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International Journal of Biological Macromolecules 80 (2015) 375–384

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac

iophysical insight into the anti-amyloidogenic behavior of taurine

umit Kumar Chaturvedia, Parvez Alama, Javed Masood Khana,ohd. Khursheed Siddiquia, Ponnusamy Kalaiarasanb, Naidu Subbaraoc,

eeshan Ahmada, Rizwan Hasan Khana,∗

Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, IndiaNational Centre of Applied Human Genetics, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, IndiaSchool of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi, India

r t i c l e i n f o

rticle history:eceived 5 February 2015eceived in revised form 15 June 2015ccepted 16 June 2015vailable online 22 June 2015

eywords:SAaurinemyloid fibril

a b s t r a c t

In this work, we investigated the inhibitory ability of taurine on the aggregation of Human serum albu-min (HSA) and also examined how it controls the kinetic parameters of the aggregation process. Wedemonstrated the structural alterations in the HSA after binding to the taurine at 65 ◦C by exploitingvarious biophysical techniques. UV–vis spectroscopy was used to check the turbidometric changes inthe protein. Thioflavin T fluorescence kinetics was subjected to explore kinetic parameters comparingthe amyloid formation in the presence of varying concentration of taurine. Further, Congo red bindingand ANS binding assays were performed to determine the inhibitory effect of taurine on HSA fibrillationprocess and surface hydrophobicity modifications occurring before and after the addition of taurine withprotein, respectively. Far UV CD and Dynamic Light Scattering (DLS) confirmed that taurine stabilized

ggregation the protein �-helical structure and formed complex with HSA which is further supported by differentialscanning calorimetry (DSC). Moreover, microscopic imaging techniques were also done to analyze themorphology of aggregation formed. Taurine is also capable of altering the cytotoxicity of the protein-aceous aggregates. Molecular docking study also deciphered the possible residues involved in proteinand drug interaction.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

Protein misfolding and aggregation is most interesting and chal-enging topic in industry and human health [1,2]. Amyloids are

ell defined protein aggregates which are responsible for morehan 20 diseases in humans such as Alzheimer, Parkinson, diabetesI and Huntington, etc. [2–5]. Soluble protein aggregates possessell cytotoxic effect and insoluble aggregates may be undesirableor drug products therefore prevention or retardation is an essen-ial task [6,7]. In addition to protein associated with diseases otherroteins such as lysozyme, serum albumins, insulin, etc. also formmyloids under suitable conditions like high temperature, low pH,n presence of surfactants, oxidation, ionic strength and crowding

gents [8–10]. Number of evidences are available that proves thatorphological and histochemical properties of disease associated

∗ Corresponding author. Tel.: +91 571 2720388; fax: +91 571 2721776.E-mail addresses: rizwanhkhan1@gmail.com, rizwanhkhan1@yahoo.com

R.H. Khan).

ttp://dx.doi.org/10.1016/j.ijbiomac.2015.06.035141-8130/© 2015 Elsevier B.V. All rights reserved.

or disease unrelated proteins are very similar which suggests thatfibril formation is the intrinsic property of all polypeptide [11].

Currently, the research is being done that provoke aggregationinhibition of proteins in vitro and in vivo. Small molecules such aspolyphenols, metal ions, vitamins, nucleotides and synthetic pep-tides have been reported as anti-aggregation agents [12–14].

Human serum albumin is a globular protein composed of 585amino acid residues and 17 disulphide bonds. It is the most abun-dant plasma protein and serves as a carrier protein for the largenumber of small molecules, fatty acids and drugs [15]. Interactionof HSA with large number of molecules is widely studied becauseof its role as a carrier molecule. Although HSA is very stable proteinbut possesses propensity to aggregate provided suitable conditionsin vitro [9]. Recently, a lot of work has been reported dealing withaggregation inhibition of serum albumin in vitro which can serveas a model for designing anti amyloidogenic drugs [16]. Here, weare reporting first time inhibitory effect of taurine on thermally

induced aggregation of human serum albumin.

Taurine (2-amino-ethanesulfonic acid) is a ubiquitous small sul-phur containing amino acid found in almost all mammals [17]. Itcan be obtained from egg, meat and seafood or alternatively can be

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76 S.K. Chaturvedi et al. / International Journal

ynthesised inside the body from methionine and cysteine in pres-nce of vitamin B6 [18]. In humans the main source of taurine isiet and endogenous synthesis is very low further its concentrationaries from 1 to 2 mM. Taurine involves in various physiologi-al functions such as balance of neurotransmitters, fat digestion,ile salt formation, calcium transport, osmoregulation and anti-

nflammation [19]. Taurine deficiency is associated with epilepsy,epression hyperactivity and anxiety [20]. Moreover, taurine and

ts analogue also have been reported to be anti-diabetic, inhibitumor cell growth, neuroprotective and it also reduces neonatal

ortality [21–23].The aim of the present study is to investigate the effect of tau-

ine on the fibrillation of human serum albumin in vitro. Diverserray of methodologies ranging from spectroscopy to imaging andolecular docking such as UV–vis spectroscopy, fluorescence spec-

roscopy, circular dichroism (CD), ThT and Congo red dye bindingssay, fluorescence microscopy, dynamic light scattering (DLS) andransmission electron microscopy (TEM) have been employed tonravel inhibitory activity of taurine against fibrillation of HSA.inally, cell cytotoxicity was measured using MTT assay; for thisurpose PC 12 and SH-SY5Y cell lines were used. The resultsemonstrated that Taurine can inhibit human serum albumin fib-il formation in vitro moreover it also reduced the cell cytotoxicffect of aggregates. We foresee that our research could pave theay for future work on the designing of suitable molecules against

ggregation associated diseases.

. Materials

Human serum albumin, Thioflavin T (ThT), (ANS), Congo red andaurine were purchased from Sigma Chemical Co. (St. Louis, MO,SA). All other reagents used were of analytical grade.

.1. Methods

.1.1. Preparation of HSA solutionA stock solution of 500 �M was made in 20 mM phosphate

uffer pH 7.4 and extensively dialyzed against the same buffer andoncentration was determined using a UV–vis spectrophotometerPerkin Elmer Lambda25) E1%

cm = 5.3 at 280 nm. For the preparationf amyloid 100 �M HSA was used and samples were incubated at5 ◦C in the presence of 50 mM NaCl for 120 h in a circulating shak-

ng water bath [24]. Rest of the study carried out diluting the proteinith the same condition to 10 �M.

