(-)-Epigallocatechin gallate (EGCG) effectively reduces the cytotoxicity of the Alzheimer's disease β-amyloid peptide (Aβ) by remodeling seeding-competent Aβ oligomers into off-pathway seeding-incompetent Aβ assemblies. However, the mechanism of EGCG-induced remodeling is not fully understood. Here we combine N andH dark-state exchange saturation transfer (DEST), relaxation, and chemical shift projection NMR analyses with fluorescence, dynamic light scattering, and electron microscopy to elucidate how EGCG remodels Aβ oligomers. We show that the remodeling adheres to a Hill-Scatchard model whereby the Aβ(1-40) self-association occurs cooperatively and generates Aβ(1-40) oligomers with multiple independent binding sites for EGCG with a K ∼10-fold lower than that for the Aβ(1-40) monomers. Upon binding to EGCG, the Aβ(1-40) oligomers become less solvent exposed, and the β-regions, which are involved in direct monomer-protofibril contacts in the absence of EGCG, undergo a direct-to-tethered contact shift. This switch toward less engaged monomer-protofibril contacts explains the seeding incompetency observed upon EGCG remodeling and suggests that EGCG interferes with secondary nucleation events known to generate toxic Aβ assemblies. Unexpectedly, the N-terminal residues experience an opposite EGCG-induced shift from tethered to direct contacts, explaining why EGCG remodeling occurs without release of Aβ(1-40) monomers. We also show that upon binding Aβ(1-40) oligomers the relative positions of the EGCG B and D rings change with respect to that of ring A. These distinct structural changes occurring in both Aβ(1-40) oligomers and EGCG during remodeling offer a foundation for understanding the molecular mechanism of EGCG as a neurotoxicity inhibitor. Furthermore, the results reported here illustrate the effectiveness of DEST-based NMR approaches in investigating the mechanism of low-molecular-weight amyloid inhibitors.

Human serum albumin (HSA) is a potent inhibitor of Aβ self-association and this novel, to our knowledge, function of HSA is of potential therapeutic interest for the treatment of Alzheimer's disease. It is known that HSA interacts with Aβ oligomers through binding sites evenly partitioned across the three albumin domains and with comparable affinities. However, as of this writing, no information is available on the HSA-Aβ interactions beyond domain resolution. Here, we map the HSA-Aβ interactions at subdomain and peptide resolution. We show that each separate subdomain of HSA domain 3 inhibits Aβ self-association. We also show that fatty acids (FAs) compete with Aβ oligomers for binding to domain 3, but the determinant of the HSA/Aβ oligomer interactions are markedly distinct from those of FAs. Although salt bridges with the FA carboxylate determine the FA binding affinities, hydrophobic contacts are pivotal for Aβ oligomer recognition. Specifically, we identified a site of Aβ oligomer recognition that spans the HSA (494-515) region and aligns with the central hydrophobic core of Aβ. The HSA (495-515) segment includes residues affected by FA binding and this segment is prone to self-associate into β-amyloids, suggesting that sites involved in fibrilization may provide a lead to develop inhibitors of Aβ self-association.

Self-association of amyloid β (Aβ) peptides is a hallmark of Alzheimer's disease and serves as a general prototype for amyloid formation. A key endogenous inhibitor of Aβ self-association is human serum albumin (HSA), which binds ∼90% of plasma Aβ. However, the exact molecular mechanism by which HSA binds Aβ monomers and protofibrils is not fully understood. Here, using dark-state exchange saturation transfer NMR and relaxation experiments complemented by morphological characterization, we mapped the HSA-Aβ interactions at atomic resolution by examining the effects of HSA on Aβ monomers and soluble high-molecular weight oligomeric protofibrils. We found that HSA binds both monomeric and protofibrillar Aβ, but the affinity of HSA for Aβ monomers is lower than for Aβ protofibrils ( values are submillimolar rather than micromolar) yet physiologically relevant because of the ∼0.6-0.7 mm plasma HSA concentration. In both Aβ protofibrils and monomers, HSA targets key Aβ self-recognition sites spanning the β strands found in cross-β protofibril structures, leading to a net switch from direct to tethered contacts between the monomeric Aβ and the protofibril surface. These HSA-Aβ interactions are isoform-specific, because the HSA affinity of Aβ monomers is lower for Aβ(1-42) than for Aβ(1-40). In addition, the HSA-induced perturbations of the monomer/protofibrils pseudo-equilibrium extend to the C-terminal residues in the Aβ(1-42) isoform but not in Aβ(1-40). These results provide an unprecedented view of how albumin interacts with Aβ and illustrate the potential of dark-state exchange saturation transfer NMR in mapping the interactions between amyloid-inhibitory proteins and amyloidogenic peptides.

Human serum albumin (HSA) serves not only as a physiological oncotic pressure regulator and a ligand carrier but also as a biomarker for pathologies ranging from ischemia to diabetes. Moreover, HSA is a biopharmaceutical with a growing repertoire of putative clinical applications from hypovolemia to Alzheimer's disease. A key determinant of the physiological, diagnostic, and therapeutic functions of HSA is the amount of long chain fatty acids (LCFAs) bound to HSA. Here, we propose to utilize (13)C-oleic acid for the NMR-based assessment of albumin-bound LCFA concentration (CONFA). (13)C-Oleic acid primes HSA for a LCFA-dependent allosteric transition that modulates the frequency separation between the two main (13)C NMR peaks of HSA-bound oleic acid (ΔνAB). On the basis of ΔνAB, the overall [(12)C-LCFA]Tot/[HSA]Tot ratio is reproducibly estimated in a manner that is only minimally sensitive to glycation, albumin concentration, or redox potential, unlike other methods to quantify HSA-bound LCFAs such as the albumin-cobalt binding assay.

Microbial growth confinement using liquid scaffolds based on an aqueous two-phase system (ATPS) is a promising technique to overcome the challenges in microbial–mammalian co-culture in vitro. To better understand the potential use of the ATPS in studying these complex interactions, the goal of this research was to characterize the effects of bacteria loading and biofilm maturation on the stability of a polyethylene glycol (PEG) and dextran (DEX) ATPS. Two ATPS formulations, consisting of 5% PEG/5% DEX and 10% PEG/10% DEX (w/v), were prepared. To test the containment limits of each ATPS formulation, Escherichia coli MG1655 overnight cultures were resuspended in DEX at optical densities (ODs) of 1, 0.3, 0.1, 0.03, and 0.01. Established E. coli colonies initially seeded at lower densities were contained within the DEX phase to a greater extent than E. coli colonies initially seeded at higher densities. Furthermore, the 10% PEG/10% DEX formulation demonstrated longer containment time of E. coli compared to the 5% PEG/5% DEX formulation. E. coli growth dynamics within the ATPS were found to be affected by the initial bacterial density, where colonies of lower initial seeding densities demonstrate more dynamic growth trends compared to colonies of higher initial seeding densities. However, the addition of DEX to the existing ATPS during the growth phase of the bacterial colony does not appear to disrupt the growth inertia of E. coli. We also observed that microbial growth can disrupt ATPS stability below the physical carrying capacity of the DEX droplets. In both E. coli and Streptococcus mutans UA159 colonies, the ATPS interfacial tensions are reduced, as suggested by the loss of fluorescein isothiocyanate (FITC)–DEX confinement and contact angel measurements, while the microbial colony remained well defined. In general, we observed that the stability of the ATPS microbial colony is proportional to polymer concentrations and inversely proportional to seeding density and culture time. These parameters can be combined as part of a toolset to control microbial growth in a heterotypic co-culture platform and should be considered in future work involving mammalian–microbial cell interactions.