Pseudomonas species export the amyloid-forming protein FapC to strengthen bacterial biofilm. P. species also produce the biosurfactant rhamnolipid (Rhl) and its outer membrane contains ...lipopolysaccharide (LPS). Given the possible contacts between FapC, Rhl and LPS, we here investigate how Rhl and LPS affect FapC fibrillation compared with SDS, known to promote fibrillation of proteins at sub-micellar concentrations. Micelles of all three surfactants help FapC bypass the nucleation lag phase, leading to rapid fibrillation, which persists even at high concentrations of micelles and incorporates almost all available FapC monomers. Fibrils formed at high micellar concentrations of Rhl and SDS seed fibrillation at low surfactant concentrations while retaining the original fibril structure. FapC interacts strongly with SDS to form a dense network of narrow fibrils. Small angle X-ray scattering (SAXS) analyses reveal that surfactants reduce the population of intermediates in the fibrillation process and detect a fast aggregation step over the first 2–4 h which precedes the main fibrillation monitored by Thioflavin T. An additional SAXS-detected rearrangement of early aggregates occurs after 4–10 h. At high Rhl concentrations, the micelles are decorated with protein fibrils. SDS induces FapC fibrillation so efficiently that epigallocatechin-3-gallate (EGCG) is unable to inhibit this process. However, EGCG stimulates FapC oligomer formation and inhibits fibrillation both on its own and in the presence of Rhl and LPS. This oligomer could be modelled as a compact core with a flexible shell. This suggests that EGCG can override the natural amyloid-stimulatory properties of these biosurfactants and thus target biofilm.
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•Pseudomonas produces functional amyloid FapC and biosurfactant rhamnolipid Rhl.•Micelles of Rhl, SDS and lipopolysaccharide LPS stimulate rapid fibrillation of FapC.•SAXS reveals that surfactants reduce formation of fibrillation intermediates.•Epigallocatechin-3-gallate stimulates FapC oligomerization with and without micelles.•EGCG overrides biosurfactants' ability to induce amyloid and may target biofilm.
α-Lactalbumin (α-LA) is the second most abundant bovine whey protein. It has been intensively studied because of its readiness to populate the molten globular (MG) state, a partially folded state ...with native levels of secondary structure but loss of tertiary structure. The MG state of α-LA exposes a significant number of hydrophobic patches that could be used to bind and stabilize small hydrophobic molecules such as vitamin D3 (vitD). Accordingly, we tested the ability of α-LA to stabilize vitD in a pH interval from 7.4 to 2; over this pH interval, α-LA transitions from the folded state to the MG state. The MG state stabilized vitD better than the folded state and was superior to the major bovine whey protein β-lactoglobulin (β-LG), which is known to stabilize vitD. At pH 7.4, β-LG and α-LA stabilized vitD to the same extent. Tryptophan fluorescence quenching measurements indicated that α-LA has one binding site at pH 7.4 but acquires an additional binding site when the pH is lowered to pH 2 to 4. Stability measurements of the vitD in the α-LA–vitD complex at different temperatures suggest that UHT processing would lead to little loss of vitD. This study demonstrates the potential of α-LA as a component in vitD fortification, particularly for low pH applications.
α-Lactalbumin (α-LA) is the second most abundant bovine whey protein. It has been intensively studied because of its readiness to populate the molten globular (MG) state, a partially folded state ...with native levels of secondary structure but loss of tertiary structure. The MG state of α-LA exposes a significant number of hydrophobic patches that could be used to bind and stabilize small hydrophobic molecules such as vitamin D
(vitD). Accordingly, we tested the ability of α-LA to stabilize vitD in a pH interval from 7.4 to 2; over this pH interval, α-LA transitions from the folded state to the MG state. The MG state stabilized vitD better than the folded state and was superior to the major bovine whey protein β-lactoglobulin (β-LG), which is known to stabilize vitD. At pH 7.4, β-LG and α-LA stabilized vitD to the same extent. Tryptophan fluorescence quenching measurements indicated that α-LA has one binding site at pH 7.4 but acquires an additional binding site when the pH is lowered to pH 2 to 4. Stability measurements of the vitD in the α-LA-vitD complex at different temperatures suggest that UHT processing would lead to little loss of vitD. This study demonstrates the potential of α-LA as a component in vitD fortification, particularly for low pH applications.
