Impedance Spectroscopy Unveiled the Surfactant-Induced Unfolding and Subsequent Refolding of Human Serum Albumin.

Protein-surfactant interaction is a dynamic interplay of electrostatic and hydrophobic forces that ensues from the folding of a protein. We employ impedance spectroscopy (IS), a label-free method, to investigate the unfolding and refolding of human serum albumin (HSA), a globular plasma protein, in the presence of two surfactants: polysorbate-20 (Tween-20), a nonionic surfactant, and sodium dodecyl sulfate (SDS), an anionic surfactant. The equivalent electrical analog circuit was predicted from impedance spectra of HSA in an aqueous solution at physiological pH and room temperature, focusing on varying the concentration of codissolved surfactants. A change in the dielectric constant (ε') and ionic conductivity (κ) is observed by comparing the surfactant-treated protein samples to the bare surfactant solutions to assess the conformational changes induced by surfactants in HSA. Far-UV circular dichroism analysis revealed a decrease in α-helices and an increase in ß-sheets and random coils upon SDS addition, which were reversed by Tween-20. Dynamic light scattering supported the findings by measuring changes in the hydrodynamic diameter (dh) of HSA. Unfolding and refolding of HSA with surfactants were also observed through photoluminescence spectroscopy by examining the microenvironment surrounding the single tryptophan (W) within the protein, and the thermodynamic parameters were obtained using the modified Stern-Volmer equation. Our research explores the intriguing domain of protein-surfactant interactions, offering insights with promising applications across diverse biological processes and IS as a suitable alternative technique for investigating protein conformational changes by studying the electrical response of the samples.

Oxidative stress induced conformational changes of human serum albumin.

Oxidative stress, generated by reactive oxygen species (ROS), is responsible for the loss of structure and functionality of proteins and is associated with several aging-related diseases. Here, we report an in vitro study to gauge the effect of ROS on the structural rearrangement of human serum albumin (HSA), a plasma protein, through metal-catalyzed oxidation (MCO) at physiological temperature through various biophysical techniques like UV-vis absorption, circular dichroism (CD), differential scanning calorimetry (DSC), MALDI-TOF, FTIR, and Raman spectroscopy. The UV-vis spectra of oxidized HSA show an early blueshift, signifying the unfolding of the protein because of ROS followed by the broadening of the absorption peak at a longer time. The DSC data corroborate the observation, revealing an exothermic transition for the oxidized sample at a longer time, suggesting in situ aggregation. The CD and FTIR spectra indicate the associated secondary structural changes occurring with time, depicting the variation of the helical content of HSA. The amide-III analysis of Raman data also complements the structural changes, and MALDI-TOF data show the mass distribution with time. Overall, this work might help determine the effect of oxidation on the biological activity of serum albumin as it can impact the physiological properties of HSA.

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.

Electric field-driven conformational changes in the elastin protein.

The formation of aggregates and amyloids, a hallmark of many protein misfolding diseases, depends on many intrinsic and extrinsic factors. Many approaches (in vitro, in vivo, and in silico) have been attempted to inhibit the aggregation process so that the progression of these diseases can be controlled. We investigate the effect of a static electric field (EF; 120 V cm-1 and 200 V cm-1) on the conformational change of elastin protein using light scattering, spectroscopy, and microscopy techniques. Laser light scattering and photoluminescence spectroscopy show the formation of fibrils of unexposed elastin with aging, whereas disruption of fibril formation with EF exposed elastin. The size of EF exposed elastin first increases and exhibits an apex, and subsequently decreases with an increasing time of exposure. We observed that a decrease in the size of EF exposed elastin depends on the strength of the EF, faster decrement at higher EF. FTIR data show that EF modifies elastin protein's secondary structures; it facilitates the interconversion of ß-sheets and turns into α-helix structures. The SEM images of unexposed and EF exposed elastin confirms the observation through light scattering and PL techniques. The effect of an EF on protein conformation and amyloids is promising to treat Parkinson's disease, a protein misfolding disease.

