Basir Ahmad

Curcumin Prevents Aggregation in α-Synuclein by Increasing Reconfiguration Rate

Background: α-Synuclein is an aggregation-prone protein that reconfigures more slowly under aggregating conditions. Results: Curcumin binds to monomeric α-synuclein, prevents aggregation, and increases the reconfiguration rate, particularly at high temperatures. Conclusion: Curcumin rescues the protein from aggregation by making the protein more diffusive. Significance: The search for aggregation inhibitors should account for changes in chain dynamics by the small molecule. α-Synuclein is a protein that is intrinsically disordered in vitro and prone to aggregation, particularly at high temperatures. In this work, we examined the ability of curcumin, a compound found in turmeric, to prevent aggregation of the protein. We found strong binding of curcumin to α-synuclein in the hydrophobic non-amyloid-β component region and complete inhibition of oligomers or fibrils. We also found that the reconfiguration rate within the unfolded protein was significantly increased at high temperatures. We conclude that α-synuclein is prone to aggregation because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs. Curcumin rescues the protein from aggregation by increasing the reconfiguration rate into a faster regime.

Interactions of thioflavin T with serum albumins: Spectroscopic analyses

The interaction of thioflavin T (ThT) with serum albumins from four different mammalian species i.e. human, bovine, porcine and rabbit, has been investigated by circular dichroism (CD), fluorescence spectroscopy and ITC. The binding constant (K) for HSA was found to be 9.9 x 10(4)M(-1), 4.3 x 10(4)M(-1) for RSA, 1.07 x 10(4)M(-1) for PSA and 0.3 x 10(4)M(-1) for BSA and the number of binding sites (n) were 1.14, 1.06, 0.94 and 0.8, respectively, which is very significant. By using unfolding pathway of HSA in the presence of urea, domain II of HSA has been assigned to possess binding site of ThT. Its binding constant is comparable to many drugs that bind at domain II of HSA, like salicylate, warfarin, digitoxin, etc. Acting force between HSA and ThT is showing that both hydrophobic and electrostatic forces have contributed for the interaction. DeltaG(binding), DeltaH and DeltaS were calculated to be -28.46 kJ mol(-1), -3.50 kJ mol(-1) and 81.04 JK(-1)mol(-1), respectively. The data described here will help to increase our understanding about the interaction of ThT with native proteins. The results also indicate that care must be taken while using ThT as a probe for detecting amyloid fibrils.

Aggregation of α-synuclein is kinetically controlled by intramolecular diffusion

We hypothesize that the first step of aggregation of disordered proteins, such as α-synuclein, is controlled by the rate of backbone reconfiguration. When reconfiguration is fast, bimolecular association is not stable, but as reconfiguration slows, association is more stable and subsequent aggregation is faster. To investigate this hypothesis, we have measured the rate of intramolecular diffusion in α-synuclein, a protein involved in Parkinson’s disease, under solvent conditions that accelerate or decelerate aggregation. Using the method of tryptophan-cysteine (Trp-Cys) quenching, the rate of intramolecular contact is measured in four different loops along the chain length. This intrinsically disordered protein is highly diffusive at low temperature at neutral pH, when aggregation is slow, and compacts and diffuses more slowly at high temperature or low pH, when aggregation is rapid. Diffusion also slows with the disease mutation A30P. This work provides unique insights into the earliest steps of α-synuclein aggregation pathway and should provide the basis for the development of drugs that can prevent aggregation at the initial stage.

Pollutant-Induced Modulation in Conformation and β-Lactamase Activity of Human Serum Albumin

Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.

Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters

Interaction of small molecule inhibitors with protein aggregates has been studied extensively, but how these inhibitors modulate aggregation kinetic parameters is little understood. In this work, we investigated the ability of two potential aggregation inhibiting drugs, curcumin and kaempferol, to control the kinetic parameters of aggregation reaction. Using thioflavin T fluorescence and static light scattering, the kinetic parameters such as amplitude, elongation rate constant and lag time of guanidine hydrochloride-induced aggregation reactions of hen egg white lysozyme were studied. We observed a contrasting effect of inhibitors on the kinetic parameters when aggregation reactions were measured by these two probes. The interactions of these inhibitors with hen egg white lysozyme were investigated using fluorescence quench titration method and molecular dynamics simulations coupled with binding free energy calculations. We conclude that both the inhibitors prolong nucleation of amyloid aggregation through binding to region of the protein which is known to form the core of the protein fibril, but once the nucleus is formed the rate of elongation is not affected by the inhibitors. This work would provide insight into the mechanism of aggregation inhibition by these potential drug molecules.

