Changes in beta-lactoglobulin conformation at the oil/water interface of emulsions studied by synchrotron radiation circular dichroism spectroscopy.

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.

Fast membrane association is a crucial factor in the peptide pep-1 translocation mechanism: a kinetic study followed by surface plasmon resonance.

The use of peptide carriers, termed "cell-penetrating peptides (CPPs)" has attracted much attention due to their potential for cellular delivery of hydrophilic molecules with pharmacological interest, overcoming the membrane barrier. These peptides are able to deliver attached cargos in a nontoxic manner, with the uptake mechanisms being either endosomally or physically driven. Pep-1 is a CPP of particular interest, not only due to outstanding delivery rates but also because its mechanism of membrane translocation is exclusively physically driven which appears to be dependent on a very high affinity for the phospholipid bilayer in the cell membrane. In this study, pep-1-lipid interactions were further explored by characterization of the pep-1-lipid association/dissociation by surface plasmon resonance. Although a high affinity of pep-1 for lipid bilayers was observed in all conditions tested, negatively charged phospholipids resulted in a larger peptide/lipid ratio. We also show that pep-1-membrane interaction is a fast process described by a multistep model initiated by peptide adsorption, primarily governed by electrostatic attractions, and followed by peptide insertion in the hydrophobic membrane core. In the context of a cell-based process, the translocation of pep-1 is a physical mechanism promoted by peptide primary amphipathicity and asymmetric properties of the membrane. This explains the high efficiency rates of pep-1 when compared with other CPPs.

Role of helix 8 in G protein-coupled receptors based on structure-function studies on the type 1 angiotensin receptor.

G protein-coupled receptors (GPCRs) are transmembrane receptors that convert extracellular stimuli to intracellular signals. The type 1 angiotensin II receptor is a widely studied GPCR with roles in blood pressure regulation,water and salt balance and cell growth. The complex molecular and structural changes that underpin receptor activation and signaling are the focus of intense research. Increasingly, there is an appreciation that the plasma membrane participates in receptor function via direct, physical interactions that reciprocally modulate both lipid and receptor and provide microdomains for specialized activities. Reversible protein:lipid interactions are commonly mediated by amphipathic -helices in proteins and one such motif - a short helix, referred to as helix VIII/8 (H8), located at the start of the carboxyl (C)-terminus of GPCRs - is gaining recognition for its importance to GPCR function. Here, we review the identification of H8 in GPCRs and examine its capacity to sense and interact with diverse proteins and lipid environment, most notably with acidic lipids that include phosphatidylinositol phosphates.

The toxicity of prion protein fragment PrP(106-126) is not mediated by membrane permeabilization as shown by a M112W substitution.

Prion diseases result from a post-translational modification of the physiological prion protein (PrP(C)) into a scrapie isoform (PrP(Sc)). The PrP(106-126) fragment is conserved among various abnormal variants and shows PrP(Sc) pathogenic properties. It has been proposed that the PrP(106-126) fragment may exhibit its toxic effects through membrane pore formation. Our previous studies showed that PrP(106-126) does not interact with membranes under physiological conditions. In the present study, PrP(106-126) affinity for membranes was increased by modifying PrP(106-126) with a M112W substitution, and pore formation was further evaluated. However, while the peptide exhibited an increased local concentration in the membrane, this did not lead to the induction of membrane permeabilization, as verified by fluorescence methodologies and surface plasmon resonance. These results further support the idea that PrP(106-126) toxicity is not a consequence of peptide-membrane interaction and pore formation.

PrP(106-126) does not interact with membranes under physiological conditions.

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.