No abstract

Transmissible spongiform encephalopathies are neurodegenerative diseases characterized by the accumulation of an abnormal isoform of the prion protein PrP(Sc). Its fragment 106-126 has been reported to maintain most of the pathological features of PrP(Sc), and a role in neurodegeneration has been proposed based on the modulation of membrane properties and channel formation. The ability of PrP(Sc) to modulate membranes and/or form channels in membranes has not been clearly demonstrated; however, if these processes are important, peptide-membrane interactions would be a key feature in the toxicity of PrP(Sc). In this work, the interaction of PrP(106-126) with model membranes comprising typical lipid identities, as well as more specialized lipids such as phosphatidylserine and GM1 ganglioside, was examined using surface plasmon resonance and fluorescence methodologies. This comprehensive study examines different parameters relevant to characterization of peptide-membrane interactions, including membrane charge, viscosity, lipid composition, pH, and ionic strength. We report that PrP(106-126) has a low affinity for lipid membranes under physiological conditions without evidence of membrane disturbances. Membrane insertion and leakage occur only under conditions in which strong electrostatic interactions operate. These results support the hypothesis that the physiological prion protein PrP(C) mediates PrP(106-126) toxic effects in neuronal cells.

The structure of proteins at interfaces is a key factor determining the stability as well as organoleptic properties of food emulsions. While it is widely believed that proteins undergo conformational changes at interfaces, the measurement of these structural changes remains a significant challenge. In this study, the conformational changes of beta-lactoglobulin (beta-Lg) upon adsorption to the interface of hexadecane oil-in-water emulsions were investigated using synchrotron radiation circular dichroism (SRCD) spectroscopy. Far-UV SRCD spectra showed that adsorption of beta-Lg to the O/W interface caused a significant increase in non-native alpha-helix structure, accompanied by a concomitant loss of beta-sheet structure. Near-UV SRCD spectra revealed that a considerable disruption of beta-Lg tertiary structure occurred upon adsorption. Moreover, heat-induced changes to the non-native beta-Lg conformation at the oil/water interface were very small compared to the dramatic loss of beta-Lg secondary structure that occurred during heating in solution, suggesting that the interface has a stabilizing effect on the structure of non-native beta-Lg. Overall, our findings provide insight into the conformational behavior of proteins at oil/water interfaces and demonstrate the applicability of SRCD spectroscopy for measuring the conformation of adsorbed proteins in optically turbid emulsions.

No abstract

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-1, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 microM. Their activities against HIV-1 protease (K(i) 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC(50) 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC(50) 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 A (1) and 1.85 A (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.