Deeper Insights into Mixed Crowding through Enzyme Activity, Dynamics, and Crowder Diffusion.

Given the fact that the cellular interior is crowded by many different kinds of macromolecules, it is important that in vitro studies be carried out in the presence of mixed crowder systems. In this regard, we have used binary crowders formed by the combination of some of the commonly used crowding agents, namely, Ficoll 70, Dextran 70, Dextran 40, and PEG 8000 (PEG 8), to study how these affect enzyme activity, dynamics, and crowder diffusion. The enzyme chosen is AK3L1, an isoform of adenylate kinase. To investigate its dynamics, we have carried out three single point mutations (A74C, A132C, and A209C) with the cysteine residues being labeled with a coumarin-based solvatochromic probe [CPM: (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin)]. Both enzyme activity and dynamics decreased in the binary mixtures as compared with the sum of the individual crowders, suggesting a reduction in excluded volume (in the mixture). To gain deeper insights into the binary mixtures, fluorescence correlation spectroscopy studies were carried out using fluorescein isothiocyanate-labeled Dextran 70 and tetramethylrhodamine-labeled AK3L1 as the diffusion probes. Diffusion in binary mixtures was observed to be much more constrained (relative to the sum of the individual crowders) for the labeled enzyme as compared to the labeled crowder showing different environments being faced by the two species. This was further confirmed during imaging of the phase-separated droplets formed in the binary mixtures having PEG as one of the crowding agents. The interior of these droplets was found to be rich in crowders and densely packed, as shown by confocal and digital holographic microscopy images, with the enzymes predominantly residing outside these droplets, that is, in the relatively less crowded regions. Taken together, our data provide important insights into various aspects of the simplest form of mixed crowding, that is, composed of just two components, and also hint at the enhanced complexity that the cellular interior presents toward having a detailed and comprehensive understanding of the same.

Bridging Soft Interaction and Excluded Volume in Crowded Milieu through Subtle Protein Dynamics.

The impact of macromolecular crowding on biological macromolecules has been elucidated through the excluded volume phenomenon and soft interactions. However, it has often been difficult to provide a clear demarcation between the two regions. Here, using temperature-dependent dynamics (local and global) of the multidomain protein human serum albumin (HSA) in the presence of commonly used synthetic crowders (Dextran 40, PEG 8, Ficoll 70, and Dextran 70), we have shown the presence of a transition that serves as a bridge between the soft and hard regimes. The bridging region is independent of the crowder identity and displays no apparent correlation with the critical overlap concentration of the polymeric crowding agents. Moreover, the dynamics of domains I and II and the protein gating motion respond differently, thereby bringing to the fore the asymmetry underlying the crowder influence on HSA. In addition, solvent-coupled and decoupled protein motions indicate the heterogeneity of the dynamic landscape in the crowded milieu. We also propose an intriguing correlation between protein stability and dynamics, with increased global stability being accompanied by eased local domain motion.

Towards the energy landscape of adenylate kinase in crowded milieu: Activity, conformation, structure and dynamics in sequence.

Enzyme function is governed by a complex network of conformational changes and internal dynamics, with the same getting more convoluted in the crowded cellular environment. Here, we have explored an intricate interplay amongst activity, structure, conformation, and dynamics of a multidomain enzyme, AK3L1 (UniProtKB: Q9UIJ7) in the crowded milieu. We have monitored changes in the enzyme landscape in response to the chemical denaturant, urea, under the influence of different concentrations of macromolecular crowders. Extensive experimental analyses using FRET-based domain displacement measurements, sub-nanosecond time scale local dynamics, and global structural changes, along with enzymatic activity studies, have been carried out to get deeper insights into the factors that may modulate the functional landscape of adenylate kinase (AK3L1). It was observed that AK3L1 gets activated at low urea concentrations, whereas higher urea concentrations unfold and thereby deactivate the enzyme. A sequential response of AK3L1 is observed towards external perturbation (urea) occurring through a series of well-defined steps. Incorporation of crowders not only shift the maximum activity of enzyme to a higher urea concentration, but also enhance domain compaction, as revealed by FRET studies. The modulation in enzyme activity and solvation dynamics acting as local response, precede global unfolding of the enzyme, indicating that the structural alterations around the active site are quite decoupled from the large amplitude global transitions.

Ocular protein optineurin shows reversibility from unfolded states and exhibits chaperone-like activity.

