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Intungamubiri z'igitagangurirwa cya poroteyine (spider proteine ​​spider) zifite uburyo bwinshi bushoboka mugutezimbere ibinyabuzima bishya, ariko imiterere yabyo ya multimodal hamwe no guhuriza hamwe bikunda kubagora kubona kandi byoroshye kuyikoresha.Hano twatangaje ko recombinant miniature spidroin proteyine kandi, icy'ingenzi, indangarubuga ya N-terminal (NT) ubwayo ikora byihuse kandi byifashisha hydrogels mu mucyo kuri 37 ° C.poroteyine zo mu bwoko bwa fusion zigizwe na NT na proteine ​​yicyatsi kibisi cyangwa purine nucleoside fosifora ikora poroteyine zuzuye zuzuye.Hydrogels.Ibisubizo byacu byerekana ko recombinant NT na proteyine za fusion zitanga umusaruro mwinshi kandi bigaha hydrogels hamwe nibintu byiza nko gukorera mu mucyo, gelation nta guhuza, hamwe na immobilisation itaziguye ya poroteyine zikora cyane.
Igitagangurirwa gifite ibice birindwi bitandukanye bya glande ya silk, buri kimwe gitanga ubwoko bwihariye bwa silik.Ubwoko burindwi bwa silike bugizwe na poroteyine yigitagangurirwa (spidroins) ibisigisigi bigera ku 6000 kandi birimo akarere kanini gisubiramo kuzengurutswe na N- na C-terminal (NT na CT) 1,2.Ubwoko bwa silike bwizwe cyane, ampulla yibanze, bukorwa na glande yibanze ya ampulla.Muri iyi glande, monolayeri yingirabuzimafatizo ya epiteliyale ikomatanya poroteyine za spidroin ikayirekura mu mitsi ya glande, aho iba iri mu buryo bworoshye (doping) ku buryo bukabije cyane (30-50% w / v) 3,4.Imitunganyirize no guhindura poroteyine nyamukuru ya ampullar spidroin muri gland byaganiriweho, ariko ibimenyetso byinshi byubushakashatsi byerekana ko hariho ihinduka rusange ryihindagurika kandi / cyangwa ridahinduka hamwe na micellar cyangwa lamellar5,6,7,8,9,10.Mugihe ibyasubiwemo bigenga imiterere yubukorikori bwa fibre yubudodo, ikora β-urupapuro rwa nanocristal nuburyo bwa amorphous11,12,13,14,15, domaine zanyuma zigenga fibre ya silike bitewe nibihinduka kumiterere ya glande ya silike 16,17,18.Mugucunga imiterere yubudodo, 19. Indangarubuga za terminale zabitswe mubwihindurize kandi imikorere yazo irashobora kuba rusange kuri proteine ​​zose za spidroin 2,20,21.Mugihe cyo kunyura muri glande, pH ya spidroine igabanuka kuva kuri 7,6 ikagera kuri <5.716 kandi ikiyongera hamwe nogosha no kurambura byahujwe no kugenda binyuze mumiyoboro igenda igabanuka.Mugukemura, CT ni α-ihindagurika igizwe na dimer17, ariko mugusubiza imbaraga za pH nimbaraga zogosha, CT irakingura kandi igahindura β-layers16, 17, birashoboka ko itera β -ibice mukarere gisubiramo Guhindura 16. NT ni monomeric munsi imiterere yerekana imiterere mumyanya ya glande no guhuza imbaraga za spidroin, ariko mugihe pH yagabanutse, protononike yiminyururu myinshi ya aside irike ya karubonike itera kugabanuka kwa NT hamwe na pKa ya 6.5, bityo bigahindura NT no gukosora spidroine muri rusange ingano.imiyoboro 16,18.Rero, NT igira uruhare runini mugushinga filament, ihinduka kuva monomer mugipfundikizo ikagera kuri dimer muri fibre 23,24,25.NT ikomeje gushonga cyane kandi ihindagurika mubihe byose byizwe kugeza ubu, 16, 18, 19, 20, 26, 27, 28, 29, byashishikarije iterambere ryayo nka label yongerera imbaraga imbaraga zo gukora proteine ​​zitandukanye.