.1.2. pH measurementpH was determined using Mettler Toledo Seven Easy pH meter

model S20) which was routinely calibrated with standard buffers.he experiments were performed in the 20 mM pH 7.4 sodiumhosphate buffer. All preparations used in the experiments wereltered through 0.45 �m Millipore Millex-HV PVDF filter.

.1.3. ThT fluorescence spectroscopic measurementsA stock solution of ThT was prepared in double distilled

ater and filtered with 0.2 �m Millipore filter. The concentra-ion of ThT was measured using molar extinction coefficientM = 36,000 M−1 cm−1 at 412 nm [25]. The protein samples10.0 �M), in the absence as well as presence of varying concen-

ration of Taurine, were incubated for 120 h at 65◦. Post incubation,amples were supplemented with 10 �M of ThT solution and wereurther incubated for 30 min in the dark. The ThT was excitedt 440 nm and spectra were recorded from 450 to 600 nm. The

logical Macromolecules 80 (2015) 375–384

excitation and emission slit widths were set at 10 nm. All data arefitted by using following equation in Sigma plot [26].

F = Fi + mit + Ff + mf t

1 + e−[t−t0

� ](1)

where F is the fluorescence intensity at time t, and t0 is the time toattain 50% of maximal fluorescence intensity. (Fi + mit) and (Ff + mft)represent the initial base line related to the induction time andfinal constant line, respectively. The apparent rate constant for fibrilgrowth is given by 1/�, and the lag time is calculated by t0 − 2�.

2.1.4. Turbidity measurementsTurbidity measurements were performed on a Perkin Elmer

double beam UV–vis spectrophotometer model lambda 25 in acuvette of 1 cm path length. The turbidity of HSA sample incu-bated in pH 7.4 phosphate buffer with 50 mM NaCl for 120 h at65 ◦C in the absence and presence of varying concentration of tau-rine, ranging from 0 to 1.5 mM was determined by monitoring thechange in absorbance at 350 nm. Respective blank corrections weredone prior to all experiments. The equilibrium data obtained fromturbidity measurements was fitted using Sigma plot 12.0 to singleexponential equation [27]:

A = A0e−�[I] (2)

where A0 and A are the turbidities at 350 nm in the absence andpresence of inhibitor, A is the inhibition constant and [I] is theconcentration of inhibitor.

2.1.5. Congo red binding assayCongo red was dissolved in a 20 mM phosphate buffer (pH

7.4) consisting of 50 mM NaCl and filtered through 0.45 �Mmembrane filter. The concentration was determined using εM45,000 M−1 cm−1 at 498 nm. The protein concentration was fixedat 10 �M. CR (10 �M) were mixed at a molar ratio of 1:1 with pro-tein in the absence and presence of Taurine (incubated for 120 h at65 ◦C) and kept for 15 min. The absorbance spectra (400–700 nm)of the samples were recorded with a UV–vis spectrophotometer(Perkin Elmer Lambda 25) in a 1 cm path length cuvette.

2.1.6. ANS fluorescence measurementsThe steady-state fluorescence measurements were performed

on Schimadzu spectrophotometer (RF-5301 PC). Both excitationand emission slits were set at 5 nm. For ANS binding experiment,protein samples (incubated for 120 h at 65 ◦C) at pH 7.4 were incu-bated with 50 fold molar excess of ANS for 30 min at 25 ◦C in dark.The excitation wavelength for ANS fluorescence was set at 380 nmand the emission spectra were recorded from 400 to 600 nm. Theprotein concentration was fixed at 10 �M.

2.1.7. Far-UV CD measurementsThe circular dichroic measurements were performed on a JASCO

spectropolarimeter (J-815) with a thermostatically controlled cellholder attached to a peltier with multitech water circulator. Theexperiments were carried out with HSA (10 �M) incubated at 65 ◦Cfor 120 h in the absence and presence of 750 �M of taurine andHSA (10 �M) incubated at 25 ◦C for 24 h in the absence and pres-ence of 750 �M of taurine. Spectra were scanned in the range of200–250 nm in a cuvette of 0.1 cm path length. Each spectrum wasan average of three scans. In all CD measurements, the HSA concen-tration was invariable. The results were expressed as MRE (meanresidue ellipticity) in deg cm2 dmol−1, which is given by:

MRE = �obs(mdeg)10 × n × Cp × l

(3)

where �obs is the observed ellipticity in degrees, Cp the molar frac-tion and l is the length of the light path in centimeter. The spectra

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S.K. Chaturvedi et al. / International Journal

ere smoothed by the Savitzky-Golay method with 25 convolutionidths.

.1.8. Dynamic light scattering measurements (DLS)The change in aggregation behavior of HSA in the presence of

arying concentration of taurine values was determined using DLS.he Rh measurements were done using a protein concentrationf 10 �M at 830 nm on a DynaPro-TC-04 dynamic light scatteringnstrument (Protein Solutions, Wyatt Technology, Santa Barbara,A) equipped with a temperature controlled microsampler. All theolutions were filtered through a 0.22 �M pore sized microfilterWhatman International, Maidstone, UK). The measured hydro-ynamic radius (Rh) was the average of 50 measurements. Theean Rh and polydispersity (Pd) were estimated, on the basis of

n autocorrelation analysis of scattered light intensity based on theranslational diffusion coefficient, from the Stokes–Einstein equa-ion:

h = kT

6��D25◦CW

(4)

here Rh is the hydrodynamic radius (nm), k the Boltzmann’s con-tant, T the absolute temperature (K), � the viscosity of water and25◦CW is the translational diffusion coefficient. All the samples were

ncubated for 120 h at 65 ◦C prior to measurements.

.1.9. Differential scanning calorimetryThe differential scanning calorimetric measurements were car-

ied out using VP-DSC micro calorimeter (Micro Cal, Northampton,A). The buffer and protein solutions were degassed under mild

acuum prior to the experiment. Samples were prepared in 20 mModium phosphate buffer, pH 7.4. The DSC measurements of HSA15 �M) in the presence of 1:50 ratio of taurine were performedrom 25 to 90 ◦C at a scan rate of 0.5 ◦C/min. Data was analyzedsing Origin software provided with the instrument to obtain theemperature at the midpoint of the unfolding transition (Tm).

