pH and alcohol induced structural transition in Ntf2 a nuclear transport factor of Saccharomyces cerevisiae.

Ntf2 is a nuclear envelope protein, which play a pivotal role in nucleocytoplasmic transport and mediates the nuclear import of RanGDP. It interacts with various nucleoporins along with Ran-GDP and part of a multicomponent system that assembles at the nuclear pore complex (NCP) during nuclear import. Here, we have described the biophysical characterization of Ntf2 from Saccharomyces cerevisiae. Recombinant Ntf2 showed increment in the ß-sheet content as well as decrement in the α-helix content from pH-7.0 to pH-4.0. A subsequent decrease in the pH led to increment in the α-helical content along with decrement in ß-sheet content. Intrinsic fluorescence studies demonstrated the unfolding of the protein below physiological pH. Ntf2 showed stabilization as well as phenomenal phase transition (ß sheet to α helix) by increase in alcohol concentration from 10% to 70%. Further increase in alcohol concentration (90%) resulted in residual secondary structure in Ntf2 protein. Presence of ammonium sulfate also stabilizes the secondary structure of Ntf2 protein. The structural characterization reveals the flexibility and the stability of Ntf2 at various conditions. These structural alterations in Ntf2 protein probably occurs in the course of nucleocytoplasmic transport when it interacts with other proteins moving towards its final destination.

An insight into structural plasticity and conformational transitions of transcriptional co-activator Sus1.

RNA biogenesis and mRNA transport are an intricate process for every eukaryotic cell. SAGA, a transcriptional coactivator and TREX-2 are the two major complexes participate in this process. Sus1 is a transcription export factor and part of both the SAGA and the TREX-2 complex. The competitive exchange of Sus1 molecule between SAGA and TREX-2 complex modulates their function which is credited to structural plasticity of Sus1. Here, we portray the biophysical characterization of Sus1 from S. cerevisiae. The recombinant Sus1 is a α-helical structure which is stable at various pH conditions. We reported the α-helix to ß-sheet transition at the low pH as well as at high pH. Sus1 showed 50% reduction in the fluorescence intensity at pH-2 as compared to native protein. The fluorescence studies demonstrated the unfolding of tertiary structure of the protein with variation in pH as compared to neutral pH. The same results were obtained in the ANS binding and acrylamide quenching studies. Similarly, the secondary structure of the Sus1 was found to be stable till 55% alcohol concentration while tertiary structure was stable up to 20% alcohol concentration. Further increase in the alcohol concentration destabilizes the secondary as well as tertiary structure. The 300 mM concentration of ammonium sulfate also stabilizes the secondary structure of the protein. The structural characterization of this protein is expected to unfold the process of the transportation of the mRNA with cooperation of different proteins.

Elucidating the inhibitory potential of Vitamin A against fibrillation and amyloid associated cytotoxicity.

Protein aggregation and amyloid fibrillation are associated with many serious human pathophysiologies like Alzheimer's, Parkinson's diseases, type II diabetes etc. A powerful strategy for controlling and understanding amyloid protein aggregation is the modulation of protein self-assembly. In this study, anti-fibrillation activity of vitamin A (VA) and its effect on the kinetics of amyloid formation of Aß-42 peptide was investigated by employing various spectroscopic, imaging and computational approaches. The present data of Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), dynamic light scattering assay, transmission electron microscopy and cell cytotoxicity assay demonstrated that vitamin A significantly inhibits fibril formation. Our experimental studies inferred that Vitamin A protects human neuroblastoma cell line (SH-SY5Y) and the neuroprotective effect against amyloid induced cytotoxicity is through modification of the amyloid formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin A interacts with Aß-42 through hydrophobic interactions as well as hydrogen bonding. Therefore, the study signifies the role of vitamin A as a potential molecule in preventing Aß-42 aggregation and associated pathophysiology. Hence, Vitamin A and related compounds can thus act as effective inhibitors in the therapeutic development to combat systemic amyloidosis.

Stabilizing proteins to prevent conformational changes required for amyloid fibril formation.

