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Communicated by Ramaswamy H. Sarma.
Assuntos
Antineoplásicos/química , Flavanonas/química , Muramidase/química , Neoplasias/tratamento farmacológico , Fenômenos Biofísicos , Dicroísmo Circular , Humanos , Muramidase/antagonistas & inibidores , Ligação Proteica/efeitos dos fármacos , Espectrometria de FluorescênciaRESUMO
Inhibition of fibrillation process and disaggregation of mature fibrils using small peptide are the promising remedial strategies to combat neurodegenerative diseases. However, designing peptide-based drugs to target ß-sheet-rich amyloid has been a major challenge. The current work describes, for the first time, the amyloid inhibitory potential of the two short peptides (selected on the basis of predisposition of their amino acid residues toward ß-sheet formation) using combination of biophysical, imaging methods, and docking approaches. Results showed that peptides employed different mechanisms to inhibit the amyloid fibrillation. Furthermore, they were also effective in blocking the amyloid fibrillation pathway. In contrary to the insulin fibrillar mesh, significantly less fibrillar species appeared in the presence of peptides, as confirmed by transmission electron microscopy. Circular dichroism analysis indicated that although peptides did not stabilize the native state of insulin, they inhibited amyloid aggregation by reducing the formation of ß-sheet rich structures. Hemolytic assay revealed the non-hemolytic nature of the species formed when insulin was co-incubated with the peptides. Therefore, despite the inherent potential to form ß-sheet structure, these peptides inhibited the amyloid formation and potentially can be used as therapeutics for the treatment of amyloid-related diseases.
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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.
Assuntos
Amiloide/química , Capreomicina/química , Insulina/química , Simulação de Acoplamento Molecular , Agregados Proteicos , Amiloide/ultraestrutura , Animais , Capreomicina/farmacologia , Bovinos , HumanosRESUMO
Protein misfolding and aggregation lead to amyloid generation that in turn may induce cell membrane disruption and leads to cell apoptosis. In an effort to prevent or treat amyloidogenesis, large number of studies has been paying attention on breakthrough of amyloid inhibitors. In the present work, we aim to access the effect of two drugs, that is, acetylsalicylic acid and 5-amino salicylic acid on insulin amyloids by using various biophysical, imaging, cell viability assay, and computational approaches. We established that both drugs reduce the turbidity, light scattering and fluorescence intensity of amyloid indicator dye thioflavin T. Premixing of drugs with insulin inhibited the nucleation phase and inhibitory potential was boosted by increasing the concentration of the drug. Moreover, addition of drugs at the studied concentrations attenuated the insulin fibril induced cytotoxicity in breast cancer cell line MDA-MB-231. Our results highlight the amino group of salicylic acid exhibited enhanced inhibitory effects on insulin fibrillation in comparison to acetyl group. It may be due to presence of amino group that helps it to prolong the nucleation phase with strong binding as well as disruption of aromatic and hydrophobic stacking that plays a key role in amyloid progression.
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Amiloide , Insulina , Mesalamina/química , Ácido Salicílico/química , Amiloide/química , Amiloide/farmacologia , Animais , Bovinos , Linhagem Celular Tumoral , Humanos , Insulina/química , Insulina/farmacologiaRESUMO
Increasing evidence proposed that amyloid deposition by proteins play a crucial role in an array of neurotoxic and degenerative disorders like Parkinson's disease, systemic amyloidosis etc, that could be controlled by anti-aggregation methodologies which either inhibit or disaggregate such toxic aggregates. The present work targets the amyloid inhibiting and disaggregating potential of promethazine (PRM) against human insulin (HI) and human lysozyme (HL) fibrillogenesis. Biophysical techniques like Rayleigh scattering measurements (RLS), Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, circular dichroism (CD) and dynamic light scattering (DLS) measurements illustrated the inhibitory action of PRM. The half maximal inhibitory concentration (IC50) of PRM for HI and HL was estimated to be 114.81±1.21µM and 186.20±1.03µM, respectively. Microscopic techniques revealed the absence of fibrillar structures when HI and HL was co-incubated with PRM. Cytoprotective behavior of PRM was investigated by cell based cytotoxicity assay performed on SH-SY5Y neuronal cell lines. The half maximal disaggregation concentration (DC50) was calculated as 21.37±0.89µM and 45.70±0.76µM, signifying that PRM is much potent to disaggregate pre formed fibrils rather than to inhibit fibrillation. Thus, PRM could be beneficial as therapeutic agent that can aid in the cure of amyloid related diseases.