.1.10. Transmission electron microscopy (TEM)TEM images were taken on Philips CM-10 transmission elec-

ron microscope operating at an accelerating voltage of 200 kV.he amyloid fibril formation was assessed by applying 6 �L of HSA10 �M) incubated for 120 h at 65 ◦C alone and with 750 �M taurinen 200-mesh copper grid (CF 200-Cu, lot no-110323) covered byarbon-stabilized formvar film. Excess of fluid was removed after

min and the grids were then negatively stained with 2% (w/v)ranyl acetate. Images were viewed at 10,000×. Before taking the

mage, all the samples were incubated overnight.

.1.11. Fluorescence microscopyTen microliter of sample containing HSA incubated at 65 ◦C for

20 h in the absence and presence of 750 �M of taurine taken andashed with 20 mM PBS, pH 7.4. The samples were finally incu-

ated with 1:1 ratio of ThT to HSA for 30 min at 37 ◦C in dark.he samples were washed thoroughly and visualized under fluo-escence microscope using a 100× oil immersion objective (Zeiss2 Imager; Zeiss, Göttingen, Germany).

.1.12. Cell cultureRat pheochromocytoma PC12 cells were maintained under

MEM medium supplemented with 5% (v/v) fetal bovine serum

FBS), 10% (v/v) horse serum, and 100 U/ml penicillin in 5% (v/v)O2/air at 37 ◦C. Similarly, SH-SY5Y cells were cultured in DMEM-12 medium in humidified 5% (v/v) CO2/air at 37 ◦C in 10% (v/v) FBSnd 100 U/ml penicillin.

logical Macromolecules 80 (2015) 375–384 377

2.1.13. MTT assayMTT (3,(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium

bromide) reduction assay was used to measure the cell viabilitiesof PC12 and SH-SY5Y. MTT in the presence of viable cells reduceto form blue formazan crystals, toxicity leads to inhibition offormazon production [28]. For the MTT reduction assays, sam-ple solutions of HSA with 250 and 750 �M concentrations oftaurine were added to the PC12/ SH-SY5Y cells in the 96-wellplates. Cells were seeded at 5000 cells/well on 96-well platesand incubated for 24 h before the treatment. The HSA samplesolutions (incubated over a period of 120 h at 65 ◦C) were incu-bated with PC12/SH-SY5Y cells for 24 h, and MTT reduction wasperformed. MTT was added to the culture medium to yield a finalconcentration of 0.5 mg/ml and incubated for 4 h at 37 ◦C in CO2incubator then removed supernatant carefully, 200 �L of DMSOwas added and mixed. After 20 h of incubation in a humidified CO2incubator, the absorbance at 585 nm was read using a Microplateabsorbance reader (Bio-Rad Instruments, iMarkTM). Cell viabilitywas compared to control cells without exposed to the HSA fibrilsolutions.

2.1.14. Molecular docking and molecular dynamic simulationstudy of HSA–taurine interaction

The molecular docking study was performed using Autodock4.2 and Autodock tools (ADT) using Lamarckian genetic algorithm.The crystal structure of HSA (PDB id: 1AO6) and three dimensionalstructure of taurine (CID: 1123) was obtained from BrookhavenProtein Data Bank and PubChem respectively. Chain A of the pro-tein was selected, water molecules and ions were removed and allhydrogen atoms were added. Then partial Kollman charges wereassigned to HSA. The protein was set to be rigid and there is noconsideration of solvent molecules on docking. The grid size wasset to be 126, 126 and 126 along X, Y and Z axes with 0.564 A gridspacing. Autodock parameters used were GA population size: 150and maximum number of energy evolutions: 2,500,000. Ten bestsolution based on docking score was retained for further analy-sis, Discovery studio 3.5 were used for visualization and for theidentification of residues involved in binding. A 20,000 ps molec-ular dynamic simulation study was performed with GROMACS4.5.3 package [29], using the GROMOS 96 [30] force field. The boxdimensions ensured that any protein atom was at least 1.5 nmaway from the wall of the box with periodic boundary condi-tions, solvated by simple point charge (spc) [31] water molecules.NaCl counter ions were added to satisfy the electro-neutrality con-dition. Energy minimization was carried out using the steepestdescent method. The energy minimized structure is taken as aninitial structure for equilibration. Berendsen temperature couplingand Parrinello–Rahman pressure coupling were used to keep thesystem in a stable environment (300 K, 1 bar), and the couplingconstants were set at 0.1 and 2.0 ps for temperature and pres-sure, respectively. The partial mesh Ewald (PME) algorithm wasemployed for electrostatic and Van der Waals interactions. Cut-off distance for the short-range VdW (rvdw) was set to 1.4 nm,where Coulomb cut-off (r coulomb) and neighbor list (rlist) werefixed at 0.9 nm. All the bond lengths were constrained using theLINCS algorithm [32], and the time step was set to 0.002 ps. Thecomplexes in medium were equilibrated for 100 ps in NVT andNPT ensembles, respectively. All trajectories were stored every 2 ps(pico-second) for further analysis. We have also carried out theMD simulation of 5 ns. Structural properties of the complex proteinwere calculated from the trajectory files with the built-in functions

of GROMACS 4.5.3. Structural analysis, such as root mean-squaredeviation (RMSD) and radius of gyration were analyzed through theuse of g rmsd and g gyrate, respectively, with the built-in functionsof GROMACS.

378 S.K. Chaturvedi et al. / International Journal of Biological Macromolecules 80 (2015) 375–384

Fig. 1. ThT fluorescence kinetics (A) and spectra (B) of HSA (10 �M) incubated at 65 ◦C after 120 h incubation in the presence of varying concentration of taurine. Inset of B:m

2

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Table 1Kinetic parameters obtained on exponential fitting of time kinetics curves of heatinduced aggregation of HSA in absence and presence of taurine.

S/N Parameters HSA HSA + 250 �Mtaurine

HSA + 750 �Mtaurine

1. Lag time 1.20 ± 0.02 19.54 ± 0.04 22.94 ± 0.052. Apparent rate

constant0.10 ± 0.001 0.08 ± 0.001 0.06 ± 0.001

3. %reduction inThT FI

– 56.60 ± 2.50 76.71 ± 4.70

Fig. 1B), respectively so both of these groups have their own poten-tial to disrupt the interaction formed during the fibrillation process[27,39–41].

Table 2Parameters obtained from fitting the turbidity and ThT fluorescence data obtainedfor inhibition of HSA aggregation by taurine.

olecular structure of taurine.

.1.15. Statistical analysisAll experiments were executed in triplicates, mean value,

tandard deviations and percent errors were calculated, whereverpplicable, using SPSS 16.0 programme for windows [33].

