Delia Picone

The α-to-β Conformational Transition of Alzheimer’s Aβ-(1–42) Peptide in Aqueous Media is Reversible: A Step by Step Conformational Analysis Suggests the Location of β Conformation Seeding

Current views of the role of β‐amyloid (Aβ) peptide fibrils range from regarding them as the cause of Alzheimers pathology to having a protective function. In the last few years, it has also been suggested that soluble oligomers might be the most important toxic species. In all cases, the study of the conformational properties of Aβ peptides in soluble form constitutes a basic approach to the design of molecules with “antiamyloid” activity. We have experimentally investigated the conformational path that can lead the Aβ‐(1–42) peptide from the native state, which is represented by an α helix embedded in the membrane, to the final state in the amyloid fibrils, which is characterized by β‐sheet structures. The conformational steps were monitored by using CD and NMR spectroscopy in media of varying polarities. This was achieved by changing the composition of water and hexafluoroisopropanol (HFIP). In the presence of HFIP, β conformations can be observed in solutions that have very high water content (up to 99 % water; v/v). These can be turned back to α helices simply by adding the appropriate amount of HFIP. The transition of Aβ‐(1–42) from α to β conformations occurs when the amount of water is higher than 80 % (v/v). The NMR structure solved in HFIP/H2O with high water content showed that, on going from very apolar to polar environments, the long N‐terminal helix is essentially retained, whereas the shorter C‐terminal helix is lost. The complete conformational path was investigated in detail with the aid of molecular‐dynamics simulations in explicit solvent, which led to the localization of residues that might seed β conformations. The structures obtained might help to find regions that are more affected by environmental conditions in vivo. This could in turn aid the design of molecules able to inhibit fibril deposition or revert oligomerization processes.

New features of the δ opioid receptor: Conformational properties of deltorphin I analogues

Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.

Does casomorphin have a functional role

Degradation of buffalo β‐casein by various physiological enzymes was studied. Digestion with gastric and pancreatic proteases plus leucine aminopeptidase did not release casomorphins but a putative precursor (procasomorphin) which was further digested by brush border peptidases into peptides differing from casomorphins.

NMR Spectroscopic Assignment of Backbone and Side-Chain Protons in Fully Protonated Proteins: Microcrystals, Sedimented Assemblies, and Amyloid Fibrils.

We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR spectroscopy with 100-111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.

NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.

Physico‐chemical features of the environment affect the protein conformation and the immunoglobulin E reactivity of kiwellin (Act d 5)

Background Allergy diagnostic systems sometimes give false positive or negative results. In this respect, the influence of protein conformational changes on the allergen–IgE interaction sites is worthy to be investigated.

Peamaclein – A new peach allergenic protein: similarities, differences and misleading features compared to Pru p 3

Among the peach‐derived allergens which are already known, the lipid transfer protein (Pru p 3) seems to be the one to exert severe allergic reactions.

Conformational properties of deltorphin: new features of the δ-opioid receptor

Deltorphin is an opioid peptide with the sequence H‐Tyr‐D‐Met‐Phe‐His‐Leu‐Met‐Asp‐NH2, recently isolated from the skin of Phyllomedusa sauvagei. Its enormous selectivity towards the δ‐opioid receptor and the similarity of the N‐terminal part of the sequence with that of dermorphin (H‐Tyr‐D‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2), a μ selective peptide isolated from the same natural source, prompted a comparative conformational study. A 1H‐NMR study in two different solvent systems showed that the conformational preferences of the N‐terminal sequences of the two peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects. Besides a general trend consistent with the role of the membrane in the preselection of the peptides, the present study demonstrates the crucial role played by charged residues in the interaction inside the receptors.

Structural characterization of the transmembrane proximal region of the hepatitis C virus E1 glycoprotein.

A detailed knowledge of the mechanism of virus entry represents one of the most promising approaches to develop new therapeutic strategies. However, viral fusion is a very complex process involving fusion glycoproteins present on the viral envelope. In the two hepatitis C virus envelope proteins, E1 and E2, several membranotropic regions with a potential role in the fusion process have been identified. Among these, we have selected the 314-342 E1 region. Circular Dichroism data indicate that the peptide exhibits a clear propensity to adopt a helical folding in different membrane mimicking media, such as mixtures of water with fluorinated alcohols and phospholipids, with a slight preference for negative charged bilayers. The 3D structure of E1(314-342) peptide, calculated by 2D-NMR in a low-polarity environment, consists of two helical stretches encompassing residues 319-323 and 329-338 respectively. The peptide, presenting a largely apolar character, interacts with liposomes, as indicated by fluorescence and electron spin resonance spectra. The strength of the interaction and the deepness of peptide insertion in the phospholipid membrane are modulated by the bilayer composition, the interaction with anionic phospholipids being among the strongest ever observed. The presence of cholesterol also affects the peptide-bilayer interaction, favoring the peptide positioning close to the bilayer surface. Overall, the experimental data support the idea that this region of E1 might be involved in membrane destabilization and viral fusion; therefore it may represent a good target to develop anti-viral molecules.

The role of the hinge loop in domain-swapping: The special case of Bovine seminal Ribonuclease

Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions. It is isolated as an equilibrium mixture of swapped (MxM) and unswapped (M=M) dimers. The interchanged N termini are hinged on the main bodies through the peptide 16–22, which changes conformation in the two isomers. At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications. Two variants, having Pro19 or the whole sequence of the hinge replaced by the corresponding residues of RNase A, show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. In comparison, the x-ray structures of MxM indicate, within a substantial constancy of the quaternary association, a greater mobility of the hinge residues. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement of Pro19 by Ala, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. Moreover, the results indirectly lend credit to the hypothesis that the major role of Pro19 resides in directing the assembly of the non-covalent dimer, the species produced by selective reduction of the interchain disulfides and considered responsible for the special biological functions of BS-RNase.