Recombinant mini spider silk protein, igizwe na NT imwe, akarere kamwe gasubiramo, CT imwe, hamwe na tagi ya His6 (His-NT2RepCT) kugirango isukure, irashonga mumazi yo mumazi nka protein yigitagangurirwa kavukire kandi yigana ibintu byingenzi biranga igitagangurirwa. .ubwishingizi 25.31.His-NT2RepCT irashobora guhindurwamo fibre ikomeza ikoresheje imashini ya biomimetike aho pH 8 ikuramo ibishishwa ikoherezwa muri pH 525,32,33,34,35.Bioreactor fermentation ya E. coli yerekana-NT2RepCT ye hanyuma nyuma yubuvuzi byavuyemo umusaruro> 14 g / L nyuma yo kwezwa.Umusaruro mwinshi, gukomera cyane, hamwe nigisubizo gihagije cya-NT2RepCT kumiterere ya acide byose biterwa na NT23, 25, 34.
Hano turatanga raporo yihuse ya hydrogels ibonerana ituruka kuri proteine ​​spidroin ya recombinant, harimo na NT yonyine, dushyiramo proteine ​​kuri 37 ° C.Twifashishije thioflavin T fluorescence (ThT), Fourier ihindura infrarafurike ya spekitroscopi (FTIR), magnetic magnetic resonance spectroscopy (NMR) hamwe na microscopi electronique (TEM), twasanze poroteyine za NT na microspider zihindura imiterere β -papuro na fibrile isa na amyloide. iyo geles ikozwe.Byongeye kandi, poroteyine zo mu bwoko bwa NT hamwe na poroteyine y'icyatsi kibisi (GFP) cyangwa purine nucleoside fosifori (PNP) ikora hydrogène ifite ibice byuzuye byo guhuza.Imvugo-yinjiza cyane muri heterologous host, hamwe no gushiraho hydrogels byihuse mubihe bya physiologique, byugurura amahirwe yo kubyara umusaruro ushimishije wa hydrogène hamwe nibikorwa byubuhanga.
Bitandukanye na poroteyine nyinshi za spombroin spombroin36, His-NT2RepCT ihagaze neza muri buffer ya Tris-HCl kuri pH 8 kandi irashobora kwibanda kuri mg / mL 500 nta mvura igwa25.Kubwibyo, twatunguwe no kubona ko iyi proteyine ikora byihuse igaragara neza, yifashisha hydrogels iyo yishyizwe kuri 37 ° C (Ishusho 1b-d).Ubushakashatsi bwakozwe bwerekanye ko iyegeranya rye-NT2RepCT ryabaye hejuru ya poroteyine nyinshi (10-300 mg / mL) kandi ko ubwo bwitonzi bwari bufitanye isano n’igihe cyo gusohora (Ishusho 1c n’inyongera Ishusho 1).Kugirango tumenye ibice bye-NT2RepCT ihuza hydrogel, noneho twasuzumye buri domeni kugiti cye no muburyo butandukanye dukoresheje flask inversion assay (Ishusho 1a, b).Ibice byose byapimwe bya spidroin ya recombinant byakozwe na geles (kuri proteine ​​ya mg / mL 300) mugihe kitarenze 1 h, usibye 2Rep yaguye (Ishusho 1b).Ibi birerekana ko NT na CT byonyine, bifatanije, cyangwa bifitanye isano no gusubiramo, bishobora kuza kuri 37 ° C kandi ko tagi ya His6 ntacyo ihindura muriki gikorwa kuburyo bugaragara.Urebye imyumvire isanzwe ivuga ko NT ari poroteyine ikemurwa cyane kandi ihamye, kandi ko raporo zabanjirije za hydrogels ya recombinant spidroin zavuze ko ingaruka ziterwa n’imiterere ihinduka ry’imihindagurikire y'ikirere mu turere dusubiramo na / cyangwa CT, NT ubwayo irashobora.Ubuvumbuzi bwa gelation ntabwo bwari butunguranye.Imbonerahamwe y'inyongera 1) 37, 38, 39. Igitangaje, NT yamaze guturika mu minota 10 ku gipimo cya mg 300 mg / mL (Ishusho 1c).Ubushakashatsi bwa Vial inversion hamwe nubushakashatsi butandukanye bwa NT bwerekanye ko kuri> 50 mg / mL igisubizo cya NT cyaje vuba kurusha His-NT2RepCT kumurongo uhuye (w / v, Ishusho 1c).