Optineurin (OPTN) is a multifunctional, ubiquitously expressed cytoplasmic protein, mutants of which are associated with primary open-angle glaucoma (POAG) and amyotrophic lateral sclerosis (ALS). The most abundant heat shock protein crystallin, known for its remarkable thermodynamic stability and chaperoning activity, allows ocular tissues to withstand stress. The presence of OPTN in ocular tissues is intriguing. Interestingly, OPTN also harbors heat shock elements in its promoter region. Sequence analysis of OPTN exhibits intrinsically disordered regions and nucleic acid binding domains. These properties hinted that OPTN might be endowed with sufficient thermodynamic stability and chaperoning activity. However, these attributes of OPTN have not yet been explored. Here, we studied these properties through thermal and chemical denaturation experiments and monitored the processes using CD, fluorimetry, differential scanning calorimetry, and dynamic light scattering. We found that upon heating, OPTN reversibly forms higher-order multimers. OPTN also displayed a chaperone-like function by reducing the thermal aggregation of bovine carbonic anhydrase. It regains its native secondary structure, RNA-binding property, and melting temperature (T m) after refolding from a thermally as well as chemically denatured state. From our data, we conclude that OPTN, with its unique ability to revert from the stress-mediated unfolded state and its unique chaperoning function, is a valuable protein of the ocular tissues.

Fluorescence by self-assembly: autofluorescent peptide vesicles and fibers.

A series of oxidized cysteinyl peptides ([P-Cys-X-OMe]2; P = Boc or H; X = Trp or Glu) showed vesicular and fibrillar assemblies. The anatomy of the self-assembled vesicles from the water-soluble cystine peptide [Cys-Trp-OMe]2 (1a) has been investigated by using various fluorescent probes such as ammonium 8-anilinonaphthalene-1-sulfonate, Nile Red and pyrene. The morphological characterization was carried out by fluorescence lifetime imaging microscopy (FLIM) and super resolution-structured illumination microscopy (SR-SIM) utilizing the autofluorescence of the vesicles stemming from the self-assembly. The self-assembled structures are also observed in solution as evident from the quantitative phase images obtained using a dual-mode digital holographic microscope (DHM) system. Present investigations show that the self-assembly is enthalpy- and entropy-driven in the aqueous medium. Based on the CD spectral studies, we proposed that 1a organizes into vesicles through the sequestration of indole units. We observed that the solutions of dipeptides 1a-b exhibit autofluorescence in the blue region upon excitation at a wavelength >350 nm. Detailed spectroscopic studies on the peptides lacking tryptophan 2a-b unequivocally showed that the autofluorescence stems exclusively from peptide aggregation. Our experimental results with appropriate controls revealed that the clustering of carbonyl chromophores is central to autofluorescence. Autofluorescence was also used to probe the vesicle formation without using any external fluorescent probe. To the best of our knowledge, this is the first report on autofluorescent vesicles formed by the spontaneous association of dipeptides. We also found that the vesicles formed by 1a can act as a host for guests like C60. The biocompatibility and biodegradability of these peptides along with the autofluorescent nature and guest binding ability of peptide-based vesicles offer numerous applications in the biomedical area.

Correlating the Local and Global Dynamics of an Enzyme in the Crowded Milieu.

Enzymes are dynamic biological macromolecules, with their catalytic function(s) being largely influenced by the changes in local fluctuations of amino acid side chains as well as global structural modulations that the enzyme undergoes. Such local and global motions can be highly affected inside the crowded physiological interior of the cell. Here, we have addressed the role of dynamic structural flexibility in affecting the activation energy barrier of a flexible multidomain enzyme adenylate kinase (AK3L1, UniProtKB: Q9UIJ7). Activation energy profiles of both local (at three different sites along the polypeptide backbone) and global dynamics of the enzyme have been monitored using solvation studies on the subnanosecond time scale and tryptophan quenching studies over the temperature range of 278-323 K, respectively, under crowded conditions (Ficoll 70, Dextran 40, Dextran 70, and PEG 8). This study not only provides the site-specific mapping of dynamics but reveals that the activation energies associated with these local motions undergo a significant decrease in the presence of macromolecular crowders, providing new insights into how crowding affects internal protein dynamics. The crowded scenario also aids in enhancing the coupling between the local and global motions of the enzyme. Moreover, select portions/regions of the enzyme when taken together can well mirror the overall dynamics of the biomolecule, showing possible energy hotspots along the polypeptide backbone.

Understanding enzyme behavior in a crowded scenario through modulation in activity, conformation and dynamics.