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.

Hierarchical cage-frame type nanostructure of CeO2 for bio sensing applications: from glucose to protein detection.

Self-assembled hierarchical nanostructures are slowly superseding their conventional counterparts for use in biosensors. These morphologies show high surface area with tunable porosity and packing density. Modulating the interfacial interactions and subsequent particle assembly occurring at the water-and-oil interface in inverse miniemulsions, are amongst the best strategies to stabilize various type of hollow nanostructures. The paper presents a successful protocol to obtain CeO2 hollow structures based biosensors that are useful for glucose to protein sensing. The fabricated glucose sensor is able to deliver high sensitivity (0.495 µA cm-2 nM-1), low detection limit (6.46 nM) and wide linear range (0 nM to 600 nM). CeO2 based bioelectrode can also be considered as a suitable candidate for protein sensors. It can detect protein concentrations varying from 0 to 30 µM, which is similar or higher than most reports in the literature. The limit of detection (LOD) for protein was ∼0.04 µM. Therefore, the hollow CeO2 electrodes, with excellent reproducibility, stability and repeatability, open a new area of application for cage-frame type particles.

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.

Glucose-induced structural changes and anomalous diffusion of elastin.

Elastin is the principal protein component of elastic fiber, which renders essential elasticity to connective tissues and organs. Here, we adopted a multi-technique approach to study the transport, viscoelastic, and structural properties of elastin exposed to various glucose concentrations (X=[gluc]/[elastin]). Laser light scattering experiments revealed an anomalous behavior (anomaly exponent, ß <0.6) of elastin. In this regime (ß <0.6), the diffusion constant decreases by 40% in the presence of glucose (X> 10), which suggests the structural change in elastin. We have observed a peculiar inverse temperature transition of elastin protein, which is a measure of structural change, at 40 °C through rheology experiments. Moreover, we observe its shift towards lower temperature with a higher X. FTIR revealed that the presence of glucose (X < 10) favors the formation of ß-sheet structure in elastin. However, for X > 10, dominative crowding effect reduces the mobility of protein and favors the increase in ß-turns and γ-turns by 25 ± 1% over the ß-sheet (ß-sheet decreases by 12 ± 0.8%) and α-helix (α-helix decreases by 13 ± 0.8%). The stiffness of protein is estimated through Flory characteristic ratio, C∞ and found to be increasing with X. These glucose-based structural changes in the elastin may explain the role of glucose in age-related issues of the skin.

Curcumin Complexed with Graphene Derivative for Breast Cancer Therapy.

In recent years, graphene-based materials complexed with drugs have been developed for application in cancer therapy, aimed at gaining synergistic effect. Here, we have prepared graphene oxide (GO) and graphene quantum dots (GQDs) with curcumin (Cur) in three different ratios (1:1, 1:3, and 1:5 w/v). We showed a successful complexation of GO and GQDs with Cur through various spectroscopy and microscopy techniques. The optical density of the complex through UV-vis spectroscopy showed less than 10% (for GQDs-Cur) and less than 20% (for GO-Cur) aggregation in 48 h, which confirms the stability of the complex. The UV-vis result estimates the loading efficiency of Cur to be 80 ± 1 and 83 ± 1% for GO-Cur and GQDs-Cur respectively. We tested the complexes GO-Cur and GQDs-Cur in different ratios as an anticancer drug against human breast cancer cell lines MCF-7 and MDA-MB-468 through the MTT assay. Following 48 h of incubation with the cell lines, a cell viability of more than 75% was observed in the case of GQDs & GO, while it was 40% in the case of Cur at a concentration of 100 µg/mL. The 1:1, 1:3, and 1:5 ratios of complexes enforced cell death ∼60, ∼80, and ∼95% at 100 µg/mL after 48 h of treatment, respectively. The optical images of cancerous cells treated with GO, GQDs, Cur, GO-Cur, and GQDs-Cur, at three different time intervals (0, 24, and 48 h), corroborated well with the results from the MTT assay in terms of the percentage of dead cells. The fluorescence images show a successful delivery of Cur drug inside the cancerous cell. The possible mechanism of killing of the cancerous cell with the complexes GO-Cur and GQDs-Cur is discussed. Moreover, this study opens a window to determine the mechanism of killing the cancerous cell.