Igishushanyo cyerekana inyubako zitandukanye za spidroin zize muriki gikorwa.b Igihe cya Gel kuri 37 ° C kuri proteine ​​zitandukanye za recombinant spidroin (300 mg / mL) zagenzuwe no guhinduranya vial.CT gel ako kanya nta incubation (<300 mg / mL), 2Rep imvura (300 mg / mL, igipimo cya mm 5).c Igihe cya Gel-NT2RepCT na NT kuri protein yerekanwe kuri 37 ° C.d Amafoto ya hydrogels ye-NT2RepCT na NT hamwe nigitagangurirwa hamwe ninyuguti "NT" yacapishijwe munsi, byombi (byombi mg / mL 200, umunzani wa mm 5).
Hydrogels ikorwa na poroteyine zitandukanye za recombinant spidroin zifite amabara atandukanye gato, kandi kwitegereza amaso byambaye ubusa byerekana uburyo butandukanye bwo gukorera mu mucyo (Ishusho 1b).NT ya geles irasobanutse bidasanzwe mugihe izindi geles ziba zitagaragara.Gele ye-NT2RepCT na NT yajugunywe mu miyoboro ya silindrike irashobora gukurwa muburyo butagaragara (Ishusho 1d).
Kugira ngo hamenyekane niba igitagangurirwa gisanzwe cyitwa silk coatings gel mubihe bimeze ubu biboneka ko bitera ihindagurika rya poroteyine za spombroin spombroin, ibyegeranyo byakusanyirijwe muri glande nini ya ampulla y’igitagangurirwa cy’ikiraro cya Suwede (Larinioides sclopetarius).Ipitingi yabitswe muri 20 mM ya Tris-HCl kuri 50 mg / mL (hashingiwe ku buremere bwumye bwapimwe), ariko nta gelation yagaragaye mu gihe cy’iminsi 21 y’ubushuhe kuri 37 ° C (Ishusho yinyongera 2a).
Kugereranya geles, ibipimo bya rheologiya birashobora gukoreshwa mukwiga inzira ya gelation no kumenya imiterere rusange yubukanishi.By'umwihariko, gukurikirana uburyo bwo kubika (elastique) ku bushyuhe bwo hejuru burashobora gutanga amakuru ku bushyuhe bwa gell kimwe n’imiterere ya viscoelastic ya coating.Ubushakashatsi bwo kuzamuka k'ubushyuhe (ukoresheje 1 ° C / min kuri 25-45 ° C, bushingiye ku bushakashatsi bwabanje ukoresheje ibisubizo by’imyenda isanzwe) 40,41 bwerekanye ko uburyo bwo kubika ibisubizo bye-NT2RepCT na NT bwiyongereye hamwe n’ubushyuhe bwiyongera.yariyongereye (Ishusho 2 n’inyongera Ishusho 3).Ikigaragara ni uko module ya NT yatangiye gukura ku bushyuhe buke ugereranije na His-NT2RepCT, ijyanye nigihe cyihuta cyagaragaye igihe NT yinjizwaga na NT -RepCT ye kuri 37 ° C (Ishusho 1).Nyuma yo kugabanuka kwubushyuhe, modulus yo kubika ntiyagarutse ku giciro cyo hasi kandi yagumye hejuru y’igihombo (reba Ishusho yinyongera 3), byerekana ubushyuhe budasubirwaho.Nyuma yo gusohora, modulus ya nyuma ya elastique yavuye kuri 15 kugeza kuri 330 kPa kuri hydrogels ye-NT2RepCT yibanda kuri mg / mL 100-500, naho modulus ya nyuma ya elastike ya hydrogel ya NT (100-500 mg / mL) yavuye kuri 2 kugeza 1400 kPa (Igishushanyo, 2 namakuru yuzuye yuzuye) reba Inyongera 3).