Macromolecular crowding, inside the physiological interior, modulates the energy landscape of biological macromolecules in multiple ways. Amongst these, enzymes occupy a special place and hence understanding the function of the same in the crowded interior is of utmost importance. In this study, we have investigated the manner in which the multidomain enzyme, AK3L1 (PDB ID: 1ZD8), an isoform of adenylate kinase, has its features affected in presence of commonly used crowders (PEG 8, Dextran 40, Dextran 70, and Ficoll 70). Michaelis Menten plots reveal that the crowders in general enhance the activity of the enzyme, with the Km and Vmax values showing significant variations. Ficoll 70, induced the maximum activity for AK3L1 at 100 g/L, beyond which the activity reduced. Ensemble FRET studies were performed to provide insights into the relative domain (LID and CORE) displacements in presence of the crowders. Solvation studies reveal that the protein matrix surrounding the probe CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin) gets restricted in presence of the crowders, with Ficoll 70 providing the maximum rigidity, the same being linked to the decrease in the activity of the enzyme. Through our multipronged approach, we have observed a distinct correlation between domain displacement, enzyme activity and associated dynamics. Thus, keeping in mind the complex nature of enzyme activity and the surrounding bath of dense soup that the biological entity remains immersed in, indeed more such approaches need to be undertaken to have a better grasp of the "enzymes in the crowd".

Influence of crowding agents on the dynamics of a multidomain protein in its denatured state: a solvation approach.

It is now well appreciated that the crowded intracellular environment significantly modulates an array of physiological processes including protein folding-unfolding, aggregation, and dynamics to name a few. In this work we have studied the dynamics of domain I of the protein human serum albumin (HSA) in its urea-induced denatured states, in the presence of a series of commonly used macromolecular crowding agents. HSA was labeled at Cys-34 (a free cysteine) in domain I with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN) to act as a solvation probe. In partially denatured states (2-6 M urea), lower crowder concentrations (~ < 125 g/L) induced faster dynamics, while the dynamics became slower beyond 150 g/L of crowders. We propose that this apparent switch in dynamics is an evidence of a crossover from soft (enthalpic) to hard-core (entropic) interactions between the protein and crowder molecules. That soft interactions are also important for the crowders used here was further confirmed by the appreciable shift in the wavelength of the emission maximum of BADAN, in particular for PEG8000 and Ficoll 70 at concentrations where the excluded volume effect is not dominant.

Exploring the potency of the naturally occurring polyphenol curcumin as a probe for protein aggregation in crowded environments.

Conventional dyes such as thioflavin T, ANS, and Congo Red etc. have mostly been used to monitor protein aggregation. In this work, we have tried the naturally occurring polyphenol, curcumin, for probing the aggregation of the serum protein bovine serum albumin (BSA) in crowded environments. The distinctive spectral profile of this polyphenol in response to varying aggregating scenario is indicative of its high sensitivity towards detection of aggregates. We have also gained possible insights into the heterogeneity of the aggregation mixture, that is, the different species that are present simultaneously at any instant of time by fitting curcumin's emission spectra to a sum of Gaussians in diverse aggregating conditions. SEM study reveals that the morphology of aggregates formed in presence of different crowding agents became independent of the nature of the crowder on prolonged incubation. To overcome the aqueous insolubility barrier of the molecular probe (curcumin), ß-cyclodextrin, which is known to complex with curcumin, was also added to the reaction mixture.

Mechanistic Insights into the Growth of Anisotropic Nanostructures Inside Reverse Micelles: A Solvation Perspective.

Reverse micelles (RMs) as soft templates have been successfully used in tailoring the structural characteristics (size and morphology) of nanomaterials that in turn have been used in various applications. In this work, we have focused on the local perturbations in the different interior domains of the cetyltrimethylammonium bromide-reverse micelle-based soft template en route to nanorod formation by monitoring the solvation response of coumarin-based solvatochromic probes (C343 and C153). We have observed an appreciable retardation of the solvent coordinate during the initial phases of nanorod growth, which we have attributed to the reorientational motion of the water molecules lodged in the interfacial region. Moreover, these rigid nanostructures leave their imprints on the soft interfacial layer as was observed from the direct correlation in the solvation response of RM-containing nanostructures and respective surfactant aggregates in supernatant solution. Supporting data from time-resolved anisotropy studies further reinforced our conclusions from the solvation experiments. Our study proves that the hydration dynamics can be a promising tool in tracking the heterogeneous growth evolution of nanostructure formation in RMs since solvent reorganization provides insights into the intrinsic, molecular-level features of the micellar assemblies.