Alkali-Induced Conformational Transition in Different Domains of Bovine Serum Albumin

Alkaline pH induced conformational changes in different domains of bovine serum albumin were studied by using domain specific ligands: chloroform, bilirubin and diazepam for domains I, II and III respectively. The effect of alkaline pH on the secondary structure of BSA was monitored by far-UV CD in the range 250 nm to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step change, one corresponding to N<-->B transition (pH 7.5 to 9.0) and the other to B --> U (pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0 and decreased thereafter reaching a value similar to one obtained with native BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH 9.5 to 11.0) suggested major loss of native conformation of domain I followed by domain III during alkaline induced unfolding of BSA. However, a significant increase in bilirubin binding showed a favorable conformational rearrangement in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete abolishment of bilirubin binding to BSA and significant loss of secondary structure around pH 12.5 indicated that domain II was more resistant to alkaline pH and unfolds only at extreme alkalinity. Taken together, these data suggest that unfolding of three domains of BSA follow the following order of susceptibility towards alkaline denaturation of BSA domain I>domain III>domain II.

Salt-induced refolding in different domains of partially folded bovine serum albumin

In our earlier communication on urea denaturation of bovine serum albumin (BSA), we showed significant unfolding of domain III along with domain I prior to intermediate formation around 4.6-5.2 M urea based on the binding results of domain specific ligands:chloroform, bilirubin and diazepam for domains I, II and III, respectively. Here, we present our results on the salt-induced refolding of the two partially folded states of BSA obtained at 4.5 M urea and at pH 3.5, respectively. Both these states were characterized by significant unfolding of both domains I and III as indicated by decreased binding of chloroform and diazepam, respectively. Salt-induced stabilization of partially folded states of BSA was accompanied by nearly complete refolding of both domains I and III as the binding isotherms of chloroform and diazepam obtained in presence of approximately 1.0 M KCl were nearly identical to that obtained with native BSA at pH 7.4. From these observations, it can be concluded that the anion binding sites on serum albumin are not only confined to domain III (C-terminal region) but few sites are also present on domain I (or N-terminal region) of the protein.

2,2,2-Trifluroethanol induces simultaneous increase in α-helicity and aggregation in alkaline unfolded state of bovine serum albumin

Little work has been done to understand the folding of proteins at alkaline conditions. BSA acquires a partially reversible unfolded state at pH 13.0, devoid of any native structure. Introduction of methanol, ethanol and 2-propanol with the alkaline unfolded protein resulted in beta-sheet-like structure formation, and 2,2,2-trifluroethanol found to enhance alpha-helical conformations with simultaneous increase in aggregation. The extent of secondary and tertiary structure formation is in the order of methanol < ethanol < 2-propanol < 2,2,2-trifluroethanol. Exposure of hydrophobic core of protein molecules in apolar environment of 2,2,2-trifluroethanol seems to promote intermolecular cluster formation. This is one of the very few reports that alpha-helical structures can also aggregate.

Searching for conditions to form stable protein oligomers with amyloid-like characteristics: The unexplored basic pH

Conversion of peptides and proteins from their native states into amyloid fibrillar aggregates is the hallmark of a number of pathological conditions, including Alzheimers disease and amyloidosis. Evidence is accumulating that soluble oligomers, as opposed to mature fibrils, mediate cellular dysfunction, ultimately leading to disease onset. In this study, we have explored the ability of alkaline pH solutions, which have remained relatively unexplored so far, to form a partially folded state of the N-terminal domain of the Escherichia coli protein HypF (HypF-N), which subsequently assembles to form stable soluble oligomers. Results showed that HypF-N unfolds at high pH via a two-state process. Characterization of the resulting alkaline-unfolded state by near- and far-UV circular dichroism, intrinsic and ANS-derived fluorescence and DLS indicated characteristics of a monomeric, premolten globule state. Interestingly, alkaline-unfolded HypF-N aggregates, at high concentration in the presence of low concentrations of TFE, into stable oligomers. These are able to bind amyloid-specific dyes, such as Congo red, ThT, and ANS, contain extensive beta-sheet structure, as detected with far-UV circular dichroism, and have a height of 2.0-3.9 nm when analysed using atomic force microscopy. This study, which complements our previous one in which morphologically, structurally, and tinctorially similar oligomers were formed at low and nearly neutral pH values by the same protein, offers opportunities to explore the fine differences existing in the mechanism of formation of these species under different conditions, in their precise molecular structure and in their ability to cause cellular dysfunction.

Thermal induced unfolding of human serum albumin isomers: assigning residual α helices to domain II.

In this study we have investigated the heat induced denaturation of HSA by utilizing spectroscopic approaches including fluorescence and circular dichroism. Thermal denaturation of N isomer (domains I-III remain intact), B isomer (loss of helical structure of interdomain contacts) and I state (domain II intact) was found to be co-operative processes while for F isomer domains unfold non-cooperatively. These finding pointed out that during N-F transition, HSA suffers more structural alterations which are not localized only to domain III. Loss of secondary structure in the temperature range 20-60 °C without effecting tertiary structure of N isomer of HSA is mainly due to loss in helical extensions connecting domain I to II and domain II to III. All the four thermally denatured states (60-96 °C) of HSA retained approximately 50% residual α-helical structures. Near-UV spectroscopy used as a probe for tertiary structure indicated that heat denatured states lost almost all of the tertiary contacts thereby forming molten globule like states. Furthermore, our results provide evidence that residual helical structures are mainly located in domain II.