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Amiloide/efeitos dos fármacos , Amiloidose/tratamento farmacológico , Prometazina/farmacologia , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/tratamento farmacológico , Amiloide/química , Proteínas Amiloidogênicas/antagonistas & inibidores , Proteínas Amiloidogênicas/química , Amiloidose/patologia , Naftalenossulfonato de Anilina/química , Benzotiazóis , Dicroísmo Circular , Difusão Dinâmica da Luz , Fluorescência , Humanos , Insulina/química , Muramidase/química , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/patologia , Tiazóis/químicaRESUMO
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.
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Fenômenos Biofísicos , Bussulfano/metabolismo , Simulação de Acoplamento Molecular , Albumina Sérica Humana/metabolismo , Sítios de Ligação , Dicroísmo Circular , Cristalografia por Raios X , Difusão Dinâmica da Luz , Humanos , Hidrodinâmica , Cinética , Ligação Proteica , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
The current study comprises of an inclusive biophysical study, enlightening the binding of L-3, 4-dihydroxyphenylalanine (l-Dopa) with human lysozyme (HL) and hen egg white lysozyme (HEWL). Spectroscopic and molecular docking tools have been utilized to study the interaction of l-Dopa with both HL and HEWL. Spectrofluorimetric measurements exhibited that l-Dopa quenched the HL and HEWL intrinsic fluorescence. A binding constant (Kb) of â¼104M-1 for both HL and HEWL was obtained, asserting a significant binding. Negative value of ΔG affirmed that the reaction between proteins and l-Dopa was spontaneous. Far-UV CD spectra revealed a boost to the proteins helical content in the presence of l-Dopa. Furthermore, DLS measurements displayed the decrease in hydrodynamic radii (Rh) of HL and HEWL in the presence of l-Dopa. Molecular docking studies established that l-Dopa formed complexes with both the proteins through hydrogen bonding and hydrophobic interaction. The present study characterizing the l-Dopa interaction with lysozyme could be noteworthy in realizing both pharmaco-dynamics and/or -kinetics of drugs used in various diseases.
Assuntos
Fenômenos Biofísicos , Levodopa/química , Muramidase/química , Animais , Dicroísmo Circular , Difusão Dinâmica da Luz , Humanos , Levodopa/metabolismo , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Muramidase/metabolismo , Ligação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Análise EspectralRESUMO
The association of protein aggregates with plentiful human diseases has fascinated studies regarding the biophysical characterization of protein misfolding and ultimately their aggregate formation mechanism. Protein-ligand interaction, their mechanism, conformational changes by ligands, and protein aggregate formation have been studied upon exploiting experimental techniques and computational methodologies. Such studies for the exploration of ligand-induced conformational changes in protein, misfolding and aggregation, has confirmed drastic progresses in the study of aggregate formation pathways. This review comprises of an inclusive description of contemporary experimental techniques as well as theoretical improvements in the interpretation of the conformational properties of protein. We have also discussed various factors responsible for the microenvironment change around protein that sequentially causes amyloidoses. Biophysical techniques and cell-based assays to gain comprehensive understandings of protein-ligand interaction, protein folding, and aggregation pathways have also been described. The promising therapeutic methods used to inhibit the protein fibrillogenesis have also been discussed.