Modulation of aggregation of silk fibroin by synergistic effect of the complex of curcumin and ß-cyclodextrin.

Amyloid aggregation has been associated with numerous human pathological diseases. A recent study has demonstrated that silk fibroin intermittently endorses amyloidogenesis in vivo. In the current study, we explored the propensity of silk fibroin to undergo amyloid-like aggregation and its prevention using an optimized concoction of curcumin with ß-cyclodextrin. Aggregation of silk fibroin resulted in the formation of fibrils with a diameter of ~3.2 nm. However, addition of the optimized concentration of curcumin and ß-cyclodextrin to silk fibroin inhibited aggregation and preserved the random coil conformation even under aggregation inducing conditions, as demonstrated by CD and FTIR spectroscopy. Benzene rings of curcumin interact with the aromatic residues of fibroin via hydrophobic interactions. However, ß-cyclodextrin preferentially interacts with the non-polar residues, which are the core components for nucleation dependent protein aggregation. The present study demonstrates the ability of the concoction of curcumin and ß-cyclodextrin in tuning the self assembly process of fibroin. It also provides a platform to explore the assembly process of nano-fibril and hierarchical structures in vitro along with a novel insight for designing clinically relevant silk-based functional biomaterials.

Crowder induced structural modulation of a multi-domain protein during its early stages of aggregation: A FRET-based and protein solvation study.

Protein aggregation has been known for long to be the prime cause for several neurological disorders in human beings. While protein aggregation is itself a complex process, understanding the same in the context of a crowded cellular medium remains a challenge. In this work, using Förster resonance energy transfer (FRET) and solvation dynamics, we have tried to gain important insights into the structural rearrangements, during the early stages of aggregation of the multidomain protein bovine serum albumin (BSA) in presence of a range of synthetic macromolecular crowding agents. FRET studies show that there is an initial compaction in the domain size (domain I) at the early time points of incubation followed by an increase in the distance between the donor-acceptor pair. Analyses of the solvent correlation traces of BADAN (labeled at free Cys-34 in domain I of BSA) reveal that the same domain becomes rigid during the initial phase of the aggregation process subsequent to which there was a gradual increase in flexibility, the latter we propose being a necessary step that allows facile addition of more protein units.

Unravelling the Intricacy of the Crowded Environment through Tryptophan Quenching in Lysozyme.

Global conformational modulation of proteins in the presence of crowding agents is well-known. In this work, using the intrinsic tryptophan (Trp) residues of lysozyme, we have studied the effect of the crowding agents on the local conformation of this biomolecule. In presence of the macromolecular crowder Dextran 6, considerable quenching of tryptophan fluorescence was observed, which we have attributed to the enhanced proximity of the surrounding charged residues arising from local perturbation of protein structure. Accessibility of the Trp residues in the presence of crowders as a function of thermal and chemical denaturation has also been monitored using the traditional quenchers, acrylamide and iodide. Quenching in the presence of the crowding agents had to be modeled predominantly using the sphere-of-action model, with the sphere-of-volume "V" being postulated to be a signature of the cage-like environments that can exist in the solutions of such polymeric macromolecular crowding agents. Moreover, percolation of the quencher molecules through the entangled crowder systems was observed to be dependent on the micropolarity of the specific crowding agent being studied, with the neutral quencher acrylamide exhibiting maximum quenching in the presence of Dextran 6, while iodide being charged, exhibits higher quenching efficiency when Dextran 70 was the crowder. Additionally, control studies with the free amino acid tryptophan suggest that the variation in quenching so observed is not only due to the changes in the conformation of lysozyme and hence accessibility of the Trp residues but is also dictated by the underlying details and complexity of the crowder solutions to an appreciable extent.

Polyphenols in combination with ß-cyclodextrin can inhibit and disaggregate α-synuclein amyloids under cell mimicking conditions: A promising therapeutic alternative.

