Investigation of interactions between zein and natamycin by fluorescence.pdf

Investigation of interactions between zein and natamycinby fluorescence spectroscopy and molecular dynamics simulationXiaochuan Wua,Xiaojian Zhaoa,Zian Denga,Xianrui Liangb,Sheng Fanga,aSchool of Food Science and Biotechnology,Zhejiang Gongshang University,Xuezheng Street No.18,Hangzhou 310018,ChinabCollege of Pharmaceutical Sciences,Zhejiang University of Technology,Hangzhou 310014,Chinaa b s t r a c ta r t i c l ei n f oArticle history:Received 19 August 2020Received in revised form 7 November 2020Accepted 23 November 2020Available online 27 November 2020Keywords:ZeinNatamycinFluorescence quenchingMolecular dynamics simulationZeinnanoparticleshavebeendemonstratedasanefficientnanocarrierfornatamycintoimproveitssolubilityandstability.This study investigated the molecular-level interactions between the complex of zein with natamycin.Thefluorescencepropertiesincludingexcitation-emissionspectra andfluorescencelifetimeofzeinwerefirstde-termined.The fluorescence quenching of zein by natamycin was analyzed by Stern-Volmer equation and wasstaticwith a rate constant Kqof 0.633 1013M1s1at303 K.The association constant(Ka)atdifferent temper-atures were calculated in order to obtain thermodynamic process parameters.The quenching process was spon-taneouswith a binding constant Kaof1.134 105M1at 303K.The positive signofH and S indicatesthat thehydrophobic interactions play a major role in the formation of the zein-natamycin complex.The interactionmechanisms between zein and natamycin were further studied using molecular dynamics simulation.The geo-metrical figurations of the most possible binding model that stabilized by both hydrophobic interaction andhydrogen-bondnetworkwerepresented.Thecomplexwasformedwithbothhydrophobicinteractionsbetweenthe conjugated tetraene group of natamycin and the Phe(183),Val(184),Leu(181)and Phe(123)residues ofzein,and hydrogen-bonds network between the OH groups of natamycin and the Val(18),Gln(116),and Ser(112)residues of zein.2020 Elsevier B.V.All rights reserved.1.IntroductionNatamycin(also known as pimaricin or E235,CAS number 7681-93-8),is a widely used food preservative that produced by Strepto-myces natalensis 1.The structure of natamycin is shown in Fig.S1.It is mainly applied to inhibit the growth of fungus and used forsurface treatments in dairy-based cheese and meat products 2.However,natamycin is hardly dissolved in water,which limits itsapplications in the food industry 3.Developing an effectivenatamycin carrier with improved solubility and stability is of utmostimportance 4.Nowadays,zein protein-based nanomaterials as novel deliveryvehicles are gaining considerable attention in pharmaceutical,food,and agriculture applications due to its biocompatibility,biodegrad-ability,low-cost and easy preparation 5,6.Zein is a plant storageprotein of maize and has a large content of hydrophobic aminoacids including alanine,leucine,phenylalanine,and proline 7.Bothof the hydrophobic and hydrophilic domains of zein protein canform interactions with cargoes that facilitate its applications as ananocarrier for hydrophobic substances 6.Recently,a natamycin-loaded zein nanoparticle stabilized by carboxymethyl chitosan withhigh antifungal activities against Botrytis cinerea was fabricated byour group 8.It was proposed that the hydrophilic and hydrophobicinteractions formed between natamycin and zein facilitated the en-capsulation process.However,up to now,there are not so many de-tailed studies about the molecular-level insight into the interactionsbetween these two molecules.Towards a more rational approach to encapsulate additives,works have been devoted to studying the interactions between pro-teins and cargoes 9,10.Joye et al.9 studied the fluorescencequenching behaviors between resveratrol and zein protein,anddemonstrated the predominantly interactions through hydrogenbonds between these two molecules.In recent years,spectroscopyanalysis combined with molecular simulations has become a power-ful tool for elucidating interaction mechanisms between proteinsand small organic molecules 1113.In this study,the molecularinteractions between natamycin and zein were investigated by fluo-rescence spectroscopy combined with molecular dynamics(MD)simulation.The fluorescence quenching effects of zein protein bynatamycin were determined and modeled.The molecular bindingmechanisms and geometrical configurations of the natamycin-zeincomplex were obtained using MD simulation.Journal of Molecular Liquids 327(2021)114873 Corresponding author.E-mail address:(S.Fang).https:/doi.org/10.1016/j.molliq.2020.1148730167-7322/2020 Elsevier B.V.All rights reserved.Contents lists available at ScienceDirectJournal of Molecular Liquidsjournal homepage: and methods2.1.MaterialsNatamycin(mass purity of 95%,food-grade)was supplied byZhejiang Silver-Elephant Bio-engineering(Zhejiang,China).Zein witha minimum protein content of 97%(mass purity,AR grade)from cornwas purchased from Macklin Co.,Ltd.