Guhindura ubushyuhe mugihe cyo gupima His-NT2RepCT (300 mg / mL) na b NT (300 mg / mL) hamwe no kunyeganyega.Imyambi yerekana ubushyuhe bwubushyuhe, kandi igicucu cyoroheje cyububiko bwa module yerekana igeragezwa ku giciro cyo hasi cya torque kubikoresho kuruta kugenwa nuwabikoze, aribyo bitera urusaku rwiyongera.c Kurangiza-module yegeranya-NT2RepCT na NT nyuma yubushyuhe bwo hejuru (100, 300, na 500 mg / mL).Ibyasomwe byose byafashwe kuri frequence ya 0.1 Hz.
Nuburyo bushoboka bwo gukora iperereza ku mpinduka zifitanye isano na gelation, twanditse FTIR yerekanwe ya-NT2RepCT na NT mbere na nyuma yo kuzunguruka kuri 37 ° C (Ishusho 3a, b).Nkuko byari byitezwe, ibisubizo bya His-NT2RepCT na NT ibisubizo byari bihuye na poroteyine zerekana α-helix / random coil random structure structure, hamwe na bande yavuzwe kuri cm 1645 cm-1.Kuri hydrogels zombi, gelation yatumye habaho amaboko abiri mugice cya I hagati ya cm 1617 cm-1 na cm 1695 cm-1 (Ishusho 3a, b), byerekana ko habayeho imiterere-yimpapuro.Izi mpinduka zirashobora kandi kugaragara neza muburyo bwa kabiri bukomokaho no gutandukanya imiterere ya gelation (Ishusho yinyongera. 4b).Amatsinda abiri ya NT β-layer yagaragaye cyane kurusha aya His-NT2RepCT, byerekana ko ibintu byose bigize β-layer band muri hydrogel ya NT byari hejuru kurenza hydrogel ya NT2RepCT.
FTIR yo kwinjiza ibintu bya His-NT2RepCT na b NT (byombi 500 mg / mL) mbere (igisubizo) na nyuma ya (gel) incubation kuri 37 ° C.c TEM amashusho yahagaritswe 50 mg / ml NT2RepCT geles na d NT.Umwanya munini 200 nm.e Diameter ya fibre ya His-NT2RepCT na NT hydrogels.n = 100 fibrile yapimwe, p <0.0001.Utubari twibeshya twerekana gutandukana bisanzwe.Hagati yikosa ryibari nicyo gisobanura.Ikizamini cya t-kidakorewe (umurizo ibiri) cyakoreshejwe mu gusesengura imibare.f Thores fluorescence ya proteine ​​zitandukanye za recombinant spidroin (100 mg / mL) kuri 37 ° C itanyeganyega.g NT (100 mg / mL) ubushakashatsi bwo gukingira kuva 100 mg / mL NT gel hamwe na 0%, 5%, 10%, nimbuto 20%.
