Alpha-Synuclein Aggregation Mechanism in the Presence of Nanomaterials.

Parkinson's disease (PD) is characterized by the toxic oligomeric and fibrillar phases formed by monomeric alpha-synuclein (α-syn). Certain nanoparticles have been demonstrated to promote protein aggregation, while other nanomaterials have been found to prevent the process. In the current work, we use nuclear magnetic resonance spectroscopy in conjunction with isothermal titration calorimetry to investigate the cause and mechanism of these opposing effects at the amino acid protein level. The interaction of α-syn with two types of nanomaterials was considered: citrate-capped gold nanoparticles (AuNPs) and graphene oxide (GO). In the presence of AuNPs, α-syn aggregation is accelerated, whereas in the presence of GO, aggregation is prevented. The study indicates that GO sequesters the NAC region of α-syn monomers through electrostatic and hydrophobic interactions, leading to a reduced elongation rate, and AuNPs leave the NAC region exposed while binding the N-terminus, leading to higher aggregation. The protein's inclination toward quicker aggregation is explained by the binding of the N-terminus of α-syn with the gold nanoparticles. Conversely, a comparatively stronger interaction with GO causes the nucleation and growth phases to be postponed and inhibits intermolecular interactions. Our finding offers novel experimental insights at the residue level regarding the aggregation of α-syn in the presence of various nanomaterials and creates new opportunities for the development of suitably functionalized nanomaterial-based therapeutic reagents against Parkinson's and other neurodegenerative diseases.

Modulation of protein-graphene oxide interactions with varying degrees of oxidation.

The degree of oxidation of graphene oxide (GO) has been shown to be important for its toxicity and drug-loading efficiency. However, the effect of its variations on GO-protein interaction remains unclear. Here, we evaluate the effect of the different oxidation degrees of GO on its interaction with human ubiquitin (8.6 kDa) using solution state nuclear magnetic resonance (NMR) spectroscopy in combination with other biophysical techniques. Our findings show that the interaction between the protein and the different GO samples is weak and electrostatic in nature. It involves fast dynamic exchange of the protein molecules from the surface of the GO. As the oxidation degree of the GO increases, the extent of the interaction with the protein changes. The interaction of the protein with GO can thus be modulated by tuning the degree of oxidation. This study opens up new avenues to design appropriate graphenic materials for use in various biomedical fields such as drug delivery, biomedical devices and imaging.

Rapid NMR assignments of intrinsically disordered proteins using two-dimensional 13C-detection based experiments.

An approach for rapid backbone resonance assignments in proteins using only two 2D NMR experiments is presented. The new method involves a combination of high-resolution 13Cα-detected NMR experiments and selective unlabeling of amino acid residues. The 13C detected 2D hNCA and 2D hNcoCA spectra of a uniformly labeled sample of the protein are analysed in concert with the 2D hNCA spectrum obtained for a selectively unlabeled sample. The combinatorial set of amino acid residues for selective unlabeling is chosen optimally to maximize the assignments. The method is useful for rapid assignment of proteins with low stability such as intrinsically disordered proteins and is applicable to deuterated proteins. This approach helped in assignments of 14.5 kDa human α-synuclein during the course of its aggregation.

Enhanced stability of an intrinsically disordered protein against proteolytic cleavage through interactions with silver nanoparticles.

Intrinsically disordered proteins (IDPs), being sensitive to proteolytic degradation both in vitro and in vivo, can be stabilized by the interactions with various binding partners. Here, we show for the first time that silver nanoparticles (AgNPs) have the ability to enhance the half-life of an IDP, thereby rendering it stable for a month against proteolytic degradation. The conjugate of the unstructured linker domain of human insulin-like growth factor binding protein-2 (L-hIGFBP2) with 10 nm citrate-capped AgNPs was studied using two-dimensional NMR spectroscopy and other biophysical techniques. Our studies reveal the extent and nature of residue-specific interactions of the IDP with AgNPs. These interactions mask proteolysis-prone sites of the IDP and stabilize it. This study opens new avenues for the design of appropriate nanoparticles targeting IDPs and for storage, stabilization and delivery of IDPs into cells in a stable form.

Evaluation of antioxidant and antibacterial activity of methanolic extracts of Gentiana kurroo royle.

In this study our objective was to evaluate the antioxidant and antimicrobial activity of methanolic extracts of leaves and roots of Gentiana kurroo. The antioxidant activities of the extracts were examined using different biochemical assays namely diphenylpicrylhydrazyl (DPPH), nitroblue tetrazolium (NBT) and ferric reducing power (FRAP). In all the assays, root extract exhibited stronger antioxidant activity than that of leaves. The antibacterial activity of the extracts was also evaluated and MIC values were calculated by broth dilution method. Although, the extracts prevented the growth of both Gram positive and Gram negative bacteria, the MIC values of methanolic extract of the leaves were higher than those of the root extract. The antibacterial and antioxidant activity of the extracts was found to be positively associated with the total phenolic and flavonoid content of the extracts.