Plasmid DNA Undergoes Two Compaction Regimes under Macromolecular Crowding.

The laser light scattering experiments were performed to explore the role of dextran (size (d): 2.6, 6.9, and 17.0 nm) in compacting the plasmids (pBS: 2.9 kbps; pCMV-Tag2B: 4.3 kbps; and pET28a: 5.3 kbps) in vitro in the volume fraction (ϕ) range 0.01 to 0.15 of the macromolecular crowder. Two compaction regimes were observed in terms of the radius of gyration (Rg) for plasmid-dextran combinations, wherein the plasmid diffusivity is governed by normal diffusion and subdiffusion, respectively. Generalized scaling, Rg ∼ ϕ-1/(1+x), where x represents the conformational geometry of plasmids, is reported. The plasmid conformation depends on the crowder's size, with larger conformational changes observed in the presence of smaller crowders. The second virial coefficient (A2) and translational diffusion coefficient (Dt) indicate that entropically driven depletion of crowders, excluded volume, and interplasmid repulsive interactions govern plasmids' conformational changes, validated herein from the scaling of Dt with molecular weight.

The catalytic core of Leishmania donovani RECQ helicase unwinds a wide spectrum of DNA substrates and is stimulated by replication protein A.

RecQ helicases are superfamily 2 (SF2) DNA helicases that unwind a wide spectrum of complex DNA structures in a 3' to 5' direction and are involved in maintaining genome stability. RecQ helicases from protozoan parasites have gained significant interest in recent times because of their involvement in cellular DNA repair pathways, making them important targets for drug development. In this study, we report biophysical and biochemical characterization of the catalytic core of a RecQ helicase from hemoflagellate protozoan parasite Leishmania donovani. Among the two putative RecQ helicases identified in L. donovani, we cloned, overexpressed and purified the catalytic core of LdRECQb. The catalytic core was found to be very efficient in unwinding a wide variety of DNA substrates like forked duplex, 3' tailed duplex and Holliday junction DNA. Interestingly, the helicase core also unwound blunt duplex with slightly less efficiency. The enzyme exhibited high level of DNA-stimulated ATPase activity with preferential stimulation by forked duplex, Holliday junction and 3' tailed duplex. Walker A motif lysine mutation severely affected the ATPase activity and significantly affected unwinding activity. Like many other RecQ helicases, L. donovani RECQb also possesses strand annealing activity. Unwinding of longer DNA substrates by LdRECQb catalytic core was found to be stimulated in the presence of replication protein A (LdRPA-1) from L. donovani. Detailed biochemical characterization and comparison of kinetic parameters indicate that L. donovani RECQb shares considerable functional similarity with human Bloom syndrome helicase.

Interactive patches over amyloid-ß oligomers mediate fractal self-assembly.

The monomeric units of intrinsically disordered proteins self-assemble into oligomers, protofilaments, and eventually fibrils which may turn into amyloid. The aggregation of these proteins is primarily studied in bulk with no restriction on their degrees of freedom. Herein we experimentally demonstrate that amyloid-ß (Aß) aggregation under diffusion-limited conditions leads to its fractal self-assembly. Confocal microscopy and scanning electron microscopy with energy dispersion x-ray analysis were used to confirm that the fractal self-assemblies were formed from Aß rather than the salt present in the two supporting media: deionized water and phosphate buffered saline. The results from the molecular docking experiments implicated that electrostatic and hydrophobic patches on the solvent-accessible surface area of the Aß oligomers mediate the fractal self-assembly. These implications were tested with laser light scattering experiments on the oligomers formed by breaking mature fibrils of Aß through sonication, which were observed to self-assemble into fractals when sonicated solutions were drop casted. The electrostatic interactions modulate the fractal morphologies with pH of the solution, which leads to a morphological phase transition observed through the variation in their fractal dimension. These transitions provide experimental evidence for the existing theoretical framework in terms of different kinetic models. The higher surface-to-volume ratio of these fractal self-assemblies may have applications in drug delivery, biosensing, and other biomedical applications.

Simultaneous Detection of Tyrosine and Structure-Specific Intrinsic Fluorescence in the Fibrillation of Alzheimer's Associated Peptides.