Parkinson's disease is characterized by the presence of insoluble and neurotoxic aggregates (amyloid fibrils) of an intrinsically disordered protein α-synuclein. In this study we have examined the effects of four naturally occurring polyphenols in combination with ß-cyclodextrin (ß-CD) on the aggregation of α-synuclein in the presence of macromolecular crowding agents. Our results reveal that even at sub-stoichiometric concentrations of the individual components, the polyphenol-ß-CD combination(s) not only inhibited the aggregation of the proteins but was also effective in disaggregating preformed fibrils. Curcumin was found to be the most efficient, followed by baicalein with (-)-epigallocatechin gallate and resveratrol coming in next, the latter two exhibiting very similar effects. Our results suggest that the efficiency of curcumin results from a balanced composition of the phenolic OH groups, benzene rings and flexibility. The latter ensures proper positioning of the functional groups to maximize the underlying interactions with both the monomeric form of α-synuclein and its aggregates. The uniqueness of ß-CD was reinforced by the observation that none of the other cyclodextrin variants [α-CD and HP-ß-CD] used was as effective, in spite of these possessing better water solubility. Moreover, the fact that the combinations remained effective under conditions of macromolecular crowding suggests that these have the potential to be developed into viable drug compositions in the near future. MTT assays on cell viability independently confirmed this hypothesis wherein these combinations (and the polyphenols alone too) appreciably impeded the toxicity of the prefibrillar α-synuclein aggregates on the mouse neuroblastoma cell lines (N2a cells).

Modulation of Self-Assembly Process of Fibroin: An Insight for Regulating the Conformation of Silk Biomaterials.

Controlling the mechanism of self-assembly in proteins has emerged as a potent tool for various biomedical applications. Silk fibroin self-assembly consists of gradual conformational transition from random coil to ß-sheet structure. In this work we elucidated the intermediate secondary conformation in the presence of Ca(2+) ions during fibroin self-assembly. The interaction of fibroin and calcium ions resulted in a predominantly α-helical intermediate conformation, which was maintained to certain extent even in the final conformation as illustrated by circular dichroism and attenuated total reflectance-Fourier transform infrared spectroscopy. Further, to elucidate the mechanism behind this interaction molecular modeling of the N-terminal region of fibroin with Ca(2+) ions was performed. Negatively charged glutamate and aspartate amino acids play a key role in the electrostatic interaction with positively charged calcium ions. Therefore, insights about modulation of self-assembly mechanism of fibroin could potentially be utilized to develop silk-based biomaterials consisting of the desired secondary conformation.

Unusual effects of crowders on heme retention in myoglobin.

Myoglobin (Mb) undergoes pronounced heme loss under denaturing conditions wherein the proximal histidine gets protonated. Our data show that macromolecular crowding agents (both synthetic and protein based) can appreciably influence the extent of heme retention in Mb. Interestingly, glucose and sucrose, the monomeric constituents of dextran and ficoll-based crowders were much more effective in preventing heme dissociation of Mb, albeit, at much higher concentrations. The protein crowders BSA and lysozyme show very interesting results with BSA bringing about the maximum heme retention amongst all the crowding agents used while lysozyme induced heme dissociation even in the native state of Mb. The stark difference that these protein crowders exhibit when interacting with the heme protein is a testament to the varied interaction potentials that a test protein might be exposed to in the physiological (crowded) milieu.

Do Macromolecular Crowding Agents Exert Only an Excluded Volume Effect? A Protein Solvation Study.

The effect of macromolecular crowding on protein structure and dynamics has mostly been explained on the basis of the excluded volume effect, its origin being entropic. In recent times a progressive shift in this view has been taking place with increasing emphasis on soft interactions that are enthalpic by nature. Using very low concentrations (1-10 g/L) of both synthetic (dextran- and poly(ethylene glycol) (PEG)-based) and protein (α-synuclein and myoglobin)-based crowders, we have shown that the solvation of probe molecule ANS (1-anilinonapthalene-8-sulfonate) bound to serum proteins bovine serum albumin (BSA) and human serum albumin (HSA) is significantly modulated in both a protein- and crowder-dependent fashion. Since under such conditions the effect of excluded volume is appreciably low, we propose that our observations are direct evidence of soft interactions between the macromolecular crowding agents used and the serum proteins. Moreover, our data reveal, that since at these low crowder concentrations major perturbations to the protein structure are unlikely to take place while minor perturbations might not be readily visible, protein solvation provides a unique spectral signature for capturing such local dynamics, thereby allowing one to decouple hard-sphere interactions from soft sphere ones. Furthermore, since fast fluctuations are known to play a major role in determining the functional characteristics of proteins and enzymes, our results suggest that such motions are prone to be modulated even when the cellular crowding conditions are quite relaxed. In other words, by the time the excluded volume effects come into the picture in the physiological milieu, modulations of functionally important protein motions that need a relatively lower activation energy have already taken place as a result of the aforementioned enthalpic (soft) interactions.

