Retroviral proteases have been shown previously to be only active as homodimers. They are essential to form the separate and active proteins from the viral precursors. Spumaretroviruses produce separate precursors for Gag and Pol, rather than a Gag and a Gag-Pol precursor. Nevertheless, processing of Pol into a PR (protease)-RT (reverse transcriptase) and integrase is essential in order to obtain infectious viral particles. We showed recently that the PR-RT from a simian foamy virus, as well as the separate PRshort (protease) domain, exhibit proteolytic activities, although only monomeric forms could be detected. In the present study, we demonstrate that PRshort and PR-RT can be inhibited by the putative dimerization inhibitor cholic acid. Various other inhibitors, including darunavir and tipranavir, known to prevent HIV-1 PR dimerization in cells, had no effect on foamy virus protease in vitro. 1H-15N HSQC (heteronuclear single quantum coherence) NMR analysis of PRshort indicates that cholic acid binds in the proposed PRshort dimerization interface and appears to impair formation of the correct dimer. NMR analysis by paramagnetic relaxation enhancement resulted in elevated transverse relaxation rates of those amino acids predicted to participate in dimer formation. Our results suggest transient PRshort homodimers are formed under native conditions but are only present as a minor transient species, which is not detectable by traditional methods.
We have studied the physico-chemical properties of high internal oil in water (o/w) emulsions, stabilized by synergistic interaction between hydrophobin and clay. As an emulsifying agent with biological background we used H Star ProteinÒ B (HPB). Its emulsifying partner, Laponite XLG, is a synthetic layered silicate. One to one aqueous mixtures of HPB and Laponite XLG resulted in homogeneous emulsions with an oil mass fraction F of 0.65 PDMS. When used separately, both systems form unstable o/w emulsions. Moreover rheological measurements indicate the weak gel-like properties of their emulsions, whereas the simultaneous use of clay and hydrophobin results in long-term stable o/w emulsions with very pronounced gel-like properties. Characteristic rheological properties are their high storage modulus G 0 (>1000 Pa), a high yield stress value and viscosity (1 Pa s at a shear rate g ¼ 100 s À1 ). Despite a low polydispersity, a certain ripening of the emulsion matrix depending on the incubation time and shear rate was observed. It is concluded that the high storage moduli in the gel-like emulsions are due to the elasticity of the clay-protein films surrounding the oil droplets forming a self-supporting three-dimensional network. Our results highlight the relevance of the novel hydrophobin-clay synergism, resulting in excellently stabilized surfactant-free emulsions.
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