. Results and discussion

.1. Aggregation kinetics study of HSA and ThT fluorescencepectroscopy-effect of taurine

Incubation of HSA under defined conditions resulted in theormation of matured amyloid fibrils. In the present study, HSA20 mg/ml) in 20 mm phosphate buffer (pH 7.4) with 50 mM NaClt 65 ◦C incubated for 120 h in the absence and presence of tau-ine. The growth of amyloid formation of HSA was monitored andharacterized by ThT fluorescence.

ThT is an amyloid specific dye and used to monitor the formationnd growth of amyloid fibrils. ThT fluorescence intensity increasespon interaction with amyloid [34]. Kinetics of amyloid formationas studied by monitoring ThT fluorescence intensity of prepara-

ions at regular time intervals (0–120 h) as depicted in Fig. 1A. HSAn the absence of taurine showed a standardized pattern of amy-oid formation, i.e., sigmoidal pattern without involving lag phaseFig. 1A) [24]. In the presence of different concentration of taurinehe aggregation profile of HSA changed dramatically. Initially at theower concentration of taurine (50 �M), decrease in ThT FI and noffect on lag phase was found which illustrates that taurine at lowoncentration starts to slow down the aggregation process but notble to interfere in nucleation process. However, beyond this con-entration of taurine (250 and 750 �M) lag phase was found to beontinuously increased to around 19 and 23 h respectively (Table 1).his delay in lag time also slows down the apparent growth rate ofmyloid formation.

Co-incubation of HSA with 750 �M of taurine resulted in aeduction of ThT fluorescence of mature fibrils to 76% (Fig. 1B andable 1). ThT fluorescence kinetics of amyloid formation consistsf distinct phases. Delay in lag time in the presence of taurine

ttributed to stabilization of the native form as reported earlier35,36]. Quinones and carnosine act in the same way and pro-ong the lag phase of insulin and lysozyme aggregation pathway,espectively [37,38].

4. %reduction inANS FI

– 61.30 ± 3.3 80.20 ± 5.20

3.2. Inhibitory effect of taurine on HSA fibril formation asmonitored by turbidity and ThT fluorescence intensitymeasurements

Fig. 2 represented the aggregation decay profile on extent ofturbidity and ThT FI of aggregated HSA with increasing concentra-tion of taurine (0–1500 �M). The turbidity and ThT FI were foundto decrease continuously with increasing concentration of taurine.Both ThT FI and turbidity were substantially reduced up to 750 �Mconcentration of taurine and then plateau achieved (Table 2). Thisindicates that taurine interfere the interactions evolving during thestart of aggregation and thus inhibits aggregation of HSA.

The data in Fig. 2 showed (best fit to Eq. (2)); suggesting thatthe light scattering intensity decreases exponentially with increas-ing concentration of the inhibitor. The parameters obtained fromdata fitting are summarized in Table 2. Taurine is a small moleculecontaining amino as well as sulphonic group at both ends (inset of

S/N ThT fluorescence Turbidity

1. � (M−1) 2 × 103 ± 0.17 1.28 × 103 ± 0.152. F0/A0 699 0.403. R2 0.991 0.99

S.K. Chaturvedi et al. / International Journal of Biological Macromolecules 80 (2015) 375–384 379

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3

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ig. 2. Aggregation decay profile of HSA as a function of increasing concentration ot 65 ◦C for 120 h. The data are averaged with standard deviation of three independ

.3. Congo red binding assay

Inhibitory effect of taurine on HSA fibrillation was observed byongo red binding assay. Similar to the ThT fluorescence results,ttenuation of HSA fibril appeared to be a function of taurine con-entration as shown in Fig. 3A. Congo red on binding with maturedbril of HSA showed increase in absorbance with significant redhift to 540 nm which is a characteristic feature of amyloid [42].

n the presence of taurine absorbance got decrease and furthereak is shifted to 494 nm depicted the inhibitory action of taurinegainst HSA fibrillation process. Similar mode of inhibition has beeneported, illustrates that carnosine inhibits the amyloidogenesis of

Fig. 3. Congo red binding and ANS fluorescence intensity of HSA (10 �M) incub

ine. Samples were incubated with different concentration of taurine (0–1500 �M)als.

lysozyme and supported by the shift in absorbance wavelength ofCongo red [38].

3.4. ANS binding studies: attenuation in surface hydrophobicity

In aggregation process, protein first undergoes to unfolding pro-cess and hydrophobic patches get exposed which further associatedto form an oligomer and finally these oligomers matured into a

fibrillar structure. ANS is a widely used extrinsic fluorescent probeto detect the exposed hydrophobic regions of protein. Prominentincrease in ANS fluorescence intensity accompanied with signif-icant blue shift compared with the native HSA referred to the

ated at 65 ◦C over120 h in the presence and absence of taurine (750 �M).

3 of Biological Macromolecules 80 (2015) 375–384

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hat

Fig. 4. Far-UV CD spectra of HSA (10 �M) incubated at 65 ◦C over a period of 120 h

Fa

80 S.K. Chaturvedi et al. / International Journal

ggregation of HSA and exposure of hydrophobic patches (Fig. 3B)9]. However in the presence of taurine ANS fluorescence intensityot decreased (61% for 250 �M and 81% for 750 �M) which depictedhe reduction in hydrophobic patches of protein that subsequentlyeads to inhibition of fibrillation (Table 1). Earlier reports have alsohown the reduction in the hydrophobic exposure, modulates theggregation process [43].

.5. Effect of taurine on secondary structural changes duringbrillation by circular dichroism spectroscopy

Far UV CD (200–250 nm) was used to determine the secondarytructural changes in HSA. In absence of taurine, we observed dualinima at 222 and 208 nm, characteristic of �-helical structure,

t pH 7.4 and 25 ◦C (Fig. 4). In the presence of 750 �M taurine at5 ◦C negative MRE value get increased which illustrated that tau-ine effectively stabilized the secondary structure of protein and it isell reported fact that secondary structure stabilizing elements are

ood inhibitor in amyloidogenic processes [27]. With the progressf incubation at 65 ◦C, secondary structural transition from �-helixo �-sheet start and after completion of aggregation process theD spectra indicated the concomitant increase in � sheet struc-ure rather than �-helix [44]. CD spectra in presence of taurineesembled to that of native spectra at 65 ◦C. This indicated thataurine promotes the native structure stabilization and delayed theucleation process of aggregation.