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Agregados Proteicos/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Proteínas/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Amiloidose/tratamento farmacológico , Amiloidose/metabolismo , Animais , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Ligantes , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Proteínas/metabolismoRESUMO
Numerous phenolic compounds have been reported in the last decade that have a good antioxidant property and interaction affinity towards mammalian serum albumins. In the present study, we have utilized mammalian serum albumins as a model protein to examine their comparative interaction property with polyphenolic compound tannic acid (TA) by using various spectroscopic and calorimetric methods We have also monitored the esterase and antioxidant activity of mammalian serum albumins in the absence and presence of TA. The obtain results recommended that the TA have a good binding affinity (â¼104 to 106M-1) towards mammalian serum albumins and shows double sequential binding sites, which depends on the concentration of TA that induced the conformational alteration which responsible for the thermal stability of proteins. Binding affinity, structural transition and thermodynamic parameters were calculated from spectroscopic and calorimetric method reveals that non-covalent interaction causes partial conformational alteration in the secondary structure of protein ie.; increase in α-helical content with decrease in ß-sheet, random coil and other structure. Meanwhile, we have found that esterase activities of serum albumins were also stabilized against hydrolysis and shows higher antioxidant activity in the presence of TA because albumins its self have an immense antioxidant activity beside TA.
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Polifenóis/química , Ligação Proteica , Albumina Sérica/química , Taninos/química , Animais , Sítios de Ligação , Fenômenos Biofísicos , Bovinos , Dicroísmo Circular , Humanos , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Estrutura Secundária de Proteína , Albumina Sérica/ultraestrutura , TermodinâmicaRESUMO
Protein aggregation and misfolding have been allied with numerous human disorders and thus inhibition of such occurrence has been center for intense research efforts against these diseases. Here, we investigated anti-fibrillation activity of cysteine and its effect on kinetics of stem bromelain amyloid fibril formation. We established the anti-fibrillation and anti aggregation activities of cysteine by using multiple approaches like turbidity measurements, dye binding assays (ThT and ANS) and structural changes were monitored by circular dichroism (CD) followed by electron microscopy. Our experimental study inferred that cysteine inhibits temperature induced fibrillation of protein in a concentration dependent way. In addition, MDA-MB-231 cell viability of pre-formed amyloid was increased in presence of cysteine as compared to the fibrils alone. Furthermore, dynamic light scattering studies of native, aggregated as well as incubated (amyloids in presence of cysteine) samples indicates that cysteine restores native like structures of stem bromelain. Isothermal titration calorimetric results revealed that hydrogen bonding between cysteine and stem bromelain plays a significant role during inhibition of stem bromelain aggregation. However, thiophilic interaction between thiol group of cysteine and aromatic amino acid residue of stem bromelain may also have noteworthy role in inhibition of amyloid formation.
Assuntos
Proteínas Amiloidogênicas/toxicidade , Cisteína/farmacologia , Citotoxinas/toxicidade , Proteínas Amiloidogênicas/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/química , Humanos , Agregados Proteicos/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacosRESUMO
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.
Assuntos
Antibacterianos/metabolismo , Ceftazidima/metabolismo , Albumina Sérica Humana/metabolismo , Antibacterianos/química , Sítios de Ligação , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Albumina Sérica Humana/química , Especificidade por SubstratoRESUMO
In spite of the fact that amyloid related neurodegenerative illnesses and non-neuropathic systemic amyloidosis have allured the research endeavors, as no cure has been announced yet apart from symptomatic treatment. Therapeutic agents which can reduce or disaggregate those toxic oligomers and fibrillar species have been studied with more compounds are on their way. The current research work describes comprehensive biophysical, computational and microscopic studies which reveal that L-3, 4-dihydroxyphenylalanine (L-Dopa) have indisputable efficacy to hinder the heat induced amyloid fibrillation of the human lysozyme (HL) and also preserve the fibril disaggregating potential. The IC50 value of L-Dopa is calculated to be 63.0±0.09µM. L-Dopa intervenes in the process of amyloid fibrillogenesis through hydrophobic interaction and hydrogen bond formation with the amino acid residues found in the amyloid fibril forming prone region of HL as clarified by molecular simulation data. L-Dopa also disaggregates the mature amyloid fibrils into some unorganized species and the DC50 value was estimated to be 19.95±0.063µM. Hence, L-Dopa and related compounds can act as effective inhibitors in the therapeutic development to combat systemic amyloidosis.