Amyloid fibrillation is associated with several human maladies, such as Alzheimer's, Parkinson's, Huntington's diseases, prions, amyotrophic lateral sclerosis, and type 2 diabetes diseases. Gaining insights into the mechanism of amyloid fibril formation and exploring novel approaches to fibrillation inhibition are crucial for preventing amyloid diseases. Here, we hypothesized that ligands capable of stabilizing the native state of query proteins might prevent protein unfolding, which, in turn, may reduce the propensity of proteins to form amyloid fibrils. We demonstrated the efficient inhibition of amyloid formation of the human serum albumin (HSA) (up to 85%) and human insulin (up to 80%) by a nonsteroidal anti-inflammatory drug, ibuprofen (IBFN). IBFN significantly increases the conformational stability of both HSA and insulin, as confirmed by differential scanning calorimetry (DSC). Moreover, increasing concentration of IBFN boosts its amyloid inhibitory propensity in a linear fashion by influencing the nucleation phase as assayed by thioflavin T fluorescence, transmission electron microscopy, and dynamic light scattering. Furthermore, circular dichroism analysis supported the DSC results, showing that IBFN binds to the native state of proteins and almost completely prevents their tendency to lose secondary and tertiary structures. Cell toxicity assay confirms that species formed in the presence of IBFN are less toxic to neuronal cells (SH-SY5Y). These results demonstrate the feasibility of using a small molecule to stabilize the native state of proteins, thereby preventing the amyloidogenic conformational changes, which appear to be the common link in several human amyloid diseases.

Capreomycin inhibits the initiation of amyloid fibrillation and suppresses amyloid induced cell toxicity.

Protein aggregation and amyloid fibrillation are responsible for several serious pathological conditions (like type II diabetes, Alzheimer's and Parkinson's diseases etc.) and protein drugs ineffectiveness. Therefore, a molecule that can inhibit the amyloid fibrillation and potentially clear amyloid fibrils is of great therapeutic value. In this manuscript, we investigated the antiamyloidogenic, fibril disaggregating, as well as cell protective effect of an anti-tuberculosis drug, Capreomycin (CN). Aggregation kinetics data, as monitored by ThT fluorescence, inferred that CN retards the insulin amyloid fibrillation by primarily targeting the fibril elongation step with little effect on lag time. Increasing the dose of CN boosted its inhibitory potency. Strikingly, CN arrested the growth of fibrils when added during the elongation phase, and disaggregated mature insulin fibrils. Our Circular Dichroism (CD) results showed that, although CN is not able to maintain the alpha helical structure of protein during fibrillation, reduces the formation of beta sheet rich structure. Furthermore, Dynamic Light Scattering (DLS) and Transmission Electronic Microscopy (TEM) analysis confirmed that CN treated samples exhibited different size distribution and morphology, respectively. In addition, molecular docking results revealed that CN interacts with insulin through hydrophobic interactions as well as hydrogen bonding, and the Hemolytic assay confirmed the non-hemolytic activity of CN on human RBCs. For future research, this study may assist in the rational designing of molecules against amyloid formation.

Investigating the site selective binding of busulfan to human serum albumin: Biophysical and molecular docking approaches.

We have studied the binding of busulfan (BN) to human serum albumin (HSA) at physiological pH 7.4 by using fluorescence, UV-vis and circular dichroism (CD) spectroscopic tools, as well as dynamic light scattering (DLS) measurements and molecular simulation approaches. HSA fluorescence quenching experiments showed that BN reduces the HSA native fluorescence intensity through the static mechanism. In addition, a single binding site on the HSA is occupied by BN with a binding constant at 298K of 1.84×103M-1. The enthalpy change (ΔH) and entropy change (ΔS) of BN-HSA interaction were calculated as -1.40kcalmol-1 and +10.14calmol-1K-1 respectively, which suggest the possible interaction mode as hydrophobic and hydrogen bonding. Moreover, the secondary structure alteration of HSA following its complexation with BN was studied and showed that α-helical content of HSA gets increased on interacting with BN. Ligand binding site to HSA was further investigated by site-specific markers in fluorescence measurements as well molecular modeling approach which indicated that BN bind to the nearby sudlow site II of HSA through hydrophobic as well as hydrogen bonding interaction. The present study will be helpful for understanding the binding mechanism of BN to human serum albumin.

Characterization of CNL like protein fragment (CNL-LPF) from mature Lageneria siceraria seeds.

Coiled coil domain-nucleotide binding site-leucine rich repeat (CC-NBS-LRR; CNL) proteins are highly conserved family of plant disease resistance proteins, remarkably comprise of coiled-coil domain, which plays significant role in plant innate immunity. The present study reports that moderately elicited oligomerization of plant CNL like protein fragment (CNL-LPF) in presence of ATP/Mg using various biophysical methods Circular dichroism (CD) results depicted a substantial increase in ß-sheet structure content of CNL-LPF. ATP/Mg induced conformational change in protein was observed by increase in blue shift with extrinsic fluorescence measurement, which indicates the exposure of hydrophobic regions of CNL-LPF and leads to self-association i.e. oligomerization. Likewise, cluster of protein oligomer and alteration in protein surface morphology were observed in presence of ATP/Mg by Transmission electron microscopy (TEM) and Atomic force microscopy (AFM), respectively. Also, augmented antiproliferation of HT1376 cells (urinary bladder cancer cell lines) was observed by CNL-LPF in presence of ATP/Mg. In conclusion, the current study illustrates that extent of CNL-LPF oligomerization was enhanced in presence of ATP/Mg (as compared to its absence). Utilization of enhanced oligomerization property of CNL-LPF as an anti-proliferative agent needs more assessment.