.6. Dynamic light scattering measurements to check theomplexation of taurine with HSA

Most proteins are certainly not spherical and their apparentydrodynamic radii may strongly vary depending upon their shapend size. Besides, water molecules bound to the proteins may affecthe diffusion and in turn affecting their hydrodynamic radii which

ig. 5. DLS pattern of HSA (10 �M) in the absence (A) and presence (B) of taurine (750 �Mbsence (C) and presence of taurine (D) (750 �M).

(5 days) in the presence and absence of Taurine (750 �M) and HSA (10 �M) in theabsence and presence of taurine (750 �M) at 25 ◦C.

can differ in some situations from the actual size of the molecule[45]. DLS was performed to understand the nature of associationand size of species during the fibrillation process of HSA in theabsence and presence of taurine. As Fig. 5A shows HSA has a hydro-dynamic radius of 3.4 nm at pH 7.4 and 25 ◦C [46]. However, inthe presence of taurine at pH 7.4 and 25 ◦C hydrodynamic radii ofHSA changed to 4.1 nm and polydispersity value decreases depictedthe complex formation between HSA and taurine leads to increasein water solvent shell around protein vicinity without affectingthe monomer population of protein (Fig. 5B) [45]. This might be

because of taurine containing two hydrogen bond donor and fourhydrogen bond acceptor promotes the water solvent shell ratherexerts perturbation. On elevating the temperature to 65 ◦C at pH

) at 25 ◦C and HSA (10 �M) incubated at 65 ◦C over a period of 120 h (5 days) in the

S.K. Chaturvedi et al. / International Journal of Biological Macromolecules 80 (2015) 375–384 381

F

7igtiarifostthrtn

on the thermal stability of protein. Fig. 6 shows the DSC thermograms for HSA:taurine in the molar ratio of 1:0, 1:50. HSA unfolds

F6

ig. 6. DSC thermo grams of HSA and HSA–taurine complex (HSA:taurine = 1:50).

.4 and incubated for 120 h the hydrodynamic radius dramaticallyncreased to 157 nm which is clear evidence of formation of aggre-ates (Fig. 5C). However, Fig. 5D shows that in the presence ofaurine under same condition, aggregated species are less densen comparison to the monomer form of HSA as clearly evident byppearance of two species having molecular weight 3.5 and 140 nm,espectively. Low polydispersity and high intensity value of peak 1ndicated the presence of monomer is significant over the aggregateorm. As it is well evidenced that hydrodynamic radius is the radiusf solvent shell around the molecule that gives the idea about thehape of molecule. As we said on complexion with taurine at roomemperature hydrodynamic radius increased. But on elevating theemperature protein gets partially unfolded and we got increase inydrodynamic radius with aggregated species. Interaction of tau-ine with the protein molecule brought conformational changes

hat either fold the protein which may be arrested in to the non-ative state or taurine interact with the unfolded protein or collapse

ig. 7. Transmission electron microscopic and fluorescence microscopic images of HSA

5 ◦C at pH 7.4 for 120 h.

Fig. 8. MTT reduction assay for cell cytotoxicity of 120-h aged HSA amyloid fibrils inthe absence and presence of different concentration of taurine individually in PC-12and SH-SY5Y cell lines. Control represents the cells without exposed to HSA fibrils.

it resulted into reduction in water solvent shell and hydrodynamicradius comes closer to native state.

3.7. Differential scanning calorimetry measurement to inferredstabilizing effect of taurine on native conformation of HSA

DSC is a well-known technique to elucidate the stabilizing ordestabilizing action of any ligand on conformation of proteins.Herein, we employed DSC to investigate the effect of taurine onthe thermal stability of HAS [47]. In DSC, �Tm is the main parame-ter which gives the information about the effect of ligand binding

cooperatively and gives a single endothermic peak with meltingtemperatures of 63 ◦C. It is observed that thermal denaturation of

fibrils (10 �M) formed in absence and presence of taurine and after incubation at

382 S.K. Chaturvedi et al. / International Journal of Biological Macromolecules 80 (2015) 375–384

F a stickd alue ot

Hoa6ttdow

3m

aci

ig. 9. Molecular docking results of HSA–taurine complex: (A) Taurine is shown inocking poses of HSA–taurine complex. (C) The root mean square deviation (nm) vhe backbone atoms of the HSA–taurine complex.

SA was found to be only partially reversible under the conditionsf this study. Under saturating conditions taurine stabilized the HSAs evidenced by escalation in melting temperature �Tm by 3.3 ◦C to6.3 ◦C. These results indicated that the taurine binding stabilizeshe protein structure linearly with the CD results and also inferredhat taurine prevent the HSA from going to the unfolded sate andecelerated the nucleation phase of aggregation. Similar to taurinether small molecules that stabilize native structure of protein areell reported [48].

.8. Fluorescence microscopy and transmission electronicroscopy

Amyloid formation and morphology of aggregates was visu-lized by using microscopic imaging techniques [49,50]. Fluores-ence microscopic images showed HSA fibril network which wasncubated at 65 ◦C at pH 7.4 for 120 h in the absence of taurine

representation, and HSA represented with ribbon model. (B) Detailed view of thef HSA–taurine complex. (D) The time dependence of the radius of gyration (Rg) for

(Fig. 7A). Although in the presence of taurine same preparationshow negligible amount of fibril network that clearly confirmedthe inhibitory effect of taurine to HSA amyloid formation (Fig. 7B).To further illustrate the morphology of amyloid TEM was utilized.Similar to the fluorescence microscopy results, TEM results alsosupport the presence of noticeable fibrillar structure of thermallyaggregated HSA and in the presence of taurine extent of fibril for-mation is reduced at significant level (Fig. 7C and D). This reductionof amyloid formation occurred on the cost of decrease in �-sheetstructure accompanied by increase in �-helical conformation in thepresence of taurine as revealed by CD spectroscopy.

3.9. Influence of taurine on fibrillar-HSA induced cytotoxicity

Oligomers and fibrils of different morphology may cause cyto-toxicity up to different extent. MTT reduction action was appliedto depict the HSA fibril toxicity against PC12 and SH-SY5Y cell lines

of Biological Macromolecules 80 (2015) 375–384 383

iVz2attatwcc7oipcfaprt

3o

afimatriribEdaibsIpaiiHctatddd7b1twhl[ta

Table 3Molecular docking parameters of HSA–taurine binding.

