively. ADAM 1 is the subunit that contains a putative fusion peptide, and we have explored the possibility that the transmembrane α-helical domain of ADAM 1 forms homotrimers, in common with other viral fusion proteins. Although this peptide was found to form various homooligomers in SDS, the infrared dichroic data obtained with the isotopically labeled peptide at specific positions is consistent with the presence of only one species in DMPC or POPC lipid bilayers. Comparison of the experimental orientational data with molecular dynamics simulations performed with sequence homologues of ADAM 1 show that the species present in lipid bilayers is only consistent with an evolutionarily conserved homotrimeric model for which we provide a backbone structure. These results support a model where ADAM 1 forms homotrimers as a step to create a fusion active intermediate

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

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

Overexpression and purification of p7 protein from <i>E. coli</i>.

<p>(A) Amino acid sequence of full-length p7, where extra residues SNAM (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078494#s2" target="_blank">Materials and Methods</a>) are present in the recombinant form and extra M in the synthetic form; (B) A band migrating near 50 kDa was observed after IPTG induction which corresponds to MBP-p7 fusion protein (arrow). BI, before IPTG induction; AI, 16 hours after IPTG induction; (C) Ni²⁺-NTA purification of MBP-p7 with close to 85% purity after elution in 500 mM imidazole. Ly, supernatant of total cell lysate; FT, flow through from Ni²⁺-NTA column; E, eluent from Ni²⁺-NTA column (D) TEV digestion results of MBP-p7 at room temperature, at time 0 and after 4 h incubation with gentle shaking; (E) RP-HPLC purification of p7 with a C3 RP-HPLC chromatography column. The peak corresponding to purified p7 is indicated by an arrow.</p

Mass spectrometry of recombinant and synthetic p7 protein.

<p>MALDI mass spectra corresponding to the HPLC fraction indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078494#pone-0078494-g001" target="_blank">Fig. 1E</a> (expected MW 7421.9 Da) (A) and synthetic p7 (expected MW 7017.5 Da) (B). In (A), a double-charged peak also appears at half the expected mass.</p

Schematic view of the proposed two forms of p7 (A and B).

<p>(1) In form B, TM2 is embedded in the lipid bilayer and the extra β-structure is contributed by exposure of TM1 to the aqueous environment. This form is able to release CF (black arrow), but not protons (grey arrow). As part of the B form extends into the extramembrane domain, it is likely to participate in fusion events or membrane destabilization; (2) Form A has two TM domains, TM1 and TM2, separated by a loop, where TM2 lines the lumen of the channel. This form is unable to release CF, but it is able to transport protons.</p