Understanding of the structural changes during their aggregation and interaction is a prerequisite for establishing the precise clinical relevance of human islet amyloid polypeptide (hIAPP) (involved in Type-II Diabetes Mellitus) in the treatment of Alzheimer's disease stemmed from beta-amyloid (Aß). Herein, we show that the steady-state emission spectra obtained from photoluminescence (PL) simultaneously capture both the tyrosine derivative (tyrosinate) and the structure-specific intrinsic fluorescence during the aggregation of Aß and hIAPP. We observe multiple peaks in the emission spectra which exist for structure-specific intrinsic fluorescence, and use the second derivative UV-Vis spectra and the shift in the tyrosine peak as a quantitative measure of the dissimilitude in the electronic states and the fibril growth. We further applied these techniques to detect the static electric field (0, 40, 120, 200 V/cm) induced promotion and inhibition of fibrillation in Aß, hIAPP and their electric field dependent role in the fibrillation of Aß : hIAPP(1 : 1). The results were corroborated by field-emission scanning electron microscopy (FESEM), and the determinations of secondary structures by Fourier transform infrared spectroscopy (FTIR). The results indicate that the emission spectrum can be used as a sensor to detect the presence of fibrils; hence for screening potential inhibitors of amyloid fibrillation.

Anisotropy versus fluctuations in the fractal self-assembly of gold nanoparticles.

In a recent report, the fractal self-assembly of gold nanoparticles (AuNPs) having a directional feature was observed in the presence of visible light. Therein, the visible light, an external parameter, was suspected to be responsible for the directional feature. Herein, we investigate the intrinsic factors, the aspect size ratio p and the size a of AuNPs, in modulating the fractal characteristics of their self-assemblies. Through light scattering experiments and microscopic imaging, we demonstrate the transition of morphologies from fractal-like to cross-shaped in gold colloidal aggregates with particles having nearly spherical and ellipsoidal shapes, respectively. The transition indicates the competitive role of anisotropy and fluctuations in deciding the morphological characteristics of the aggregates. By taking noise-reduced diffusion-limited aggregation (NRDLA) as a model system, we address the shape and size induced noise of the particles in the colloidal systems which are prone to form fractal aggregates. We qualitatively relate the noise due to the particles having a distinct aspect size ratio p and size a with the noise reduction parameter m of NRDLA. The realistic nature of the experimental systems, where the particles of different p and a are present during the growth process, is incorporated by introducing the Gaussian noise reduction in diffusion-limited aggregation (DLA). The morphological phase transition in Gaussian noise reduced DLA is characterized, and its relevance for accounting the shape and size originated noise fluctuations during the fractal growth process is discussed. The results of the present study may be used for tailored applications of AuNPs in drug delivery, biomedicine, biosensing, and cancer nanotechnology.

Fibril growth captured by electrical properties of amyloid-ß and human islet amyloid polypeptide.

The aggregation of amyloid-ß (Aß) and human islet amyloid polypeptide (hIAPP) proteins have attracted considerable attention because of their involvement in protein misfolding diseases. These proteins have mostly been investigated using atomic force microscopy, transmission electron microscopy, and fluorescence microscopy to study the directional growth of fibrils both perpendicular to and along the fibril axis. Here, we demonstrate the real-time monitoring of the directional growth of fibrils in terms of activation energy of proton transfer using an impedance spectroscopy technique. The activation energy is used to quantify the sensitivity of proton conduction to the different stages of protein aggregation. The decrement (increment) in activation energy is related to the fibril growth along (perpendicular to) the fibril axis in intrinsic protein aggregation. The entire aggregation process shows different phases of the directional growth for Aß and hIAPP, indicating different pathways for their aggregation. The activation energy for hIAPP is found to be smaller than the activation energy of Aß during the aggregation process. The oscillatory behavior of the activation energy of hIAPP reflects a rapid change in the directional growth of the protofilaments of hIAPP. The results indicate higher aggregation propensity of Aß than hIAPP. In the presence of resveratrol, hIAPP exhibits slower aggregation compared to Aß. Methods of this study may in general be used to reveal the modulated aggregation pathway of proteins in the presence of different ligands.

Fractal self-assembly and aggregation of human amylin.

Human amylin is an intrinsically disordered protein believed to have a central role in Type-II diabetes mellitus (T2DM). The formation of intermediate oligomers is a seminal event in the eventual self-assembled fibril structures of amylin. However, the recent experimental investigations have shown the presence of different self-assembled (oligomers, protofilaments, and fibrils) and aggregated structures (amorphous aggregates) of amylin formed during its aggregation. Here, we show that amylin under diffusion-limited conditions leads to fractal self-assembly. The pH and solvent sensitive fractal self-assemblies of amylin were observed using an optical microscope. Confocal microscopy and scanning electron microscopy (SEM) with energy dispersion X-ray analysis (EDAX) were used to confirm the fractal self-assembly of amylin in water and PBS buffer, respectively. The fractal characteristics of the self-assemblies and the aggregates formed during the aggregation of amylin under different pH conditions were investigated using laser light scattering. The hydropathy and the docking study indicated the interactions between the anisotropically distributed hydrophobic residues and polar/ionic residues on the solvent-accessible surface of the protein as the crucial interaction hot-spots for driving the self-assembly and aggregation of human amylin. The simultaneous presence of various self-assemblies of human amylin was observed through different microscopy techniques. The present study may help in designing different fractal-like nanomaterials with potential applications in drug delivery, sensing, and tissue engineering.