Amino acid residues Interactionsinvolved

Binding energy(kcal M−1)

bindingconstant (M−1)

Asn 18 H-bonding andhydrophobic

−4.27 1.36 × 103

Glu 17

[

[

S.K. Chaturvedi et al. / International Journal

n the absence and presence of varying concentration of taurine.iable cells reduce the MTT and form a blue colored product forma-on which is used to measure cell viability. 120 h aged HSA fibrils on4 h incubation showed significant decline in cell viability to 44%nd 50% relative to both control cell lines means HSA fibrils ableo induce cell mortality and reduced the cell survival by damaginghe membrane of cells. Dye binding and imaging results showed thenti-amyloidogenic behavior of taurine against HSA fibrils. Furthero check the effect of taurine on amyloid mediated cell cytotoxicitye incubated 250 and 750 �M of taurine with HSA and added to

ell culture. Results in Fig. 8 depicted that cell viability of PC12ells were regained to 58% and 71% in the presence of 250 and50 �M of taurine concentration, respectively. Similar effects werebserved in SH-S5H5 cells, i.e., survivability of cells progressivelyncreases in dose dependent manner. We have also checked therotective effect of taurine on cell lines and for this we incubatedell line with taurine and then exposed to 120 h aged HSA fibrils. Weound cells pre-incubated with taurine not showing any resistancegainst fibrils and nearly same viability was found as in cell withoutre-incubated with taurine which further support the results thategain of cell viability is solely due to anti-amyloidogenic action ofaurine (data not shown).

.10. Molecular docking and molecular dynamic simulation studyf HSA–taurine interaction

Molecular docking study was done to check which aminocid residue involves in the HSA–taurine interaction and to con-rm whether the taurine binds on HSA or not. Sometimes smallolecule with non-specific binding shows inhibitory effect on

ggregation which might be due to environmental changes aroundhe protein molecules such as by solvation effect. The principleegions of ligand binding on HSA are located in hydrophobic cav-ties in subdomain II and III which correspond to site I and site II,espectively. Autodock 4.2 program was used to examine the bind-ng mode of taurine to HSA. The docking results showed that taurineinds within the binding pocket of subdomain IB (Fig. 9A and B).arlier studies reported several endogenous compounds and somerugs also bind to subdomain IB [51,52]. Subdomain IB (108–197mino acid residues) of HSA is a third major binding site for var-ous exogenous and endogenous molecules such as drugs, hemin,ilirubin and fatty acids, etc., apart from two conventional Sudlowites that is site I and site II. According to He and Carter, subdomainB is made up of four helices and an N-terminal portion of extendedolypeptide with turns that create a folding topology resembling

simple up-down helical bundle. The binding of drug moleculess mainly the result of cation–� interactions with Arg114, polarnteraction with Lys190, and electrostatic attraction to Asp187 [53].SA in reversibly and non-covalent manner interacts with diverselass of molecules within subdomain IB. Subdomain IB actively par-icipates in binding with natural products (limonene, aristolochiccid, glycyrrhetinic acid) [47] and anticancer drugs and formula-ions (cytosine �-d arabinofuranoside, doxorubicin, camptothecin,aunorubicin, teniposide, suramin) [54]. Moreover, anticoagulanticoumarol, steroids (bile acids, carbenoxolone), and syntheticyes (azocarmine B, methyl orange), trans-feruloyl maslinic acid,-hydroxy-4-methyl coumarin derivatives and lidocaine stronglyind to the IB domain [55]. Glu 17, Asn 18, Leu 135 and Lys62 are the key amino acid residues located in the vicinity ofaurine–HSA complex and involved in the interaction of taurineith HSA (Table 3). HSA–taurine complex is mainly stabilized byydrogen bonding and hydrophobic interactions. Molecular simu-

ation study was further performed to support the docking study56]. The structure properties, such as root mean-square devia-ion (RMSD) and radius of gyration of the backbone and proteintoms were calculated, respectively. The results show the protein

[

Leu 135Lys 162

is well stabilized within 2 ns and radius of gyration shows the pro-tein become more compact at the end of 5 ns. The compound wellfit into the protein throughout the simulation (Fig. 9C and D).

4. Conclusion

In view of all results this can be concluded that taurine is anefficient anti amyloidogenic agent against HSA fibrillation and theprocess is concentration dependent of taurine. Thus from the subse-quent work we delineates that taurine forced to both secondary aswell as tertiary structure to get stabilized and affects aggregationkinetic parameters by slowing down lag time in a concentrationdependent manner, means nucleation process of amyloid aggrega-tion was interrupted. Nevertheless sulphonic and amino functionalgroup attached to taurine make it efficient to easily interfere withthe head to head and side by side elongation of fibrils during fibrilla-tion process. This study may open the door for new pharmaceuticalformulations that would be able to abrogate the amyloid fibril for-mation through native state stabilization.

Acknowledgement

S.K. Chaturvedi, P. Alam, J.M. Khan acknowledge Council ofScientific and Industrial Research (CSIR), New Delhi, India for pro-viding financial assistance.

References

[1] A.L. Fink, Protein aggregation: folding aggregates, inclusion bodies andamyloid, Folding Des. 3 (1998), R9-R23.

[2] D.P. Raleigh, Guilt by association: the physical chemistry and biology ofprotein aggregation, J. Phys. Chem. Lett. 5 (2014) 2012–2014.

[3] C. Haass, D.J. Selkoe, Soluble protein oligomers in neurodegeneration: lessonsfrom the Alzheimer’s amyloid �-peptide, Nat. Rev. Mol. Cell Biol. 8 (2007)101–112.

[4] M. Maheshwari, S. Shekhar, B.K. Singh, I. Jamal, N. Vatsa, V. Kumar, A. Sharma,N.R. Jana, Deficiency of Ube3a in Huntington’s disease mice brain increasesaggregate load and accelerates disease pathology, Hum. Mol. Genet. (2014)ddu343.

[5] C.A. Ross, M.A. Poirier, Protein aggregation and neurodegenerative disease,Nat. Med. (2004).

[6] N. Shigihara, A. Fukunaka, A. Hara, K. Komiya, A. Honda, T. Uchida, H. Abe, Y.Toyofuku, M. Tamaki, T. Ogihara, Human IAPP-induced pancreatic b celltoxicity and its regulation by autophagy, J. Clin. Investig. 124 (2014) 3634.

