RESUMEN
The assembly of proteins into amyloid fibrils has become linked not only with the progression of myriad human diseases, but also important biological functions. Understanding and controlling the formation, structure, and stability of amyloid fibrils is therefore a major scientific goal. Here we utilize electron microscopy-based approaches combined with quantitative statistical analysis to show how recently developed kind of amyloid modulators-multivalent polymer-peptide conjugates (mPPCs)-can be applied to control the structure and stability of amyloid fibrils. In doing so, we demonstrate that mPPCs are able to convert 40-residue amyloid beta fibrils into ordered nanostructures through a combination of fragmentation and bundling. Fragmentation is shown to be consistent with a model where the rate constant of fibril breakage is independent of the fibril length, suggesting a local and specific interaction between fibrils and mPPCs. Subsequent bundling, which was previously not observed, leads to the formation of sheet-like nanostructures which are surprisingly much more uniform than the starting fibrils. These nanostructures have dimensions independent of the molecular weight of the mPPC and retain the molecular-level ordering of the starting amyloid fibrils. Collectively, we reveal quantitative and nanoscopic understanding of how mPPCs can be applied to control amyloid structure and stability, and demonstrate approaches to elucidate nanoscale amyloid phase behavior in the presence of functional macromolecules and other modulators.
RESUMEN
Protein aggregation is implicated in multiple deposition diseases including Alzheimer's Disease, which features the formation of toxic aggregates of amyloid beta (Aß) peptides. Many inhibitors have been developed to impede or reverse Aß aggregation. Multivalent inhibitors, however, have been largely overlooked despite the promise of high inhibition efficiency endowed by the multivalent nature of Aß aggregates. In this work, we report the success of multivalent polymer-peptide conjugates (mPPCs) as a general class of inhibitors of the aggregation of Aß40. Significantly delayed onset of fibril formation was realized using mPPCs prepared from three peptide/peptoid ligands covering a range of polymer molecular weights (MWs) and ligand loadings. Dose dependence studies showed that the nature of the ligands is a key factor in mPPC inhibition potency. The negatively charged ligand LPFFD (LD) leads to more efficient mPPCs compared to the neutral ligands, and is most effective at 7% ligand loading across different MWs. Molecular dynamics simulations along with dynamic light scattering experiments suggest that mPPCs form globular structures in solution due to ligand-ligand interactions. Such interactions are key to the spatial proximity of ligands and thus to the multivalency effect of mPPC inhibition. Excess ligand-ligand interactions, however, reduce the accessibility of mPPC ligands to Aß peptides, and impair the overall inhibition potency.
RESUMEN
Amyloid aggregation and deposition are associated with many intractable human diseases. Although the inhibition of amyloid protein aggregation has been well-studied, the disaggregation and dissolution of existing amyloid fibrils is less known. Taking a fibrillar assembly of amyloid ß (Aß) peptide as the model system, here we report multivalent polymer-peptide conjugates (mPPCs) that disassemble preformed Aß fibrils into dispersible sub-100 nm structures. Atomic force microscopy and dynamic light scattering studies show that the disassembly rate of preformed Aß fibrils is controlled by the molecular weight of mPPCs. Rate equations on fibril disappearance are deduced from a simple model, which indicate that the disassembly reaction is first-order in the concentration of Aß fibrils and a pseudo-first-order reaction in the concentration of peptide moieties on mPPCs, respectively. We eliminate the possibility that the disassembly occurs by the association between mPPCs and Aß monomer/oligomers based on circular dichroism and Thioflavin T fluorescence assays. It is mostly likely that the mPPCs disassemble Aß fibrils through a direct interaction. The mPPCs may thus offer a general macromolecular design concept that breaks down existing amyloid fibrils in a predictable fashion.
Asunto(s)
Péptidos beta-Amiloides/química , Polímeros/química , Humanos , Conformación Molecular , Peso MolecularRESUMEN
Manipulating the size and shape of noncovalent multivalent assemblies is an ongoing challenge in the field of supramolecular polymers. Following a mechanistic approach, we reasoned that nucleation-elongation kinetics presents unique opportunities for controlled growth since the final outcome is likely to depend on the structure and dynamics of critical-nucleus formation. Taking fibrillar assembly of amyloid ß (Aß) peptide as the model system of nucleation-dependent supramolecular polymerization, here we report multivalent polymer-peptide conjugates (mPPCs) that redirect fibrillar assembly of Aß to form discrete nanostructures. The mPPCs were rationally designed to target Aß intermediates formed prior to critical nucleation. Atomic force microscopy and transmission electron microscopy studies show that in the presence of mPPCs, Aß self-assembles into zero-dimensional discrete nanostructures with lateral dimensions approximately in 5-35 nm, while Aß alone self-assembles into one-dimensional fibrils in micrometer. Thioflavin T kinetics fluorescence assays demonstrate that mPPCs suppress Aß fibrillogenesis. The mPPCs may thus represent a prototypical molecular design of multivalent macromolecules able to control the final shape of supramolecular polymers assembled via a nucleation-dependent mechanism.