Liprotides are complexes between lipids and partially denatured proteins in which the protein forms a stabilizing shell around a fatty acid micelle core. We have previously shown that liprotides ...stabilize small aliphatic molecules such as retinal and tocopherol by sequestering these molecules in the fatty acid core. This opens up the use of liprotides to formulate food additives. Here, we expand our investigations to the large and bulky molecule vitamin D3 (vitD), motivated by the population-wide occurrence of vitD deficiency. We prepared liprotides using different proteins and fatty acids and evaluated their ability to protect vitD upon exposure to heating or intense UV light. Additionally, we determined the stability of liprotides toward pH, Ca2+, and BSA. The best results were obtained with liprotides made from α-lactalbumin and oleate. These liprotides were able to completely solubilize vitD, increase the stability toward UV light 9-fold, and increase the long-term stability at 37°C up to 1,000-fold. Native α-lactalbumin binds Ca2+, making Ca2+ potentially disruptive toward liprotides. However, liprotides prepared by incubation at 80°C were stable toward Ca2+, in contrast to those made at 20°C. Nevertheless, the fatty acid binding protein BSA reduced the ability of both liprotides to protect vitD; the amount of vitD remianing after 20d at 20°C decreased from 79±3% in the absence of BSA to 49±4 and 23±3% in the presence of BSA for liprotides made at 80 and 20°C, respectively. Both classes of liprotides were able to release their vitD content, as demonstrated by the transfer of vitD encapsulated in liprotides to phospholipid vesicles. Importantly, liprotides were not stable at pH 6 and below, limiting the useful pH range of the liprotides to >pH 6. Our results indicate that vitD may be encapsulated and stabilized for enrichment of clear beverages at neutral pH to improve the intake and bioavailability of vitD.
Empirically, α-helical membrane protein folding stability in surfactant micelles can be tuned by varying the mole fraction MFSDS of anionic (sodium dodecyl sulfate (SDS)) relative to nonionic (e.g., ...dodecyl maltoside (DDM)) surfactant, but we lack a satisfying physical explanation of this phenomenon. Cysteine labeling (CL) has thus far only been used to study the topology of membrane proteins, not their stability or folding behavior. Here, we use CL to investigate membrane protein folding in mixed DDM-SDS micelles. Labeling kinetics of the intramembrane protease GlpG are consistent with simple two-state unfolding-and-exchange rates for seven single-Cys GlpG variants over most of the explored MFSDS range, along with exchange from the native state at low MFSDS (which inconveniently precludes measurement of unfolding kinetics under native conditions). However, for two mutants, labeling rates decline with MFSDS at 0–0.2 MFSDS (i.e., native conditions). Thus, an increase in MFSDS seems to be a protective factor for these two positions, but not for the five others. We propose different scenarios to explain this and find the most plausible ones to involve preferential binding of SDS monomers to the site of CL (based on computational simulations) along with changes in size and shape of the mixed micelle with changing MFSDS (based on SAXS studies). These nonlinear impacts on protein stability highlights a multifaceted role for SDS in membrane protein denaturation, involving both direct interactions of monomeric SDS and changes in micelle size and shape along with the general effects on protein stability of changes in micelle composition.
α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinsonâs disease patients. ...Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of â2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.
DIBMA nanodiscs keep α-synuclein folded Adão, Regina; Cruz, Pedro F.; Vaz, Daniela C. ...
Biochimica et biophysica acta. Biomembranes,
09/2020, Letnik:
1862, Številka:
9
Journal Article
Recenzirano
Odprti dostop
α-Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane ...enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on α-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on αsyn secondary-structure formation, using human-, elephant- and whale -αsyn. Our results confirm that negatively charged lipids induce αsyn folding in h-αsyn and e-αsyn but not in w-αsyn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45 °C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying αsyn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as αsyn into nontoxic α-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and α-helix formation by αsyn, paramount to the proposal of new methods to avoid protein aggregation and disease.