Assuntos
Amiloidose/tratamento farmacológico , Levodopa/farmacologia , Transtornos Parkinsonianos/tratamento farmacológico , Amiloidose/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Levodopa/metabolismo , Levodopa/uso terapêutico , Simulação de Acoplamento Molecular , Muramidase/química , Muramidase/metabolismo , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacosRESUMO
Nowadays, understanding of interface between protein and drugs has become an active research area of interest. These types of interactions provide structural guidelines in drug design with greater clinical efficacy. Thus, structural changes in catalase induced by clofazimine were monitored by various biophysical techniques including UV-visible spectrometer, fluorescence spectroscopy, circular dichroism, and dynamic light scattering techniques. Increase in absorption spectra (UV-visible spectrum) confers the complex formation between drug and protein. Fluorescence quenching with a binding constants of 2.47 × 104 M-1 revealed that clofazimine binds with protein. Using fluorescence resonance energy transfer, the distance (r) between the protein (donor) and drug (acceptor) was found to be 2.89 nm. Negative Gibbs free energy change (ΔG°) revealed that binding process is spontaneous. In addition, an increase in α-helicity was observed by far-UV circular dichroism spectra by adding clofazimine to protein. Dynamic light scattering results indicate that topology of bovine liver catalase was slightly altered in the presence of clofazimine. Hydrophobic interactions are the main forces between clofazimine and catalase interaction as depicted by molecular docking studies. Apart from hydrophobic interactions, some hydrogen bonding was also observed during docking method. The results obtained from the present study may establish abundant in optimizing the properties of ligand-protein mixtures relevant for numerous formulations.
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Catalase/química , Clofazimina/química , Fígado/química , Simulação de Acoplamento Molecular , Animais , Sítios de Ligação , Catalase/isolamento & purificação , Bovinos , Cristalografia por Raios X , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fígado/enzimologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Análise Espectral/métodos , TermodinâmicaRESUMO
Drug and protein interaction provides a structural guideline in the rational drug designing and in the synthesis of new and improved drugs with greater efficacy. We have examined here the interaction tendency and mechanism of nintedanib (NTB), an anticancer drug (tyrosine kinase inhibitor) with bovine serum albumin (BSA), by spectroscopic techniques. The decline in Stern-Volmer quenching constants and binding constant with the temperature rise suggests that BSA forms a complex with NTB. Binding constant obtained by modified Stern-Volmer equation at 3 temperatures was realized to be of the order of ~104 M-1. Negative ΔG (~-5.93 kcal mol-1), ΔH (-3.74 kcal mol-1), and ΔS (-1.50 kcal mol-1) values exhibited a spontaneous and exothermic reaction between BSA and NTB. NTB molecule interacts with BSA by forming hydrogen bonds, as elucidated by fluorescence results. Moreover, a minor increment in the helical conformation of BSA upon its binding to NTB was observed by circular dichroism spectroscopy. The modification in protein's symmetry and a decline in hydrodynamic radii were observed in the presence of NTB (from ~3.6 to ~3 nm) as obtained by the dynamic light scattering measurement results.
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Indóis/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Soroalbumina Bovina/metabolismo , Animais , Sítios de Ligação , Bovinos , Dicroísmo Circular , Difusão Dinâmica da Luz , Indóis/química , Ligação Proteica , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Soroalbumina Bovina/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
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.
