Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion on the millisecond timescale.

Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a 'visible' major conformer exchanges with two 'invisible' minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate-fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide 15N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although 15N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, 15N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of 15N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale ( k e x , F I 1 ~ 550 s-1, k e x , F I 2 ~ 1200 s-1, k e x , I 1 I 2 ~ 5000 s-1), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15 °C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.

Dependence of Exchange Bias on Interparticle Interactions in Co/CoO Core/Shell Nanostructures.

This article reports the dependence of exchange bias (EB) effect on interparticle interactions in nanocrystalline Co/CoO core/shell structures, synthesized using the conventional sol-gel technique. Analysis via powder X-Ray diffraction (PXRD) studies and transmission electron microscope (TEM) images confirm the presence of crystalline phases of core/shell Co/CoO with average particle size ≈ 18 nm. Volume fraction (φ) is varied (from 20% to 1%) by the introduction of a stoichiometric amount of non-magnetic amorphous silica matrix (SiO2) which leads to a change in interparticle interaction (separation). The influence of exchange and dipolar interactions on the EB effect, caused by the variation in interparticle interaction (separation) is studied for a series of Co/CoO core/shell nanoparticle systems. Studies of thermal variation of magnetization (M-T) and magnetic hysteresis loops (M-H) for the series point towards strong dependence of magnetic properties on dipolar interaction in concentrated assemblies whereas individual nanoparticle response is dominant in isolated nanoparticle systems. The analysis of the EB effect reveals a monotonic increase of coercivity (HC) and EB field (HE) with increasing volume fraction. When the nanoparticles are close enough and the interparticle interaction is significant, collective behavior leads to an increase in the effective antiferromagnetic (AFM) CoO shell thickness which results in high HC and HE. Moreover, in concentrated assemblies, the dipolar field superposes to the local exchange field and enhances the EB effect contributing as an additional source of unidirectional anisotropy.

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.

The A39G FF domain folds on a volcano-shaped free energy surface via separate pathways.

Conformational dynamics play critical roles in protein folding, misfolding, function, misfunction, and aggregation. While detecting and studying the different conformational states populated by protein molecules on their free energy surfaces (FESs) remain a challenge, NMR spectroscopy has emerged as an invaluable experimental tool to explore the FES of a protein, as conformational dynamics can be probed at atomic resolution over a wide range of timescales. Here, we use chemical exchange saturation transfer (CEST) to detect "invisible" minor states on the energy landscape of the A39G mutant FF domain that exhibited "two-state" folding kinetics in traditional experiments. Although CEST has mostly been limited to studies of processes with rates between ∼5 to 300 s-1 involving sparse states with populations as low as ∼1%, we show that the line broadening that is often associated with minor state dips in CEST profiles can be exploited to inform on additional conformers, with lifetimes an order of magnitude shorter and populations close to 10-fold smaller than what typically is characterized. Our analysis of CEST profiles that exploits the minor state linewidths of the 71-residue A39G FF domain establishes a folding mechanism that can be described in terms of a four-state exchange process between interconverting states spanning over two orders of magnitude in timescale from ∼100 to ∼15,000 µs. A similar folding scheme is established for the wild-type domain as well. The study shows that the folding of this small domain proceeds through a pair of sparse, partially structured intermediates via two discrete pathways on a volcano-shaped FES.

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.

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.

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.