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•Impact of lipid size on α-syn structuring, LUVs vs lipid-bilayer nanodiscs encapsulated by DIBMA.•DIBMALPs are suitable nanoscale membrane mimics for studying αsyn secondary-structure.•In DIBMALPS folding was independent of L/P ratio, in contrast with LUVs.•New and promising application of polymer-encapsulated lipid-bilayer nanodiscs.•Human, elephant and whale αsyn were compared for α-helix formation in the presence of lipids.
The interaction between nanoparticles (NPs) and the small intrinsically disordered protein α-synuclein (αSN), whose aggregation is central in the development of Parkinson's disease, is of great ...relevance in biomedical applications of NPs as drug carriers. Here we showed using a combination of different techniques that αSN interacts strongly with positively charged polyethylenimine-coated human serum albumin (PEI-HSA) NPs, leading to a significant alteration in the αSN secondary structure. In contrast, the weak interactions of αSN with HSA NPs allowed αSN to remain unfolded. These different levels of interactions had different effects on αSN aggregation. While the weakly interacting HSA NPs did not alter the aggregation kinetic parameters of αSN, the rate of primary nucleation increased in the presence of PEI-HSA NPs. The aggregation rate changed in a PEI-HSA NP-concentration dependent and size independent manner and led to fibrils which were covered with small aggregates. Furthermore, PEI-HSA NPs reduced the level of membrane-perturbing oligomers and reduced oligomer toxicity in cell assays, highlighting a potential role for NPs in reducing αSN pathogenicity in vivo. Collectively, our results highlight the fact that a simple modification of NPs can strongly modulate interactions with target proteins, which may have important and positive implications in NP safety.
Chemical glycosylation of proteins is a powerful tool applied widely in biomedicine and biotechnology. However, it is a challenging undertaking and typically relies on recombinant proteins and ...site‐specific conjugations. The scope and utility of this nature‐inspired methodology would be broadened tremendously by the advent of facile, scalable techniques in glycosylation, which are currently missing. In this work, we investigated a one‐pot aqueous protocol to achieve indiscriminate, surface‐wide glycosylation of the surface accessible amines (lysines and/or N‐terminus). We reveal that this approach afforded minimal if any change in the protein activity and recognition events in biochemical and cell culture assays, but at the same time provided a significant benefit of stabilizing proteins against aggregation and fibrillation ‐ as demonstrated on serum proteins (albumins and immunoglobulin G, IgG), an enzyme (uricase), and proteins involved in neurodegenerative disease (α‐synuclein) and diabetes (insulin). Most importantly, this highly advantageous result was achieved via a one‐pot aqueous protocol performed on native proteins, bypassing the use of complex chemical methodologies and recombinant proteins.
Chemical glycosylation of proteins is achieved via a one‐pot aqueous protocol, to pursue indiscriminate modification of the surface accessible amines. This facile method, drastically simpler than site‐specific glycosylation, afforded a highly desired stabilization of proteins against aggregation and fibrillation, with a minimal change to the protein activity.
The surface of a carboxylate‐enriched octuple mutant of Bacillus subtilis lipase A (8M) is chemically anionized to produce core (8M)‐shell (cationic polymer surfactants) bionanoconjugates in protein ...liquid form, which are termed anion‐type biofluids. The resultant lipase biofluids exhibit a 2.5‐fold increase in hydrolytic activity when compared with analogous lipase biofluids based on anionic polymer surfactants. In addition, the applicability of the anion‐type biofluid using Myoglobin (Mb) that is well studied in anion‐type solvent‐free liquid proteins is evaluated. Although anionization resulted in the complete unfolding of Mb, the active α‐helix level is partially recovered in the anion‐type biofluids, and the effect is accentuated in the cation‐type Mb biofluids. These highly active anion‐type solvent‐free liquid enzymes exhibit increased thermal stability and provide a new direction in solvent‐free liquid protein research.
Novel cationic polymer surfactant is synthesized and applied for the preparation of anionic solvent‐free liquid lipases and myoglobin. The anionic lipase biofluids exhibit a 2.5‐fold increase in hydrolytic activity compared with analogous anionic lipase biofluids. For myoglobin enhanced structure is observed upon being electrostatically conjugated with cationic polymer surfactants following anionization. This work provides a new direction in solvent‐free liquid protein research to obtain highly active thermostable enzyme biofluids.
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