Asunto(s)
Péptidos beta-Amiloides/química , Nanoestructuras/química , Polímeros/química , Sustancias Macromoleculares/química , Estructura Molecular , Tamaño de la PartículaRESUMEN
This article addresses the identification and quantification of the chemical species resulting in resonances at 2.17 and 2.25 ppm in the (1)H nuclear magnetic resonance (NMR) spectrum of pharmaceutical-grade heparin sodium. The NMR signals in question were first confirmed to arise from chemical moieties covalently attached to the heparin molecule through NMR diffusion experiments as well as chemical treatment of heparin active pharmaceutical ingredient (API) containing the resonances. The material responsible for the extra NMR signals was then demonstrated by NMR spiking studies to be something other than oversulfated chondroitin sulfate and was finally identified as an O-acetylation product of heparin through (13)C labeling experiments with subsequent NMR analysis. The extent of O-acetylation was quantified using three orthogonal techniques: (1)H NMR, ion chromatography, and headspace gas chromatography/mass spectrometry. The results of this work showed good agreement between the three quantitative methods developed to analyze the signals in the United States Pharmacopeia-specified region of 2.12-3.00 ppm for heparin API.
Asunto(s)
Anticoagulantes/química , Heparina/química , Espectroscopía de Resonancia Magnética/métodos , Acetilación , Sulfatos de Condroitina/análisis , Ácido Nitroso/química , PolimerizacionRESUMEN
The thermodynamics and kinetics of the reaction DeoxyHb-Fe(2+)<-->MetHb-Fe(3+) for human hemoglobin A (HbA), alpha- and beta-fumarate crosslinked hemoglobins were investigated by spectroelectrochemistry. Information from this study is used to determine what structural features and experimental conditions stabilize ferrous vs. ferric form of hemoglobin, and what implications this stabilization may have on the autoxidation reaction. Alpha- and beta-fumarate crosslinked hemoglobins, alphaXL-HbA and betaXL-HbA, were obtained by crosslinking deoxyhemoglobin and oxyhemoglobin, respectively, with bis(3,5-dibromosalicyl) fumarate (DBSF). Formal redox potentials, E(0), and reduction/oxidation rates were measured in the presence of mediator, hexammineruthenium(III) chloride. It was found that E(0) shifted positive for the alpha-, and negative for the beta-fumarate crosslinked hemoglobin compared to HbA for all experimental conditions investigated. This shift was consistent with stabilization of the tense (positive shift) or relaxed conformation (negative shift) conferred by crosslinking. Formal redox potentials shifted positive with addition of nitrate and chloride ions for alphaXL-HbA, indicating additional stabilization of the T quaternary. The slopes of the Nernst plots showed evidence of cooperativity as expressed by n(max). The data points (E(0), n(max)) were fitted by the MWC model which states that the electron transfer and the addition/removal of water are concerted. The set of K(R) and c values, where the parameter c is the ratio K(R)/K(T) and K(R) and K(T) are the ligand (water molecule and an electron-hole) dissociation constants for the R and T states, for the beta-crosslinked hemoglobin compared to that of HbA and alpha-crosslinked hemoglobin indicated that crosslinking of oxyhemoglobin affected differently the inner-coordination sphere at the heme site. By modulating the electrolyte concentration the reduction rates were measured as a function of DeltaE(0), the difference in E(0) between hemoglobin molecules and mediator. Linearization of the Marcus cross-relationship (based on the concerted water and electron transfer) was good for HbA, and poor for alphaXL-HbA and betaXL-HbA, consistent with results obtained by the MWC analysis. This may imply that the reduction of HbA is controlled by the driving force, DeltaE(0), whereas the reduction of alphaXL-HbA and betaXL-HbA occurs by a non-concerted mechanism controlled by structural features brought about by crosslinking. The autoxidation reaction, conversion of oxygen-bound ferrous hemoglobin to ferric hemoglobin, was found independent of E(0). Alpha-fumarate crosslinked hemoglobin showed the highest autoxidation rate despite its positive shift in formal redox potential as compared to HbA, followed by beta-fumarate crosslinked hemoglobin, and by native hemoglobin. These data suggest that the chemical mechanism of oxygen dissociation and accessibility of water and oxygen radicals to heme site control autoxidation.