Probing the interaction of cephalosporin antibiotic-ceftazidime with human serum albumin: A biophysical investigation.

The fate of drug administered to a living organism depends on drug's pharmacokinetics as well as pharmacological behavior. Serum albumins (proteins in blood plasma of human) act as a carrier molecule to deliver the drug at specific site. In the present study, we have explored the mechanism of interaction between cephalosporin antibiotic-ceftazidime (CFD) and human serum albumin (HSA) by spectroscopic and molecular docking studies. Quenching of HSA fluorescence by CFD inferred that it binds to HSA through static quenching mechanism; with binding affinity in order of 104M-1. Fluorescence resonance energy transfer (FRET) results shows that donor and acceptor molecule are at 2.08nm apart and also reflects the high probability of energy transfer between HSA and CFD. Change in secondary structure as well as microenvironment around both tryptophan and tyrosine residue, were monitored by Circular Dichroism (CD) and Synchronous fluorescence spectroscopy respectively; confirms that CFD increases the alpha helical secondary structure as well as altered the environment around tryptophan and tyrosine. The specific binding site of CFD on HSA was determined by site-specific markers and molecular docking methods. CFD preferably bind to subdomain IIIA (Sudlow site II) on HSA.

Ascorbic acid inhibits human insulin aggregation and protects against amyloid induced cytotoxicity.

Protein aggregation into oligomers and fibrils are associated with many human pathophysiologies. Compounds that modulate protein aggregation and interact with preformed fibrils and convert them to less toxic species, expect to serve as promising drug candidates and aid to the drug development efforts against aggregation diseases. In present study, the kinetics of amyloid fibril formation by human insulin (HI) and the anti-amyloidogenic activity of ascorbic acid (AA) were investigated by employing various spectroscopic, imaging and computational approaches. We demonstrate that ascorbic acid significantly inhibits the fibrillation of HI in a dose-dependent manner. Interestingly ascorbic acid destabilise the preformed amyloid fibrils and protects human neuroblastoma cell line (SH- SY5Y) against amyloid induced cytotoxicity. The present data signifies the role of ascorbic acid that can serve as potential molecule in preventing human insulin aggregation and associated pathophysiologies.

Vitamin B12 offers neuronal cell protection by inhibiting Aß-42 amyloid fibrillation.

Protein misfolding and aggregation has been implicated as the cause of more than 20 diseases in humans such as Alzheimer's and Parkinson's and systemic amyloidosis. Retardation of Aß- 42 aggregation is considered as a promising and challenging strategy for developing effective therapeutics against Alzheimer's disease. Herein, we demonstrated the effect of vitamin B12 (VB) on inhibiting amyloid formation by employing ThT fluorescence assay, circular dichroism, ANS fluorescence assay, dynamic light scattering measurements and transmission electron microscopy and cell viability assay. Our results demonstrate that vitamin B12 (VB), inhibits Aß- 42 aggregation in a concentration dependent manner. Further VB also provide protection against amyloid induced cytotoxicity in human neuronal cell line. This study points towards a promising strategy to combat Aß- 42 aggregation and may have broader implication for targeting other neurological disorders whose distinct hallmark is also amyloid formation.

Biophysical insights into the interaction of hen egg white lysozyme with therapeutic dye clofazimine: modulation of activity and SDS induced aggregation of model protein.

The present study details the binding process of clofazimine to hen egg white lysozyme (HEWL) using spectroscopy, dynamic light scattering, transmission electron microscopy (TEM), and molecular docking techniques. Clofazimine binds to the protein with binding constant (Kb) in the order of 1.57 × 104 at 298 K. Binding process is spontaneous and exothermic. Molecular docking results suggested the involvement of hydrogen bonding and hydrophobic interactions in the binding process. Bacterial cell lytic activity in the presence of clofazimine increased to more than 40% of the value obtained with HEWL only. Interaction of the drug with HEWL induced ordered secondary structure in the protein and molecular compaction. Clofazimine also effectively inhibited the sodium dodecyl sulfate (SDS) induced amyloid formation in HEWL and caused disaggregation of preformed fibrils, reinforcing the notion that there is involvement of hydrophobic interactions and hydrogen bonding in the binding process of clofazimine with HEWL and clofazimine destabilizes the mature fibrils. Further, TEM images confirmed that fibrillar species were absent in the samples where amyloid induction was performed in the presence of clofazimine. As clofazimine is a drug less explored for the inhibition of fibril formation of the proteins, this study reports the inhibition of SDS-induced amyloid formation of HEWL by clofazimine, which will help in the development of clofazimine-related molecules for the treatment of amyloidosis.