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.

Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein.

The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

Energy landscape analysis of native folding of the prion protein yields the diffusion constant, transition path time, and rates.

Protein folding is described conceptually in terms of diffusion over a configurational free-energy landscape, typically reduced to a one-dimensional profile along a reaction coordinate. In principle, kinetic properties can be predicted directly from the landscape profile using Kramers theory for diffusive barrier crossing, including the folding rates and the transition time for crossing the barrier. Landscape theory has been widely applied to interpret the time scales for protein conformational dynamics, but protein folding rates and transition times have not been calculated directly from experimentally measured free-energy profiles. We characterized the energy landscape for native folding of the prion protein using force spectroscopy, measuring the change in extension of a single protein molecule at high resolution as it unfolded/refolded under tension. Key parameters describing the landscape profile were first recovered from the distributions of unfolding and refolding forces, allowing the diffusion constant for barrier crossing and the transition path time across the barrier to be calculated. The full landscape profile was then reconstructed from force-extension curves, revealing a double-well potential with an extended, partially unfolded transition state. The barrier height and position were consistent with the previous results. Finally, Kramers theory was used to predict the folding rates from the landscape profile, recovering the values observed experimentally both under tension and at zero force in ensemble experiments. These results demonstrate how advances in single-molecule theory and experiment are harnessing the power of landscape formalisms to describe quantitatively the mechanics of folding.

Phthalocyanine tetrasulfonates bind to multiple sites on natively-folded prion protein.

The phthalocyanine tetrasulfonates (PcTS), a class of cyclic tetrapyrroles, bind to the mammalian prion protein, PrP. Remarkably, they can act as anti-scrapie agents to prevent the formation and spread of infectious, misfolded PrP. While the effects of phthalocyanines on the diseased state have been investigated, the interaction between PcTS and PrP has not yet been extensively characterized. Here we use multiple, complementary assays (surface plasmon resonance, isothermal titration calorimetry, fluorescence correlation spectroscopy, and tryptophan fluorescence quenching) to characterize the binding of PcTS to natively-folded hamster PrP(90-232), in order to determine binding constants, ligand stoichiometry, influence of buffer ionic strength, and the effects of chelated metal ions. We found that binding strength depends strongly on chelated metal ions, with Al(3+)-PcTS binding the weakest and free-base PcTS the strongest of the three types tested (Al(3+), Zn(2+), and free-base). Buffer ionic strength also affected the binding, with K(d) increasing along with salt concentration. The binding isotherms indicated the presence of at least two different binding sites with micromolar affinities and a total stoichiometry of ~4-5 PcTS molecules per PrP molecule.

Direct observation of multiple misfolding pathways in a single prion protein molecule.

Protein misfolding is a ubiquitous phenomenon associated with a wide range of diseases. Single-molecule approaches offer a powerful tool for deciphering the mechanisms of misfolding by measuring the conformational fluctuations of a protein with high sensitivity. We applied single-molecule force spectroscopy to observe directly the misfolding of the prion protein PrP, a protein notable for having an infectious misfolded state that is able to propagate by recruiting natively folded PrP. By measuring folding trajectories of single PrP molecules held under tension in a high-resolution optical trap, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. Instead, frequent but fleeting transitions were observed into off-pathway intermediates. Three different misfolding pathways were detected, all starting from the unfolded state. Remarkably, the misfolding rate was even higher than the rate for native folding. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. These measurements of individual misfolding trajectories demonstrate the power of single-molecule approaches for characterizing misfolding directly by mapping out nonnative folding pathways.