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Amiloide/química , Clofazimina/química , Hansenostáticos/química , Muramidase/química , Dodecilsulfato de Sódio/química , Amiloide/antagonistas & inibidores , Animais , Benzotiazóis , Sítios de Ligação , Galinhas , Clara de Ovo/química , Corantes Fluorescentes/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Muramidase/antagonistas & inibidores , Agregados Proteicos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Tiazóis/químicaRESUMO
The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 104 M-1, and with the increase in temperature, Stern-Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV-visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug-albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (Ro) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA.
Assuntos
Antineoplásicos/química , Antituberculosos/química , Clofazimina/química , Modelos Moleculares , Albumina Sérica/química , Análise Espectral , Sítios de Ligação , Dicroísmo Circular , Transferência Ressonante de Energia de Fluorescência , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Espectrometria de FluorescênciaRESUMO
Neurodegenerative disorders are mainly associated with amyloid fibril formation of different proteins. Stem bromelain (SB), a cysteine protease, is known to exist as a molten globule state at pH 10.0. It passes through the identical surrounding (pH 10.0) in the gut epithelium of intestine upon oral administration. Protein-surfactant complexes are widely employed as drug carriers, so the nature of surfactant toward protein is of great interest. The present work describes the effect of cationic surfactants (CTAB & DTAB) and their hydrophobic behavior toward amyloidogenesis behavior of SB at pH 10.0. Multiple approaches including light scattering, far UV-CD, turbidity measurements, and dye binding assay (ThT, Congo red and ANS) were performed to measure the aggregation propensity of SB. Further, we monitored the hydrodynamic radii of aggregates formed using dynamic light scattering technique. Structure of fibrils was also visualized through fluorescence microscopy as well as TEM. At pH 10.0, low concentration of CTAB (0-200 µM) induced amyloid formation in SB as evident from a prominent increase in turbidity and light scattering, gain in ß-sheet content, and enhanced ThT fluorescence intensity. However, further increase in CTAB concentration suppressed the fibrillation phenomenon. In contrast, DTAB did not induce fibril formation at any concentration used (0-500 µM) due to lower hydrophobicity. Net negative charge developed on protein at high pH (10.0) might have facilitated amyloid formation at low concentration of cationic surfactant (CTAB) due to electrostatic and hydrophobic interactions.
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Amiloide/química , Bromelaínas/química , Compostos de Cetrimônio/química , Compostos de Amônio Quaternário/química , Tensoativos/química , Amiloide/ultraestrutura , Naftalenossulfonato de Anilina/química , Benzotiazóis , Cetrimônio , Corantes/química , Vermelho Congo/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Agregados Proteicos , Ligação Proteica , Eletricidade Estática , Tiazóis/químicaRESUMO
Studying amyloid associated neurodegenerative diseases is an active area of research. Cure for these diseases are still to be discovered. In the present study we have performed comprehensive biophysical and computational experiments showing levodopa not only significantly inhibits heat induced fibrillization of human serum albumin but also disaggregates preformed fibrils. Thioflavin T (ThT) binding assay was used to monitor the fibrillation process of human serum albumin (HSA) at 65°C in the presence and absence of levodopa. Binding of levodopa was studied using isothermal titration calorimetry (ITC), binding constant was found to be 3.6×103M-1. Thermal stabilization effect of levodopa on HSA was studied using differential scanning calorimetry (DSC). Microscopic imaging techniques were employed to analyze the morphology of aggregates and effect of levodopa on aggregation. Further, molecular docking study was also utilized to decipher the amino acid residues involved in the binding interaction of levodopa with HSA. Levodopa interferes in the Fibrillogenesis of HSA by interacting with the amino acid residues near to drug binding site II on the HSA with the binding constant of the order of 103 and stabilizes the protein. The results are indicative of the potential use of levodopa as a therapeutic agent for the treatment of amyloid diseases.