Isesengura rya gel ukoresheje microscopi ya electronique (TEM) ryerekanye ko hydrogel igizwe na fibrile imeze nka amyloide (Ishusho 3c, 3d).Fibrile yakozwe na NT yararambuye (5-12 nm ya diametre) kandi idashamiwe, mugihe fibrile ye-NT2RepCT yari ngufi mu burebure kandi yagutse cyane kuri diameter (7-16 nm) (Ishusho 3e).Ibisubizo byatwemereye gukurikiza kinetics ya fibrosis dukoresheje thioflavin T (ThT).Kuri poroteyine zose za recombinant spidroin, ibimenyetso bya fluorescent byiyongereye mugihe ingero zashyizwe kuri 37 ° C (Ishusho 3f, Ishusho yinyongera 5a).Dukurikije ubu bushakashatsi, isuzuma rya microscopique kuri NT na His-NT2RepCT mu bihe bya gelling ryerekanye ko kwiyongera kwa ThT fluorescence nta gukusanyiriza hamwe kw’ibintu bya ThT-byiza (Ishusho yinyongera 5b, c).Ishirwaho rya fibrile ya ThT-nziza ntabwo ryaherekejwe no kwiyongera kwa NT hamwe na N-NTCT (Igicapo cyinyongera.Imbuto wongeyeho fibrile nkeya yabanje gukorwa irashobora kwihutisha cyane fibril kwibumbira muri amyloide 42,43,44 ariko ukongeraho 5%, 10% cyangwa 20% (w / w) NT kubisubizo bya hydrocoagulants ya NT.Ingaruka yo gutera imbuto (Ishusho 3g).Ahari ibi biterwa nuko fibrile muri hydrogel ihagaze neza kandi ntishobora gukoreshwa nkimbuto.
Imyitwarire itunguranye ya poroteyine ya spombroin ya recombinant ku bushyuhe bwinshi yatumye ubushakashatsi bwa magnetiki resonance (NMR) bwerekana ubushakashatsi bwerekana impinduka zifitanye isano no gukora gel.NMR yerekana ibisubizo bye-NT2RepCT yanditswe mugihe cya 37 ° C yerekanaga ko CT yari ikikubye igice, mugihe ibimenyetso bya NT na 2Rep byari byarazimiye (Ishusho 4a), byerekana ko ahanini NT na 2Rep ari byo byagenzuye igice cyo gushinga- NT2RepCT hydrogel.Ikimenyetso cya CT nacyo cyahujwe na 20% yububasha bwacyo bwa mbere, byerekana ko CT nayo ikosowe kandi ikinjizwa mumiterere ya hydrogel.Kubice bito bya CT, bigendanwa nkibisanzwe byateganijwe bityo bikagaragazwa nigisubizo NMR, spekrike ibura ibimenyetso kubisigisigi 10 byambere byubatswe, bishoboka ko biterwa no kwimuka bigoye igice cyometse kuri His-NT2Rep.Ikirangantego cya NMR cya -state ya hydrogels -NT2RepCT yerekanye ko yiganjemo α-helices na β -ibice kandi, ku rugero ruto, guhinduranya ibishishwa bidasanzwe (Ishusho 4b).Isesengura ryimiti yibisigazwa bya methionine biboneka muri NT gusa byerekanye ko iyi domeni yahinduwe β -urupapuro.Ikirangantego gishingiye ku gihe cya NT mu gisubizo cyerekanaga igabanuka rimwe ry’uburemere bwibimenyetso (Igicapo 4c), kandi NMR-ikomeye ya NMR ya hydrogels ya NT yerekanaga ko ibisigazwa byinshi bya NT byahinduwe muburyo bw'urupapuro (Ishusho 4d).Ihinduka rya 2Rep ntirishobora kugenwa ukundi bitewe nuburyo bwo guteranya.Nyamara, leta ihamye ya NMR ya hydrogels ya NTCT na His-NT2RepCT yasaga cyane (Igishusho 4b; Igishushanyo cya 6b), byerekana ko 2Rep yagize uruhare runini mubice byubaka hydrogel ye-NT2RepCT.Kuri hydrogels ya CT, α-helices, ets -impapuro, hamwe nuburyo bwa kabiri bwa tekiniki ya kabiri wasangaga ibaho (Ishusho yinyongera. 6d).Ibi birerekana ko ibice bimwe bya CT bikomeza α-helices mugihe ibindi bihinduka β -impapuro.Rero, ibisubizo bya NMR spectroscopy byerekana ko NT ari ingenzi mu gushiraho hydrogel kandi ikanahinduka β -impapuro zimaze guhinduka hamwe na 2Rep na CT.Dufatiye kuri ibi, duherutse kubona ko zipper zo mu bwoko bwa amyloide zishobora kuba mu bice bitanu byose bigize domaine ya NT, kandi algorithm ya Waltz yahanuye akarere ka amyloidogenic muri helix 1 (Ishusho 4e).