[7] S.P. Schwendeman, Recent advances in the stabilization of proteinsencapsulated in injectable PLGA delivery systems, Crit. Rev. Ther. Drug CarrierSyst. 19 (2002).

[8] J.M. Khan, S.K. Chaturvedi, S.K. Rahman, M. Ishtikhar, A. Qadeer, E. Ahmad,R.H. Khan, Protonation favors aggregation of lysozyme with SDS, Soft Matter10 (2014) 2591–2599.

[9] G. Sancataldo, V. Vetri, V. FoderÃ, G. Di Cara, V. Militello, M. Leone, Oxidationenhances human serum albumin thermal stability and changes the routes ofamyloid fibril formation, PloS One 9 (2014) e84552.

10] L. Breydo, K.D. Reddy, A. Piai, I.C. Felli, R. Pierattelli, V.N. Uversky, The crowdyou’re in with: effects of different types of crowding agents on proteinaggregation, Biochim. Biophys. Acta (BBA)-Proteins Proteomics 1844 (2014)346–357.

11] T.P.J. Knowles, M. Vendruscolo, C.M. Dobson, The amyloid state and itsassociation with protein misfolding diseases, Nat. Rev. Mol. Cell Biol. 15

(2014) 384–396.

12] C. Regitz, L. Marie DuÃYling, U. Wenzel, Amyloid-beta (Ab-42) inducedparalysis in Caenorhabditis elegans is inhibited by the polyphenol quercetinthrough activation of protein degradation pathways, Mol. Nutr. Food Res.(2014).

3 of Bio

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

84 S.K. Chaturvedi et al. / International Journal

13] J.N. Abraham, D. Kedracki, E. Prado, C. Gourmel, P. Maroni, C. Nardin, Effect ofthe interaction of the amyloid b (1–42) peptide with short single strandedsynthetic nucleotide sequences: morphological characterization of theinhibition of fibrils formation and fibrils disassembly, Biomacromolecules(2014).

14] K.C. Nadimpally, A. Paul, B. Mandal, Reversal of aggregation using b-breakerdipeptide containing peptides: application to Ab (1–40) self-assembly and itsinhibition, ACS Chem. Neurosci. 5 (2014) 400–408.

15] A. Varshney, Y. Ansari, N. Zaidi, E. Ahmad, G. Badr, P. Alam, R.H. Khan, Analysisof binding interaction between antibacterial ciprofloxacin and human serumalbumin by spectroscopic techniques, Cell Biochem. Biophys. 70 (2014)93–101.

16] S. Bhattacharya, N.K. Pandey, A. Roy, S. Dasgupta, Effect of(−)-epigallocatechin gallate on the fibrillation of human serum albumin, Int. J.Biol. Macromol. 70 (2014) 312–319.

17] R.J. Huxtable, Physiological actions of taurine, Physiol. Rev. 72 (1992)101–163.

18] N. Froger, L. Moutsimilli, L. Cadetti, F. Jammoul, Q.-P. Wang, Y. Fan, D. Gaucher,S.G. Rosolen, N. Neveux, L. Cynober, Taurine: the comeback of a neutraceuticalin the prevention of retinal degenerations, Prog. Retinal Eye Res. (2014).

19] J. Menzie, C. Pan, H. Prentice, J.-Y. Wu, Taurine and central nervous systemdisorders, Amino Acids 46 (2014) 31–46.

20] S.W. Schaffer, T. Ito, J. Azuma, Clinical significance of taurine, Amino Acids 46(2014) 1–5.

21] M. Imae, T. Asano, S. Murakami, Potential role of taurine in the prevention ofdiabetes and metabolic syndrome, Amino Acids 46 (2014) 81–88.

22] S. Venkatachalam, P. Kuppusamy, B. Kuppusamy, S. Dhanapal, The potency ofessential nutrient taurine on boosting the antioxidant status andchemopreventive effect against benzo (a) pyrene induced experimental lungcancer, Biomed. Prevent. Nutr. 4 (2014) 251–255.

23] E. Park, S.Y. Park, C. Dobkin, G. Schuller-Levis, Development of a novelcysteine sulfinic acid decarboxylase knockout mouse: dietary taurine reducesneonatal mortality, J. Amino Acids 2014 (2014).

24] J. Juarez, P. Taboada, V. Mosquera, Existence of different structuralintermediates on the fibrillation pathway of human serum albumin, Biophys.J. 96 (2009) 2353–2370.

25] S. Freire, M.H. de Araujo, W. Al-Soufi, M. Novo, Photophysical study ofThioflavin T as fluorescence marker of amyloid fibrils, Dyes Pigments (2014).

26] S.S.S. Wang, K.-N. Liu, C.-H. Wu, J.-K. Lai, Investigating the influences of redoxbuffer compositions on the amyloid fibrillogenesis of hen egg-whitelysozyme, Biochim. Biophys. Acta (BBA)—Proteins Proteomics 1794 (2009)1663–1672.

27] A. Qadeer, E. Ahmad, M. Zaman, M.W. Khan, J.M. Khan, G. Rabbani, K.F.Tarique, G. Sharma, S. Gourinath, S. Nadeem, G. Badr, R.H. Khan,Concentration-dependent antagonistic persuasion of SDS and naphthalenederivatives on the fibrillation of stem bromelain, Arch Biochem. Biophys. 540(2013) 101–116.

28] G. Hopping, J. Kellock, R.P. Barnwal, P. Law, J. Bryers, G. Varani, B. Caughey, V.Daggett, Designed alpha-sheet peptides inhibit amyloid formation bytargeting toxic oligomers, Elife 3 (2014) e01681.

29] D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A.E. Mark, H.J. Berendsen,GROMACS: fast, flexible, and free, J. Comput. Chem. 26 (2005) 1701–1718.

30] W.R.P. Scott, P.H. Hünenberger, I.G. Tironi, A.E. Mark, S.R. Billeter, J. Fennen,A.E. Torda, T. Huber, P. Krüger, W.F. van Gunsteren, The GROMOSbiomolecular simulation program package, J. Phys. Chem. A 103 (1999)3596–3607.

31] B. Pullman, H.J.C. Berendsen, J.P.M. Postma, W.F. van Gunsteren, J. Hermans,Interaction Models for Water in Relation to Protein Hydration IntermolecularForces, vol. 14, Springer, Netherlands, 1981, pp. 331–342.

32] B. Hess, H. Bekker, H.J.C. Berendsen, J.G.E.M. Fraaije, LINCS: a linear constraintsolver for molecular simulations, J. Comput. Chem. 18 (1997) 1463–1472.