Controlling the Microstructure of Reverse Micelles and Their Templating Effect on Shaping Nanostructures.

Reverse micelles as nanoreactors have been most successful in designing nanostructures of different sizes and shapes. Nevertheless, important questions regarding the explicit roles of intrinsic parameters in modifying soft colloid templates which eventually give rise to variety of nanostructures are still unresolved. In this paper, we have focused on this challenging aspect of microemulsion based synthesis of nanostructures, i.e., how the tunable parameters like water to surfactant molar ratio, solvent properties, and surfactant structure modify the microstructure (size/shape) of reverse micelles (surfactant/cosurfactant/oil/water). Further, we have elucidated the correlation between these nanoreactors with the size and morphology of the evolving nanostructures within the aqueous core (using in situ studies) as well as the finally obtained nanostructures. We have employed fluorescence correlation spectroscopy (FCS), small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) to obtain details on the microstructural transformation of reverse micelles and their templating behavior on designing nanostructures, at (near) single droplet level and in an ensemble. The structure (size/shape) of nanoreactors, i.e., reverse micelles, finally guides the size and shape of nanostructures. As the water content increases, it induces the micellar growth and subsequently the growth of nanostructures develops linearly up to a critical value beyond which the finite bending modulus of surfactant film triggers the structural rearrangement of microemulsion droplets (MEDs) and the linear plot shows deviation. Bulkiness of the solvent molecules modulates the ME droplets, and MEDs encapsulates nanostructures by influencing the curvature and rigidity of the surfactant film and results in smaller dimensions of the micellar core, which leads to nanostructures with large aspect ratio. The origin of this structural evolution may be explained in terms of solvent molecular structure, which affects the penetrability of solvent molecules into the surfactant tail region. Interestingly, MEDs with cationic surfactants feature the onset of one-dimensional micellar growth and the shape evolves into a nearly prolate spheriod. Consequently, the growth of the micellar core and dynamical exchange in an anisotropic manner leads to the formation of nanorods. The implication of such studies could be far reaching due to the geometry-dependent novel properties and potential applications of anisotropic nanostructures.

Unusual domain movement in a multidomain protein in the presence of macromolecular crowders.

Domain movements play a fundamental and critical role in the specific biological function that multidomain proteins have evolved to perform. A significant amount of research has been carried out to investigate the effects of macromolecular crowding agents, mostly on single domain proteins, thereby furthering our appreciation for the crowding phenomenon. However similar studies on proteins having multiple domains are relatively scarce. Using the plasma protein human serum albumin (HSA) as the protein of interest, we have probed the influence of dextran based crowding agents (Dextran 6, Dextran 40, and Dextran 70) on the relative movements of domains I and II using FRET, with Trp-214 in domain II acting as the donor and acrylodan (Ac) covalently attached to Cys-34 of domain I as the acceptor. Amongst the higher molecular weight crowders, while both Dextran 70 and Dextran 40 induced a significant decrease in the distance between the aforesaid domains, however for the latter macromolecular crowder (Dextran 40), beyond 50 g L(-1), no change in domain separation was observed even up to concentrations of 175 g L(-1). On the other hand, contrary to our expectations, Dextran 6, having the highest packing density by virtue of it being the smallest crowding agent used, provided an asymmetric excluded volume which resulted in forced elongation of HSA along the Trp-Ac FRET axis. Additionally both chemical and thermal studies performed at varying concentrations of the chemical denaturant, urea, reveal unusual movements of the two domains, an aspect that can have important implications with regard to HSA being an avid transporter of fatty acids, with the binding of latter being known to invoke appreciable domain displacements. We hypothesise that we see a distinct crossover from entropy dominated depletion effects in the case of Dextran 6 to significant enthalpic contribution for Dextran 70 with Dextran 40 lying midway between these two crowders, having characteristics of both.

ß-cyclodextrin and curcumin, a potent cocktail for disaggregating and/or inhibiting amyloids: a case study with α-synuclein.

Aggregation of α-synuclein has been implicated in Parkinson's disease (PD). While many compounds are known to inhibit α-synuclein aggregation, dissolution of aggregates into their constituent monomers cannot be readily achieved. In this study, using a range of techniques, we have shown that an optimized cocktail of curcumin and ß-cyclodextrin, at appreciably low concentrations, not only inhibited aggregation but also broke up the preformed aggregates almost completely. We propose that these compounds exhibit synergy in their action and thus provide us with the exciting prospect of working toward the development of a suitable drug candidate for prevention and treatment of PD.