(Shanghai,China).Absolute etha-nol was of analytical grade and purchased from Xilong Chemicals(Zhejiang,China).2.2.3D fluorescence measurementsAcertainamountofzeinornatamycinwasdissolvedinaqueouseth-anol solution(80%,v/v).The final concentrations of zein and natamycinsolutions were 12.5 M(the molecular weight of zein is set at 22 kDa)and 17.5 M,respectively.The 3D fluorescence of samples was deter-mined using a fluorescence spectrophotometer(Model F-7000,Hitachi,Japan)at 298 K.The excitation and emission wavelength range waschanged from 250 to 400 nm with a step of 5 nm,the slit width of exci-tation and emission was 5 nm.2.3.Time-resolved fluorescence spectroscopyZein was dissolved in aqueous ethanol solution(80%,v/v)and thefinal concentration was 12.5 M.The fluorescence quenching lifetimeof zein was determined with the method of time-resolved fluorescenceby an FLS1000 fluorescence spectrometer(Edinburgh Instruments,Liv-ingston,Scotland,UK).Anultrafastnanosecondflashlampwasequippedand operated at 280 nm as a light source of fluorescence excitation.Ad-ditionally,emission wavelength was 305 nm and time resolution was19.53 ps.2.4.Fluorescence quenching measurementsAcertainamountofzeinornatamycinwasdissolvedinaqueouseth-anol solution(80%,v/v).The two solutions were mixed according to acertain ratio to prepare series quenching solutions.The final concentra-tionofzeinproteinwasfixedat12.5M(themolecularweightofzeinisset at 22 kDa)7 with natamycin varied from 0 to 17.5 M and a gradi-entof2.5M.Thesamplesweremeasuredusingafluorescencespectro-photometer(Model F-7000,Hitachi,Japan)at 293,303,and 313 K withan excitation wavelength of 280 nm.The solution was maintained atleast 20 min for temperature equilibrium.The wavelength range wasfrom 290 to 400 nm under 1.0 nm slit widths.The quenching parame-ters and binding constants were calculated by fluorescence intensityat the maximum fluorescence emission wavelength.2.5.Molecular dynamics simulationTheMDsimulationwasperformedusingGROMACS2018.1softwarepackage 14.The initial coordinates of natamycin(pimaricin)wasbased on the crystal structure 15.The-zein structure is constructedbasedonmolecularinformationfromtheliterature16.CHARMMGen-eral Force Field(CGenFF)and Charmm36 force field were used to de-scribe the coordination of the ligand and protein,respectively.The unit cell of 8 nm 8 nm 8 nm standard cubic box wasestablished and the complex was placed in the center of the box.Thesystem was simulated three times with different random seed andstart positions.For the equilibrium of charges,Na+and Clions wereadded to offset charges from the complex.Transferable IntermolecularPotential 3 Point model(TIP3P)was used to model water molecule be-havior.Thesystemtemperature andpressure were maintainedat 298 Kand 1 bar using V-rescale and Parrinello-Rahman method,respectively17,18.BondlengthsandangleswerefixedfollowingLINCSconstraints.The system was geometry optimized using the steepest descent algo-rithm.Equation of motion was integrated using Leapfrog integratorwith a time step of 2 fs using the Verlet leapfrog algorithm.The short-range electrostatic cut-off was 1.0 nm and the long-range electrostaticforce was described using the Particle Mesh Ewald method 19.NVTand NPT ensemble was carried out for 100 ps.Finally,50 ns MD simula-tions were performed under the periodic boundary condition and theMD trajectories were analyzed with GROMACS.2.6.Statistical analysisAllmeasurements were carried out intriplicate.Thevalues were de-scribed as the mean STD.The data were analyzed according to theanalysis of variance(ANOVA).The significance was defined at the 95%confidence level.3.Results and discussion3.1.3D fluorescence spectroscopy of zein and natamycinThe fluorescence intensity contour maps of zein and natamycin areshown in Fig.1.The characteristic peak of zein appears at an excitationwavelength of approximately 270 nm and a maximum emission wave-length of 305 nm.It is known that the intrinsic fluorescence property ofprotein mainly comes from the tryptophan(Trp),tyrosine(Tyr),andphenylalanine(Phe)residues.The absorption at 305 nm of zein mainlycomes from its tyrosine residues,since the content of tryptophan resi-due is very low,and the tyrosine residue content accounts for almost5%(w/w)of the entire protein.Joye et al.9 studied the fluorescencequenching of zein and gliadin by resveratrol binding.They attributedthe emission peak at 304 nm of zein and 336 nm of gliadin to the tyro-sine and tryptophan residues,respectively.Therefore,the fluorescenceintensity of the maximum wavelength at 305 nm was adapted to calcu-latetheparameters of fluorescence quenchingmodel20.Additionally,natamycin has no any emission peaks(below 400 nm)at the excitationwavelengthregionbetween280and320nmasshowninFig.1(B).Itin-dicatesthatthefluorescencequenchingofzeinwillnotbeinfluencedbythe background fluorescence of natamycin at this region.Therefore,theexcitation wavelength of 280 nm was selected for the determination ofquenching process 9.3.2.Time-resolved fluorescence spectroscopy of zeinThefluorescencedecaycurveofzeinispresentedinFig.2(A)andan-alyzedaccordingtothemethodofMirdaetal.21.Thedecaycurvewasfitted by Eq.