Quantification of protein aggregation rates and quenching effects of amylin-inhibitor complexes.

The formation of amyloid aggregates is the hallmark of many protein misfolding diseases, including Type-II diabetes mellitus, which is caused by the fibrillation of amylin protein. It is established that nano-sized ligands such as curcumin, resveratrol and graphene quantum dots can modify protein aggregation rates. In this article, we report a comparative study of these ligands to estimate their protein aggregation rates and fluorescence quenching using various experimental techniques. Through light scattering experiments, the RH of bare amylin was found to increase at a rate of 43% per day, whereas in the presence of the ligands in different molar ratios (A1C10, A1R10 and A1GQDs20), the sizes of the complexes were found to grow at rates of 7%, 8% and 13% per day, respectively. We observed fluorescence quenching using photoluminescence experiments for all three protein-ligand complexes. The protein aggregation rate and fluorescence quenching exhibited a concentration-dependent competitive role in the inhibition process. Interestingly, for graphene quantum dots, the protein aggregation rate is more affected at lower concentrations, while fluorescence quenching dominates at higher concentrations; this is in contrast to curcumin and resveratrol, where fluorescence quenching dominates at all concentrations of the ligands in the complex. The FTIR data showed appreciable conversion of ß-sheets into less aggregation-prone secondary structures for all three amylin-ligand ratios; however, the inhibition performance of curcumin overshadowed those of the other two inhibitors. The inhibition behavior of these three ligands was corroborated by analysis of analytical and high-resolution TEM images of the fibrils.

Aggregation of amylin: Spectroscopic investigation.

Apart from its relevance to pathology, protein misfolding disease like Type-II Diabetes Mellitus, caused by amyloids of amylin protein has attracted more attention due to structural changes occurring during the aggregation process. We report extensive spectroscopy data of amylin during fibril formation through Raman, FTIR, CD, UV-vis absorption and photoluminescence (PL) spectroscopy. UV-vis and PL spectrum showed the sigmoidal growth of fibril with a lag time of ~2 days, which is consistent with earlier reported work using dynamic light scattering (DLS). Raman spectra revealed the formation of parallel and anti-parallel ß-sheet from 0% to 20% with ageing (1st day to 21st day) at pH 6.5 ±â€¯0.1. The results are corroborated by CD and FTIR data. These show the change in ß-sheet by 23% at pH 6.5 ±â€¯0.1, 26% at pH = 1.0 ±â€¯0.1 and 30% at pH = 12 ±â€¯0.1. It is also shown that the formation and conversion of other secondary structures into ß-sheet is very sensitive towards the pH and ageing. The study may be used for the development of therapeutic strategies that could inhibit or even reverse the process of aggregation.

DNA supported graphene quantum dots for Ag ion sensing.

The use of graphene quantum dots can be extended for bio-sensing and metal ion detection. Synergistic combination of graphene quantum dots (GQDs) with DNA leads to high performance Ag-ion detection system. The thoroughly characterized GQDs were found to have spherical morphology, with dimensions in the range of 5-10 nm. The atomic force microscopy studies proved that the synthesized GQDs were mostly comprised of two to four graphene layers. To make the system biocompatible, GQDs/NGQDs were combined with DNA. Two properties of DNA were exploited, capacity to provide nitrogen to GQDs; and to synergistically contribute to Ag+ detection. In addition to Ag+, the strong green photoluminescence (PL) of GQDs showed significant quenching, owing to the appearance of associated Förster resonance energy transfer processes. This led to high sensing efficiencies.

Repulsive interaction induces fibril formation and their growth.

In a type-II diabetes disease, amylin protein takes an incorrect structure that leads to the formation of the amyloid fibril. The conversion mechanism of amyloid fibril is not well understood. We have observed a repulsive interaction, in terms of second virial co-efficient (A2), between protein molecules in their native state in the PBS buffer through laser light scattering technique. The A2 switches from repulsive (positive A2) to attractive (negative A2) interactions with elapsed time favoring the formation and growth of the fibril. We report aggregation and fibril growth kinetics of amylin protein in different environmental conditions. The measurement of shape factor (ρ) through light scattering experiment shows a transition from coil-like structure to rod-like growth. In addition to rod-like growth, sheet-like growth of fibril is also observed through analytical and high-resolution TEM imaging techniques. The nucleation leading to elongation of fibrils as well as stacking of individual fibril perpendicular to the fibril axis is held by hydrogen bonding observed through high-resolution TEM.