The aggregation of α-synuclein (A-syn) has been implicated in the pathogenesis of Parkinson's disease (PD). Although the early events of aggregation and not the matured amyloid fibrils are believed to be responsible for the toxicity, it has been difficult to probe the formation of early oligomers experimentally. We studied the effect of Fe3O4 nanoparticle (NP) in the early stage of aggregation of A-syn using fluorescence correlation spectroscopy (FCS) and laser scanning microscopy. The binding between the monomeric protein and NPs was also studied using FCS at single-molecule resolution. Our data showed that the addition of bare Fe3O4 NPs accelerated the rate of early aggregation, and it did not bind the monomeric A-syn. In contrast, L-lysine (Lys)-coated Fe3O4 NPs showed strong binding with the monomeric A-syn, inhibiting the early events of aggregation. Lys-coated Fe3O4 NPs showed significantly less cell toxicity compared with bare Fe3O4 NPs and can be explored as a possible strategy to develop therapeutic application against PD. To the best of our knowledge, this report is the first example of using a small molecule to attenuate the early (and arguably the most relevant in terms of PD pathogenesis) events of A-syn aggregation.

The aggregation of α-synuclein (A-syn) has been implicated strongly in Parkinson's disease (PD). In vitro studies established A-syn to be a member of the intrinsically disordered protein (IDP) family. This protein undergoes structural interconversion between an extended and a compact state, and this equilibrium influences the mechanism of its aggregation. A combination of fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) has been used to study the membrane induced conformational reorganization and aggregation of A-syn. Different structural and conformational events, including the early collapse, the formation of the secondary structure, and aggregation have been identified and characterized using FCS and other biophysical methods. In addition, the concentrations of glycerol and urea have been varied to study the effect of solution conditions on the above conformational events. Further, we have extended this study on a number of A-syn mutants, namely, A30P, A53T, and E46K. These mutants are chosen because of their known implications in the disease pathology. The variation of solution conditions and mutational analyses suggest a strong correlation between the extent of early collapse and the onset of aggregation in PD.

Anatase TiO2 and Ag nanoparticles (NPs) codoped SiO2 films were prepared by the sol-gel method. Proportionate amounts of 3-(glycidoxypropyl)trimethoxysilane (GLYMO), tetraethylorthosilicate (TEOS) and 3-(methacryloxypropyl)trimethoxysilane (MEMO) derived inorganic-organic silica sol, commercially available dispersed anatase TiO2 NPs, and AgNO3 were used to prepare the sols. The films were prepared on ZrO2 (cubic) precoated soda-lime glass substrates by a single-dipping technique and heat-treated at 450 °C in air and H2/Ar atmosphere to obtain hard, relatively porous, and transparent coatings of thickness>600 nm. The ZrO2 barrier layer was previously applied on soda-lime glass to restrict the diffusion of Ag into the substrate. The Ag-TiO2 NPs incorporated SiO2 films were intense yellow in color and found to be fairly stable at ambient condition for several days under fluorescent light. These films show a considerable growth inhibition on contact with the gram negative bacteria E. coli.

Fluorescence correlation spectroscopy (FCS) has been commonly used to study the diffusional and conformational fluctuations of labeled molecules at single-molecule resolution. Here, we explored the applications of FCS inside a polyacrylamide gel to study the effects of molecular weight and molecular shape in a crowded environment. To understand the effect of molecular weight, we carried out FCS experiments with four model systems of different molecular weights in the presence of varying concentrations of acrylamide. The correlation curves were fit adequately using a model containing two diffusing components: one representing unhindered diffusion and one representing slow hindered diffusion in the gel phase. A large number of measurements carried out at different randomly chosen spots on a gel were used to determine the most probable diffusion time values using Gaussian distribution analysis. The variation of the diffusivity with the molecular weight of the model systems could be represented well using the effective medium model. This model assumes a combination of hydrodynamic and steric effects on solute diffusivity. To study the effects of solute shape, FCS experiments were carried inside a urea gradient gel to probe the urea-induced unfolding transition of Alexa488Maleimide-labeled bovine serum albumin. We showed that the scaling behavior, relating the hydrodynamic radius and the number of amino acids, changes inside an acrylamide gel for both folded and unfolded proteins. We showed further that crowding induced by a polyacrylamide gel increases the resolution of measuring the difference in hydrodynamic radii between the unfolded and folded states.

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