2D yerekanwe ya 15N-HSQC 10 mg / mL Igisubizo cye-NT2RepCT mbere (ubururu) namasaha 19 nyuma ya incububasi (umutuku) kuri 37 ° C.Umusaraba ku giti cye hejuru yumutuku na F24, G136, polyA mubururu bwubururu bigaragazwa ninyuguti imwe ibimenyetso bya aside amine hamwe numubare usigaye.Ibyinjijwe byerekana kwishingira ubukana bwikimenyetso mugihe kubisigisigi byatoranijwe kuva NT, 2Rep, na CT.b Imirasire ikomeye ya radiofrequency (RFDR) ya hydrogels ye-NT2RepCT.Isano ry’ibisigisigi bya Cα / Cβ byagaragaye muri spekure ya RFDR byagenwe ugereranije n’imihindagurikire y’imiti ya peptide y’indangagaciro hamwe n’indangagaciro zikomoka ku mibare 82,83 n’inzego zabo za kabiri.SSB - kuzenguruka kuruhande.c Ikigereranyo kimwe cya 15N-HSQC 10 mg / mL NT igisubizo mugihe cya incubation kuri 37 ° C mumasaha 36.Inet yerekana ubukana bwa volumetric nigihe.d Leta ikomeye RFDR yerekanwe ya hydrogels ya NT.Isano ryibisigisigi bya Cα / Cβ hamwe nuburyo bwabo bwa kabiri bwagaragaye muri RFDR yerekanwe.e Ukurikije umwirondoro wa NT45.79 fibrillation yerekana kuva muri Zipper base (https://services.mbi.ucla.edu/zipperdb/).Ingufu za Rosetta yumurabyo uhinduranya idirishya rya hexapeptide irerekanwa muri kcal / mol.Utubari dutukura twerekana hexapeptides ifite fibrosis nyinshi (ingufu za Rosetta munsi -23 kcal / mol; munsi yumurongo utudomo).Icyatsi kibisi cyerekana ibice bifite ingufu za Rosetta hejuru yurugero bityo ntibishoboka gukora zipper steric.Ibice birimo protine ntibyakuwe mubisesengura (nta nkingi).Ibibanza byerekana uduce twa amyloidose byahanuwe na algorithm ya Waltz (https://waltz.switchlab.org).Urukurikirane rwibisigisigi bya aside amine ya NT iri hejuru, kandi ubwoko bwibisigisigi biboneka murwego β rwa kabiri (byagenwe na NMR spectroscopy ikomeye) byerekanwe mumutuku.Imyanya ya batanu NT α-helices yagenwe nka (H1-H5) 28.
Kuri pH <6.5, HT iragabanuka, irwanya ubushyuhe- cyangwa urea iterwa no gutandukana18.Kugirango usobanure uburyo NT dimerisation na stabilite bigira ingaruka kumyuka, ibisubizo birimo 100 mg / ml NT byagenzuwe kuri pH 8, 7, na 6 hakoreshejwe ikizamini cya inversion.Ingero za NT zashyizwe kuri pH 8 na 7 zashushe nyuma yiminota 30 kuri 37 ° C, ariko gel ya pH 8 yagumye isobanutse, mugihe gel ya pH 7 yerekanaga imvura igaragara (Ishusho 5a).Ibinyuranye, igisubizo kirimo HT kuri pH 6 ntabwo cyakoze gel, kandi imvura nini yashoboraga kuboneka nyuma yiminota 20 kuri 37 ° C.Ibi birerekana ko ibipimo ubwabyo na / cyangwa umutekano wabo muremure ugereranije na monomers birinda gelation.Ishirwaho ryimvura ya NT kuri pH 7 na 6 ntabwo byari byitezwe, kubera ko byavuzwe ko NT ikemuka 200 mg / ml27, igahinduka byoroshye nyuma yubushyuhe, kandi ikagumana α-helix ku giciro cyo hasi cya pH 18. Ibisobanuro bishoboka kuri uku kunyuranya ni uko ubushakashatsi bwatangajwe mbere bwakorewe ku bushyuhe bwicyumba cyangwa munsi yacyo, cyangwa kuri proteine ​​nkeya ugereranije 16,18,19.
