Specific protein-protein and protein-nucleic acid interactions are required for successful assembly of a large variety of biologically important macromolecular complexes, including viruses. We used the bacteriophage MS2 as a model for the study of such interactions. MS2 is a member of a large group of small RNA phages that infect Escherichia coli [1]. Its icosahedral shell consists of 180 copies of coat protein (M r 13 728) arranged in a T ¼ 3 quasi-equivalent surface lattice surrounding the ssRNA genome. Each virion also contains one copy of the maturase (or A) protein, responsible for attachment of the virus to E. coli through the F-pilus. Coat protein folds as a dimer of identical subunits and consists of a 10-stranded antiparallel b-sheet facing the interior of the phage particle, with antiparallel, interdigitating a-helical segments on the virus' To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation ⁄ denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.Abbreviations ANS, 8-anilinonaphthalene-1-sulfonate; GdnHCl, guanidine hydrochloride; VLP, virus-like particle.

Understanding how Nep-like proteins (NLPs) behave during the cell cycle and disease progression of plant pathogenic oomycetes, fungi and bacteria is crucial in light of compelling evidence that these proteins play a role in Witches` Broom Disease (WBD) of Theobroma cacao, one of the most important phytopathological problems to afflict the Southern Hemisphere. The crystal structure of MpNep2, a member of the NLP family and the causal agent of WBD, revealed the key elements for its activity. This protein has the ability to refold after heating and was believed to act as a monomer in solution, in contrast to the related homologs MpNep1 and NPP from the oomyceteous fungus Phytophthora parasitica. Here, we identify and characterize a metastable MpNep2 dimer upon over-expression in Escherichia coli using different biochemical and structural approaches. We found using ultra-fast liquid chromatography that the MpNep2 dimer can be dissociated by heating but not by dilution, oxidation or high ionic strength. Small-angle X-ray scattering revealed a possible tail-to-tail interaction between monomers, and nuclear magnetic resonance measurements identified perturbed residues involved in the putative interface of interaction. We also explored the ability of the MpNep2 monomer to refold after heating or chemical denaturation. We observed that MpNep2 has a low stability and cooperative fold that could be an explanation for its structure and activity recovery after stress. These results can provide new insights into the mechanism for MpNep2′s action in dicot plants during the progression of WBD and may open new avenues for the involvement of NLP- oligomeric species in phytopathological disorders.

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Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP). Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus–cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLAG (98DRGWGNGCGLFGK110) and FLAH (98DRGWGNHCGLFGK110), and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD) simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLAG and FLAH were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs.

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Lactoferrin (Lf) is an iron-binding glycoprotein and a component of many external secretions with a wide diversity of functions. Structural studies are important to understand the mechanisms employed by Lf to exert so varied functions. Here, we used guanidine hydrochloride and high hydrostatic pressure to cause perturbations in the structure of bovine Lf (bLf) in apo and holo (unsaturated and iron-saturated, respectively) forms, and analyzed conformational changes by intrinsic and extrinsic fluorescence spectroscopy. Our results showed that the iron binding promotes changes on tertiary structure of bLf and increases its structural stability. In addition, we evaluated the effects of bLf structural change on the kinetics of bLf internalization in Vero cells by confocal fluorescence microscopy, and observed that the holo form was faster than the apo form. This finding may indicate that structural changes promoted by iron binding may play a key role in the intracellular traffic of bLf. Altogether, our data improve the comprehension of bLf stability and uptake, adding knowledge to its potential use as a biopharmaceutical.