Gold Nanoparticles as a Probe for Amyloid-ß Oligomer and Amyloid Formation.

The process of amyloid-ß (Aß) amyloid formation is pathologically linked to Alzheimer's disease (AD). The identification of Aß amyloids and intermediates that are crucial players in the pathology of AD is critical for exploring the underlying mechanism of Aß aggregation and the diagnosis of the disease. Herein, we performed a gold nanoparticle (AuNP)-based study to detect the formation of Aß amyloid fibrils and oligomers. Our results demonstrate that the intensity of the surface plasmon resonance (SPR) absorption band of the AuNPs is sensitive to the quantity of Aß40 amyloids. This allows the SPR assay to be used for detection and semi-quantification of Aß40 amyloids, and characterization of the kinetics of Aß amyloid formation. Furthermore, our study demonstrates that the SPR band intensity of the AuNPs is sensitive to the presence of oligomers of both Aß40 and an Aß40 mutant, which forms more stable oligomers. The kinetics of the stable oligomer formation of the Aß40 mutant can also be monitored following the SPR band intensity change of AuNPs. Our results indicate that this nanoparticle based method can be used for mechanistic studies of early protein self-assembly and fibrillogenesis.

Suppression of amyloid fibrils using the GroEL apical domain.

In E. coli cells, rescue of non-native proteins and promotion of native state structure is assisted by the chaperonin GroEL. An important key to this activity lies in the structure of the apical domain of GroEL (GroEL-AD) (residue 191-376), which recognizes and binds non-native protein molecules through hydrophobic interactions. In this study, we investigated the effects of GroEL-AD on the aggregation of various client proteins (α-Synuclein, Aß42, and GroES) that lead to the formation of distinct protein fibrils in vitro. We found that GroEL-AD effectively inhibited the fibril formation of these three proteins when added at concentrations above a critical threshold; the specific ratio differed for each client protein, reflecting the relative affinities. The effect of GroEL-AD in all three cases was to decrease the concentration of aggregate-forming unfolded client protein or its early intermediates in solution, thereby preventing aggregation and fibrillation. Binding affinity assays revealed some differences in the binding mechanisms of GroEL-AD toward each client. Our findings suggest a possible applicability of this minimal functioning derivative of the chaperonins (the "minichaperones") as protein fibrillation modulators and detectors.

Positively Charged Chitosan and N-Trimethyl Chitosan Inhibit Aß40 Fibrillogenesis.

Amyloid fibrils, formed by aggregation of improperly folded or intrinsically disordered proteins, are closely related with the pathology of a wide range of neurodegenerative diseases. Hence, there is a great deal of interest in developing molecules that can bind and inhibit amyloid formation. In this regard, we have investigated the effect of two positively charged polysaccharides, chitosan (CHT) and its quarternary derivative N-trimethyl chitosan chloride (TMC), on the aggregation of Aß40 peptide. Our aggregation kinetics and atomic force microscopy (AFM) studies show that both CHT and TMC exhibit a concentration-dependent inhibiting activity on Aß40 fibrillogenesis. Systematic pH-dependent studies demonstrate that the attractive electrostatic interactions between the positively charged moieties in CHT/TMC and the negatively charged residues in Aß40 play a key role in this inhibiting activity. The stronger inhibiting activity of TMC than CHT further suggests the importance of charge density of the polymer chain in interacting with Aß40 and blocking the fibril formation. The possible interactions between CHT/TMC and Aß40 are also revealed at the atomic level by molecular docking simulation, showing that the Aß40 monomer could be primarily stabilized by electrostatic interactions with charged amines of CHT and quaternary amines of TMC, respectively. Binding of CHT/TMC on the central hydrophobic core region of Aß40 peptide may be responsible for blocking the propagation of the nucleus to form fibrillar structures. These results suggest that incorporation of sugar units such as d-glucosamine and N-trimethyl-d-glucosamine into polymer structural template may serve as a new strategy for designing novel antiamyloid molecules.

Poly(4-styrenesulfonate) as an inhibitor of Aß40 amyloid fibril formation.

The formation of amyloid, a cross-ß-sheet fibrillar aggregate of proteins, is associated with a variety of neurodegenerative diseases. Amyloidogenic proteins such as ß-amyloid (Aß) are known to exist with a large amount of polyelectrolyte macromolecules in vivo. The exact nature of Aß-polyelectrolyte interactions and their roles in Aß-aggregation are largely unknown. In this regard, we report the inhibiting effect of an anionic polyelectrolyte poly(4-styrenesulfonate) (PSS) on the aggregation of Aß40 peptide. The results demonstrate the strong inhibition potential of PSS on the aggregation of Aß40 and imply the dominant role of hydrophobicity of the polyelectrolyte in reducing the propensity of Aß40 amyloid formation. Additional studies with poly(vinyl sulfate) (PVS) and p-toluenesulfonate (PTS), which share similar charge density with PSS except the former lacking the nonpolar aromatic side chain and the latter the aliphatic hydrocarbon backbone, reveal that the presence of both aliphatic backbone and aromatic side chain group in PSS is essential for its Aß-aggregation inhibition activity. The interactions involved in the Aß40-PSS complex were further investigated using molecular dynamics (MD) simulation. Our results provide new insights into the structural interplay between polyelectrolytes and Aß peptide, facilitating the ultimate understanding of amyloid formation in Alzheimer's disease. The results should assist in developing novel polyelectrolytes as potential chemical tools to study amyloid aggregation.

Study protein folding and aggregation using nonnatural amino acid p-cyanophenylalanine as a sensitive optical probe.