Mechanisms of protein aggregation and inhibition.

Protein and peptide aggregation raises keen interest due to their involvement in number of pathological conditions ranging from neurodegenerative disorders to systemic amyloidosis. Here, we have reviewed recent advances in mechanisms of aggregation, emerging technologies towards exploration, characterization of aggregate structures, detection at molecular level and the strategies to combat the phenomenon of aggregation both in cellular and in vitro conditions. In consistence, we have illustrated almost all factors that influence the protein aggregation both in vitro and in vivo environments. In addition, we have discussed a detailed journey of protein aggregation phenomenon that starts with very first events of protein aggregation. We had also described advancement in current scenarios, present aspects of fibril association to several life threatening disorders and current experimental strategies that are employed to oppose or reverse the amyloid formation.

Attenuation of amyloid fibrillation in presence of Warfarin: A biophysical investigation.

Protein misfolding and aggregation are associated with more than twenty diseases, such as neurodegenerative diseases. The amyloid oligomers and fibrils may induce cell membrane disruption and lead to cell apoptosis. A great number of studies have focused on discovery of amyloid inhibitors which may prevent or treat amyloidosis. In this study, we used human serum albumin (HSA) as an amyloid model to test the anti-amyloid effects of warfarin (WFN), a very well-known drug for treatment of thrombosis and also used by biophysicists to characterize the specific binding site on HSA (site I of subdomain IIA). We have used a combination of different biophysical, spectroscopic and imaging techniques to prove the anti-amyloidogenic behavior of WFN. Our results demonstrated that WFN is capable enough to inhibit the HSA fibrillation. Exposed HSA surface hydrophobicity was decreased by 50% as judged by ANS analysis. Moreover, anti-amyloidegenic behavior of WFN was found to be concentration dependent as supported by decreased ThT fluorescence by 22.4% and 46% at WFN concentrations of 500 and 1000µM, respectively. Circular dichroism technique showed the change in secondary structure of native HSA as well as in presence of WFN. These results suggests that WFN is capable of inhibiting amyloid aggregation, hence, WFN related compounds may thus be further explored for designing effective anti-amyloidosis compounds.

Anti-amyloidogenic behavior and interaction of Diallylsulfide with Human Serum Albumin.

In this work, binding of garlic component-Diallysulfide (DAS) with major human blood transport protein, Human Serum Albumin (HSA) and its anti- amyloidogenic behavior has been studied by utilizing various spectroscopic and molecular docking strategies. The HSA exhibit significant reduction in fluorescence intensity upon interaction with DAS. DAS quenches the fluorescence of HSA in concentration dependent manner with binding affinity of 1.14×103M-1. UV-visible spectroscopy results confirm the formation of DAS-HSA complex and secondary structure of HSA get stabilized upon complexation with DAS as observed by far UV CD spectroscopy and Differential Scanning Calorimetry. The topology of HSA in absence and presence of DAS was monitored through Dynamic Light Scattering (DLS) technique, inferred that protein becomes more compact in presence of DAS. Further, molecular docking study shows that DAS bind to the nearby site II in subdomain III of HSA. Moreover, effect of DAS was studied on HSA fibrillation process. ThT binding, ANS fluorescence assay, CD measurement, DLS and Transmission Electron Microscopy (TEM) results altogether confirm the anti-amyloidogenic property of DAS. This work will provide biophysical insight into the interaction of DAS with HSA and will help in designing more potential therapeutic strategies against protein aggregation by exploiting other related compounds.

Amyloidogenic behavior of different intermediate state of stem bromelain: A biophysical insight.