(1)and the average fluorescence quenching lifetime wascalculated by Eq.(2):F t iiexp t=i10ii?2iii?i2where,iis the fluorescence lifetime,iis the fractional contribution ofi,0is the average lifetime of fluorescence decay,and F(t)is a fittedequation of hypothetical multi-exponential decay.The fitted curve isshown in Fig.2(A)and the residual plot that evaluates the accuracy ofthe regression is shown in Fig.2(B).The values of 0and 2are18.5 1.3 ns and 1.1 0.1,respectively.The results are similar withprevious study about the fluorescence lifetime of protein 22.The 0value is meaningful for determining whether the quenching process ofzein by different quenchers is dynamic or static 23.3.3.Fluorescence quenching and mechanismsThe interactions between zein protein and natamycin were studiedby fluorescence quenching methods.The results are shown in Fig.3.ItX.Wu,X.Zhao,Z.Deng et al.Journal of Molecular Liquids 327(2021)1148732can be seen that the fluorescence intensity of zein gradually decreasesas the concentration of natamycin increases.Moreover,the wavelengthof fluorescence emission peak tends to blueshift with the increase ofnatamycin which can be attributed to the existence of intermolecularinteractions 24.Fluorescence quenching can be divided into dynamic and staticquenching which shows different patterns of temperature impact andexcited-state lifetime 25,26.The fluorescence quenching data wereanalyzed by the Stern-Volmer equation as:F0=F 1 Kq0N?1 KSVN?3where F and F0represent the fluorescence intensities of zein proteinwith or without the quencher(natamycin),respectively;N meansthe concentration of the natamycin,Kqis the quenching rate constantof a biomolecule,Ksvis the Stern-Volmer dynamic quenching constant,0represents the average excited-state lifetime of the zein in the ab-sence of quencher.In this study,the fluorescence quenching lifetimeFig.1.The 3D fluorescence spectroscopy of(A)zein and(B)natamycin.Fig.2.(A)Fluorescence decay curve of zein(orange line)and fitting curve(red line)against measure time(ns);(B)Residual plot of the fluorescence decay profile.X.Wu,X.Zhao,Z.Deng et al.Journal of Molecular Liquids 327(2021)1148733of zein was 1.85 108s.The values of Ksv,Kq,and the regression coef-ficient(R2)at three temperatures(20,30,and 40 C)are listed inTable 1.The values of R2are about 0.99 which indicates thelinear equa-tion is appropriate for quenching modeling.The Ksvvalues are near1.2 105M1,and not changed too much as the temperatureincreasing.from 293 to 313 K.Furthermore,the values of Kqat three tempera-tures surpass three orders of the scatter collision rate constant forkinds of quenchers with biopolymer molecular 26.These results sug-gest that the fluorescence quenching of zein protein by natamycin isstatic(Kq 2 1010M1s1)with the formation of the complex bystrong binding affinity.Our results are similar to literature about inter-actions between protein and small organic molecules 2729.For ex-ample,the interaction between-casein and p-coumaric acid wasdrivenbystatic quenchingwiththeKqvalue of 2.2 1013M1s127.3.4.Binding constants and thermodynamic parametersThe equilibrium characteristics of the complex between zein andnatamycin during static quenching can be quantitatively described bythe association constant(Ka).The value of Kacan be obtained from theLineweaver-Burk equation as:logF0FF?nlogN?log Ka4where n means the number of the binding site.The regression is showninFig.4 andresults are listed inTable 1.The values ofKaare in theorderof105M1,indicatingastronginteractionbetweennatamycinandzein.The Kavalue obtained is similar to that for some proteins with smallorganic molecules,such as 13.19 105M1for bovine serum albumin(BSA)withafatinibat288K,and1.19105M1forhumanserumalbu-min(HSA)with anlotinib at 288 K 30,31.On the other hand,n valuesare found to be larger than 1 that indicates the existence of at least onebinding site between zein and natamycin 30,32.The temperature-dependent of Kacan be fitted by the Vant Hoffequation as:ln Ka HRTSR5where H is the enthalpy change,S is the entropy change,and R is theuniversal gas constant.These values can be calculated by the linearequation between ln(Ka)and 1/T.Then the free energy change(G)ofthebindingprocessiscalculatedfromtheGibbs-Helmholtzequationas:G HT?S6The thermodynamic parameters(H,S,and G)for binding pro-cess of zein and natamycin are listed in Table 1.The G is negativewhich suggests that the formation of complex is an endothermic anddriven by interaction forces spontaneously 31.The interactions be-tween small molecules and macromolecule include electrostatic inter-actions,hydrophobic force,van der Waals force,and hydrogen bonds,etc.Ross and Subramania have presented a relationship between thesigns of thermodynamic parameters and interactions that might occur,which can be briefly described as:(a)if