Incorporation of nonnatural amino acids with a variety of special side groups into protein sequences has substantially expanded the experimental means of exploring protein structures and functions. Recently, p-cyanophenylalanine (PheCN), the nitrile analogue of phenylalanine, has been used as a novel optical probe for protein binding and folding studies. The fluorescence emission of PheCN is sensitive to solvent and local environment of the residue, making it a useful fluorescent probe of protein structural change at residue-specific resolution. Moreover, the utility of PheCN is increased by its ability to excite tryptophan fluorescence via the mechanism of fluorescence resonance energy transfer. PheCN could be applied to study a variety of biological problems, e.g., protein folding/unfolding and protein aggregation.

A novel amphiphilic thiosemicarbazone derivative for binding and selective sensing of human serum albumin.

The interaction of ligands and drug molecules with protein is of major interest in drug pharmacokinetics and pharmacodynamics. In this study, we synthesized a novel thiosemicarbazone-based amphiphilic molecule for selective binding and detection of human serum albumin (HSA) with significant increase in fluorescence intensity. The compound 5-(octyloxy) naphthalene substituted salicylaldehyde thiosemicarbazone was designed to interact with site I of HSA. The weak fluorescence of the probes in aqueous solution showed a dramatic increase in fluorescence intensity upon binding with HSA, while the responses to various other proteins and enzymes were negligible under similar experimental conditions. Changes in fluorescence intensity and formation of a new emission maximum of the compound in the presence of HSA as well as an increase in steady-state anisotropy values reflected well the nature of binding and location of the probe inside the protein environment.

Environment-sensitive amphiphilic fluorophore for selective sensing of protein.

Here we report the selective sensing of BSA (bovine serum albumin) by 8-(alkoxy)quinoline-based fluorescent probes, via non-covalent interactions. The weak fluorescence of these probes in aqueous solution showed a dramatic increase in quantum yield and lifetime upon binding with BSA, while the responses to various other proteins/enzymes used were negligible under similar experimental conditions. The emission of the probe is affected by the interplay with BSA but not with tryptophan amino acid suggesting that the microenvironment created by the macromolecule induces some change in their excited-state properties. Binding site assignment by a known site-selective binding ligand enabled us to conclude that the compounds predominantly bind at site I of BSA. The changes in fluorescence intensity and the position of emission maxima of compounds in presence of BSA along with the increase in steady state anisotropy values well reflect the nature of binding and location of the probe inside the protein environment. Compounds interact with BSA efficiently and exhibit site selectivity and thus have the potentiality to serve as an efficient and selective sensor of protein.

Role of hydrophobic and polar interactions for BSA-amphiphile composites.

To evaluate the role of hydrophobic and electrostatic or other polar interactions for protein-ligand binding, we have studied the interactions of bovine serum albumin (BSA) with 2-alkylmalonic acid and 2-alkylbenzimidazole amphiphiles having different head group and alkyl chain length. The binding affinity for the protein-amphiphile interactions is found to depend predominantly on the length of hydrocarbon chain, suggesting the crucial role of hydrophobic forces, supported by polar interactions at the protein surface. The BSA fluorescence exhibits appreciable hypsochromic shift along with a reduction in fluorescence intensity and mean lifetime upon binding with 2-alkylmalonic acid. UV-visible, steady state and time-resolved fluorescence measurements were performed to compare the effects of amphiphiles on BSA as a function of the amphiphiles head group and alkyl chain length.

2-Alkylmalonic acid: amphiphilic chelator and a potent inhibitor of metalloenzyme.

The present investigation demonstrates the potential of 2-alkylmalonic acid amphiphile as inhibitor of metalloenzymes like Taq DNA polymerase and alpha-amylase. A dose-dependent inhibition of Taq DNA polymerase was observed when a polymerase chain reaction (PCR) was performed in the presence of amphiphiles while in the case of alpha-amylase the inhibition was found to be independent of the inhibitor concentration. Control experiments revealed that both the chelating as well as the amphiphilic nature of the inhibitor was essential for enzyme inhibition. The fluorescence intensity and lifetime of alpha-amylase were also found to decrease in the presence of the amphiphiles. Steady-state fluorescence quenching studies suggested that removal of the metal ion from the enzyme leads to a decrease in the solvent accessibility of tryptophans, indicating change in the tertiary structure of the protein. It is proposed that removal of metal ion from the active sites of the enzyme by the amphiphilic compound possibly leads to disruption of the native conformation of the enzyme which is responsible for loss of its activity.

Artificial amphiphilic scaffolds for the selective sensing of protein based on hydrophobicity.

Here we report, novel amphiphilic fluorescent probes for selective detection of BSA (bovine serum albumin), via non-covalent interactions. The intrinsic weak fluorescence of the probes in an aqueous medium increased dramatically after addition of BSA, while responses to various other proteins/enzymes are negligible.

The interaction of 5-(alkoxy)naphthalen-1-amine with bovine serum albumin and its effect on the conformation of protein.

The effects of binding and conformational changes induced by the neutral amphiphilic ligand [5-(alkoxy)naphthalen-1-amine] with different alkyl chain lengths on bovine serum albumin (BSA) have been studied using UV-visible and fluorescence spectroscopic methods. The BSA fluorescence exhibits appreciable bathochromic shift along with a reduction in fluorescence intensity and fluorescence lifetime upon binding with ligands. Ligand quenches the fluorescence of BSA in a concentration-dependent manner and deviates positively from the linear Stern-Volmer equation. The calculated quenching rate constants and binding constants were shown to depend entirely on the alkyl chain length of the ligands. After binding of probes with protein, the distance between the donor and acceptor was calculated using Forster theory. Ligands bind near Trp-134 in the subdomain IA of the native BSA and they become accessible to Trp-212 when BSA gets unfolded. The spectral data well supports the idea that BSA changes its three-dimensional conformation incrementally during its unfolding.