33] N. Zaidi, M.R. Ajmal, G. Rabbani, E. Ahmad, R.H. Khan, A comprehensive insight

into binding of hippuric acid to human serum albumin: a study to uncover itsimpaired elimination through hemodialysis, PloS One 8 (2013) e71422.

34] R. Khurana, C. Coleman, C. Ionescu-Zanetti, S.A. Carter, V. Krishna, R.K. Grover,R. Roy, S. Singh, Mechanism of thioflavin T binding to amyloid fibrils, J. Struct.Biol. 151 (2005) 229–238.

[

logical Macromolecules 80 (2015) 375–384

35] S. Li, L. Wang, C.C. Chusuei, V.M. Suarez, P.L. Blackwelder, M. Micic, J.Orbulescu, R.M. Leblanc, Nontoxic carbon dots potently inhibit human insulinfibrillation, Chem. Mater. 27 (2015) 1764–1771.

36] H.-Y. Lian, H. Zhang, Z.-R. Zhang, H.t.M. Loovers, G.W. Jones, P.J.E. Rowling, L.S.Itzhaki, J.-M. Zhou, S. Perrett, Hsp40 interacts directly with the native state ofthe yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils, J.Biol. Chem. 282 (2007) 11931–11940.

37] H. Gong, Z. He, A. Peng, X. Zhang, B. Cheng, Y. Sun, L. Zheng, K. Huang, Effectsof several quinones on insulin aggregation, Sci. Rep. 4 (2014).

38] J.W. Wu, K.-N. Liu, S.-C. How, W.-A. Chen, C.-M. Lai, H.-S. Liu, C.-J. Hu, S.S.S.Wang, Carnosine’s effect on amyloid fibril formation and induced cytotoxicityof lysozyme, PloS One 8 (2013) e81982.

39] R. Kisilevsky, L.J. Lemieux, P.E. Fraser, X. Kong, P.G. Hultin, W.A. Szarek,Arresting amyloidosis in vivo using small-molecule anionic sulphonates orsulphates: implications for Alzheimer’s disease, Nat. Med. 1 (1995)143–148.

40] M. Owczarz, P. Arosio, Sulfate anion delays the self-assembly of human insulinby modifying the aggregation pathway, Biophys. J. 107 (2014) 197–207.

41] K. Ikeda, T. Okada, S.-i. Sawada, K. Akiyoshi, K. Matsuzaki, Inhibition of theformation of amyloid Î2-protein fibrils using biocompatible nanogels asartificial chaperones, FEBS Lett. 580 (2006) 6587–6595.

42] P. Taboada, S. Barbosa, E. Castro, V.c. Mosquera, Amyloid fibril formation andother aggregate species formed by human serum albumin association, J. Phys.Chem. B 110 (2006) 20733–20736.

43] S. Ghosh, N.K. Pandey, P. Banerjee, K.N. Chaudhury, r. Veronika Nagy, S.Dasgupta, Copper (II) directs formation of toxic amorphous aggregatesresulting in inhibition of hen egg white lysozyme fibrillation under alkalinesalt mediated conditions, J. Biomol. Struct. Dyn. (2014) 1–54.

44] S. Sen, S. Konar, A. Pathak Mahanty, S. Dasgupta, S. DasGupta, Effect offunctionalized magnetic MnFe2O4 nanoparticles on fibrillation of humanserum albumin, J. Phys. Chem. B 118 (2014) 11667–11676.

45] J.K. Armstrong, R.B. Wenby, H.J. Meiselman, T.C. Fisher, The hydrodynamicradii of macromolecules and their effect on red blood cell aggregation,Biophys. J. 87 (2004) 4259–4270.

46] J.M. Khan, S.K. Chaturvedi, R.H. Khan, Elucidating the mode of action of ureaon mammalian serum albumins and protective effect of sodium dodecylsulfate, Biochem. Biophys. Res. Commun. 441 (2014) 681–688.

47] S.K. Chaturvedi, E. Ahmad, J.M. Khan, P. Alam, M. Ishtikhar, R.H. Khan,Elucidating the interaction of limonene with bovine serum albumin: amulti-technique approach, Mol. BioSyst. (2014).

48] S.K. Chaturvedi, E. Ahmad, J.M. Khan, P. Alam, M. Ishtikhar, R.H. Khan,Elucidating the interaction of limonene with bovine serum albumin: amulti-technique approach, Mol. BioSyst. 11 (2015) 307–316.

49] W. Hoyer, T. Antony, D. Cherny, G. Heim, T.M. Jovin, V. Subramaniam,Dependence of alpha-synuclein aggregate morphology on solutionconditions, J. Mol. Biol. 322 (2002) 383–393.

50] S. Ghosh, N.K. Pandey, S. Dasgupta, Crowded milieu prevents fibrillation ofhen egg white lysozyme with retention of enzymatic activity, J. Photochem.Photobiol. B: Biol. 138 (2014) 8–16.

51] A. Garg, D.M. Manidhar, M. Gokara, C. Malleda, C. Suresh Reddy, R.Subramanyam, Elucidation of the binding mechanism of coumarin derivativeswith human serum albumin, PLoS One 8 (2013) e63805.

52] F. Zsila, Subdomain IB is the third major drug binding region of human serumalbumin: toward the three-sites model, Mol. Pharm. 10 (2013) 1668–1682.

53] K.L. Hein, U. Kragh-Hansen, J.P. Morth, M.D. Jeppesen, D. Otzen, J.V. MÃ, P. ller,Nissen, Crystallographic analysis reveals a unique lidocaine binding site onhuman serum albumin, J. Struct. Biol. 171 (2010) 353–360.

54] P. Alam, S.K. Chaturvedi, T. Anwar, M.K. Siddiqi, M.R. Ajmal, G. Badr, M.H.Mahmoud, R. Hasan Khan, Biophysical and molecular docking insight into theinteraction of cytosine �-d arabinofuranoside with human serum albumin, J.Lumin. 164 (2015) 123–130.

55] D.P. Yeggoni, R. Subramanyam, Binding studies of

l-3,4-dihydroxyphenylalanine with human serum albumin, Mol. BioSyst. 10(2014) 3101–3110.

56] C. Malleda, N. Ahalawat, M. Gokara, R. Subramanyam, Molecular dynamicssimulation studies of betulinic acid with human serum albumin, J. Mol. Model18 (2014) 2589–2597.