Stem bromelain, a cysteine proteases from Ananas comosus is a widely accepted therapeutic drug with broad medicinal application. It exists as intermediate states at pH 2.0 and 10.0, where it encountered in gastrointestinal tract during adsorption (acidic pH) and in gut epithelium (alkaline pH), respectively. In this study, we monitored the thermal aggregation/amyloid formation of SB at different pH intermediate states. Thermal treatment of stem bromelain at pH 10.0 favors the fibrillation in which the extent of aggregation increases with increase in protein concentration. However, no fibril formation in stem bromelain at pH 2.0 was found at all the concentration used at pH 10.0. The fibril formation was confirmed by various techniques such as turbidity measurements, Rayleigh light scattering, dye binding assays and far UV circular dichroism. The Dynamic light scattering confirmed the formation of aggregates by measuring the hydrodynamic radii pattern. Moreover, microscopic techniques were performed to analyze the morphology of fibrils. The aggregation behavior may be due to variation in number of charged amino acid residues. The less negative charge developed at pH 10.0 may be responsible for aggregation. This work helps to overcome the aggregation related problems of stem bromelain during formulations in pharmaceutical industry.

Peroxynitrite-induced structural perturbations in human IgG: A physicochemical study.

IgG is an important defence protein. To exhibit optimum function the molecule must maintain its native structure. Peroxynitrite is a potent oxidizing and nitrating agent produced in vivo under pathophysiological conditions. It can oxidize and/or nitrate various amino acids causing changes in the structure and function of proteins. Such proteins may be involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis. In the present work, peroxynitrite-induced structural changes in IgG have been studied by UV-visible, fluorescence, CD, FT-IR, DLS spectroscopy and DSC as well as by SDS-PAGE. Peroxynitrite-modified IgG exhibited hyperchromicity at 280 nm, quenching of tryptophan fluorescence, increase in ANS fluorescence, loss of ß-sheet, shift in the positions of amide I and amide II bands, appearance of new peak in FT-IR, attachment of nitro residues and increase in melting temperature, compared to native IgG. Furthermore, peroxynitrite-modified IgG exhibited an additional peak at 420 nm, quenching in tyrosine fluorescence and enhancement in dityrosine fluorescence compared to native IgG. Generation of nitrotyrosine, dityrosine and nitrotryptophan was also observed in peroxynitrite-modified IgG. Gross structural changes in IgG caused by peroxynitrite and observed in vitro may favour autoantibodies induction in vivo under similar conditions.

Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases.

Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer's, Parkinson's and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aß-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aß-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

Biophysical and molecular docking insight into interaction mechanism and thermal stability of human serum albumin isoforms with a semi-synthetic water-soluble camptothecin analog irinotecan hydrochloride.

In the present work, we have examined the binding parameters, thermodynamics, and stability of human serum albumin (HSA) isoforms at pH 7.4 and 9.0, using spectroscopic, calorimetric, and molecular docking methods in the presence of water-soluble camptothecin analog irinotecan hydrochloride (CPT-11). We observed that CPT-11 binds to HSA through a static quenching procedure of ground-state complex formation with N-isoform and B-isoform. Hydrogen bond and hydrophobic interactions are the major governing forces that participating in the formation of protein-drug complex. To determine the binding site of CPT-11 within HSA molecules, we also have performed molecular docking experiments. We explored the CPT-11-mediated stability and modulation of HSA by performing dynamic light scattering (DLS) and differential scanning calorimetry (DSC) experiments. DLS and DSC techniques are used to determine the size and the melting point (Tm) of HSA, which was decreased in the presence of CPT-11. Therefore, CPT-11 plays an important role in HSA stability and protein-ligand interactions. The present study provides valuable information in the field of pharmacokinetics, pharmaco-dynamics, and drug discovery.

Unraveling Comparative Anti-Amyloidogenic Behavior of Pyrazinamide and D-Cycloserine: A Mechanistic Biophysical Insight.

Amyloid fibril formation by proteins leads to variety of degenerative disorders called amyloidosis. While these disorders are topic of extensive research, effective treatments are still unavailable. Thus in present study, two anti-tuberculosis drugs, i.e., pyrazinamide (PYZ) and D-cycloserine (DCS), also known for treatment for Alzheimer's dementia, were checked for the anti-aggregation and anti-amyloidogenic ability on Aß-42 peptide and hen egg white lysozyme. Results demonstrated that both drugs inhibit the heat induced aggregation; however, PYZ was more potent and decelerated the nucleation phase as observed from various spectroscopic and microscopic techniques. Furthermore, pre-formed amyloid fibrils incubated with these drugs also increased the PC12/SH-SY5Y cell viability as compare to the amyloid fibrils alone; however, the increase was more pronounced for PYZ as confirmed by MTT assay. Additionally, molecular docking study suggested that the greater inhibitory potential of PYZ as compare to DCS may be due to strong binding affinity and more occupancy of hydrophobic patches of HEWL, which is known to form the core of the protein fibrils.