The non-covalent interactions between the monomeric phenolic compound chlorogenic acid (5-CQA) and bovine serum albumin (BSA), lysozyme, and alpha-lactalbumin were characterized, and their effect on protein properties was examined. 5-CQA had a low affinity for all three proteins, and these interactions seemed to show a negative cooperativity. 5-CQA-BSA binding decreased with increasing temperature, whereas pH (pH 3.0 compared to pH 7.0) and ionic strength had no pronounced effect. At high 5-CQA/protein molar ratios, both the denaturation enthalpy and temperature of BSA increased; however, covalent bonds were created at high temperatures. The presence of 5-CQA had no effect on the solubility of BSA and alpha-lactalbumin as a function of pH, whereas it decreased lysozyme solubility at alkaline pH due to covalent interactions. These results indicate that the non-covalent interactions with 5-CQA do not have pronounced effects on the functional properties of globular proteins in food systems.

Aggregation and misfolding of the prion protein (PrP) are thought to be the cause of a family of lethal neurodegenerative diseases affecting humans and other animals. Although the structures of PrP from several species have been solved, still little is known about the mechanisms that lead to the misfolded species. Here, we show that the region of PrP comprising the hairpin formed by the helices H2 and H3 is a stable independently folded unit able to retain its secondary and tertiary structure also in the absence of the rest of the sequence. We also prove that the isolated H2H3 is highly fibrillogenic and forms amyloid fibers morphologically similar to those obtained for the full-length protein. Fibrillization of H2H3 but not of full-length PrP is concomitant with formation of aggregates. These observations suggest a "banana-peeling" mechanism for misfolding of PrP in which H2H3 is the aggregation seed that needs to be first exposed to promote conversion from a helical to a ␤-rich structure.Transmissible spongiform encephalopathies are fatal neurodegenerative pathologies that affect humans as well as several other mammalian species. They are thought to be caused by the aggregation and misfolding of the prion protein (PrP).7 According to the "protein-only" hypothesis (1-3), PrP undergoes an ␣-to-␤ transition from its native state (PrP c ) to a misfolded species (PrP sc ), which is believed to act as a template to "infect" and misfold other PrP copies. As in other misfolding pathologies such as Alzheimer and Parkinson diseases, the neurotoxicity of PrP Sc is thought to be associated to an oligomeric form of the protein rather than to the mature aggregates (4).One of the crucial questions that remains unanswered concerns which region(s) of PrP promotes the polymerization process; this information would be both the key for understanding cross-species infectivity and help in decoding the bases of the aggregation process. Different regions have been proposed to be the fibrillogenic seed. PrP c consists of an unstructured N-terminal tail and a folded C-terminal domain formed by three helices (H1, H2, and H3) and a short-stranded ␤-sheet (formed by S1 and S2). H2 and H3 are connected through a disulfide bridge (5). A common view suggests the S1H1S2 region is crucial for ␤-sheet seeding and PrP Sc formation (6, 7). H1 has been implicated as a primary interaction site between PrP Sc and PrP c (8, 9), whereas the loop between S2 and H2, a rigid loop stabilized by its long range interactions with H3 (10), and the C terminus of H3 has been suggested to be recognized by a "Protein-X" that would affect the conversion of PrP c into PrP Sc (11). A study based on intrachain distance estimation performed on tagged PrP amyloid fibrils obtained under chaotropic treatment suggests the involvement of the H2H3 domain of PrP in amyloid formation (12). H/D exchange studies of the amyloid fibrils from human PrP reveal that the ␤-sheet core of PrP amyloids is formed by H2, the major part of H3, and the loop between them (13, 14).We have fol...

The propensity of the prion protein (PrP) to adopt different structures is a clue to its pathological behavior. The determination of the region involved in the PrP(C) to PrP(Sc) conversion is fundamental for the understanding of the mechanisms underlying this process at the molecular level. In this paper, the polymerization of the helical H2H3 domain of ovine PrP (OvPrP) was compared to the full-length construct (using chromatography and light scattering). We show that the oligomerization patterns are identical, although the H2H3 domain has a higher polymerization rate. Furthermore, the depolymerization kinetics of purified H2H3 oligomers compared to those purified from the full-length PrP reveal that regions outside H2H3 do not significantly contribute to the oligomerization process. By combining rational mutagenesis and molecular dynamics to investigate the early stages of H2H3 oligomerization, we observe a conformationally stable beta-sheet structure that we propose as a possible nucleus for oligomerization; we also show that single point mutations in H2 and H3 present structural polymorphisms and oligomerization properties that could constitute the basis of species or strain variability.

Protein polymerization consists in the aggregation of single monomers into polymers that may fragment. Fibrils assembly is a key process in amyloid diseases. Up to now, protein aggregation was commonly mathematically simulated by a polymer size-structured ordinary differential equations (ODE) system, which is infinite by definition and therefore leads to high computational costs. Moreover, this Ordinary Differential Equation-based modeling approach implies biological assumptions that may be difficult to justify in the general case. For example, whereas several ordinary differential equation models use the assumption that polymerization would occur at a constant rate independently of polymer size, it cannot be applied to certain protein aggregation mechanisms. Here, we propose a novel and efficient analytical method, capable of modelling and simulating amyloid aggregation processes. This alternative approach consists of an integro-Partial Differential Equation (PDE) model of coalescence-fragmentation type that was mathematically derived from the infinite differential system by asymptotic analysis. To illustrate the efficiency of our approach, we applied it to aggregation experiments on polyglutamine polymers that are involved in Huntington’s disease. Our model demonstrates the existence of a monomeric structural intermediate acting as a nucleus and deriving from a non polymerizing monomer (). Furthermore, we compared our model to previously published works carried out in different contexts and proved its accuracy to describe other amyloid aggregation processes.

BACKGROUND: The interactions between phenolic compounds and proteins can modify protein properties important in the food industry. To understand the effects of these interactions, the covalent interactions between caffeoylquinic acid (chlorogenic acid, CQA) oxidised by polyphenol oxidase (PPO) at acidic pH 6 (pH 6) and αlactalbumin, lysozyme and bovine serum albumin (BSA) were compared with non-enzymatically induced covalent interactions at alkaline pH (pH 9). The effects of these modifications on protein properties were examined.RESULTS: Both ways of modification seemed to result in protein modification mainly via dimeric rather than monomeric CQA quinones. These modifications led to a decrease in the number of free primary amino groups of the proteins. Modification with CQA alone induced a low degree of protein dimerisation, which also occurred through the action of PPO alone. Modification drastically reduced the solubility of lysozyme over a broad pH range, whereas that of α-lactalbumin was strongly reduced only at pH values close to its pI. The solubility of BSA was much less affected than that of the other proteins and only at acidic pH. CONCLUSION: These results indicate some similarities between modifications at pH 6 and 9 and that both modifications clearly change the functional properties of globular proteins.