Ikizamini cya NT vial inversion (100 mg / mL) kuri pH 8, 7, 6 na 154 mM NaCl (pH 8) nyuma yubushakashatsi kuri 37 ° C.b NT CD yerekanwe hamwe na 154 mM NaF na 154 mM NaCl, kimwe.Imirasire ya molar kuri 222 nm ihindurwa mukigereranyo cyimiterere karemano.c NT ihinduka (100 mg / mL) NT * (37 ° C na 60 ° C), NTA72R (37 ° C), na NT-L6 (37 ° C na 60 ° C).d CD yerekanwe ya NT ihinduka NT *, NTA72R, na His-NT-L6.Imirasire ya molar kuri 222 nm ihindurwa mukigereranyo cyimiterere karemano.e Inversion ikizamini cya NTFlSp, NTMiSp no kugabanya NTMiSp (100 mg / mL).Umwanya muto wa mm 5.f CD yerekanwe ya NT, NTFlSp, NTMiSp kandi yagabanije NTMiSp.Imirasire ya molar kuri 222 nm ihindurwa mukigereranyo cyimiterere karemano.NT yuzuye ya 25 ° C na 95 ° C irerekanwa mugice cya 8.
Imyunyu ngugu ya physiologique igena imikoranire ya electrostatike hagati ya NT subunits no kugabanya igihe cyo kwimura NT kuri pH18 yo hepfo.Twabonye ko kuba 154 mM NaCl na NaF byabujije rwose kuzunguruka, (Ishusho 5a, b; Igishushanyo cya 2b) kandi ko iyo myunyu yongereye ubushyuhe bwumuriro wa NT monomers (Ishusho 5b, Igishushanyo cya 8) .Irerekana kandi ko kongera umutekano, aho kugabanuka, birinda gukora gel.
Kugirango turusheho gucukumbura uruhare rwa protein dimerisation no gutuza muri gelation, twakoresheje mutant ebyiri, NT * na NTA72R, nazo zikomeza kuba monomeric kuri pH28.30.NT * ni inshuro ebyiri zisubiza inyuma mutant aho bigaragara ko dipolar yishyurwa rya monomer iringaniye, irinda dimerisation kandi byongera cyane monomer ituje.NTA72R ni dipole yishyuzwa, ariko Ala yasimbuwe na Ala iherereye kumupaka wa dimer, bityo ihinduka rikabangamira imikoranire ya subunit isabwa kugirango dimerisation.Iyo inkubasi iri kuri 37 ° C, NT * ntabwo yakoze hydrogel, mugihe NTA72R yakoze gel itagaragara muminota 15 (Ishusho 5c).Kubera ko NT * na NTA72R byombi bidashobora gucika intege ariko bitandukanye muburyo butajegajega bwa monomer (Igishusho 5d), ibisubizo birerekana neza ko ituze ryinshi rya termodinamike ribuza NT gutera.Ibi kandi bishyigikirwa nuko HT * ikora gel mugihe idahindagurika mubushyuhe bwinshi (nyuma yiminota 8 kuri 60 ° C; Ishusho 5c).Byerekanwe mbere ko ibintu byinshi bya methionine muri NT bihindura uburyo busanzwe kandi ko bitandatu Met to Leu byasimbuwe (bivugwa hano nka His-NT-L6) bihamye cyane monomer ya NT46.Dushingiye ku gitekerezo kivuga ko imiterere ihindagurika ikenewe kugirango NT gel ikorwe, twasanze ihinduka ryayo-NT-L6 itajegajega kuri 37 ° C (Ishusho 5c, d).Nyamara, His-NT-L6 yakoze kandi gel mugihe cyo gukuramo 60 ° С muminota 60 (Ishusho 5c).
Ubushobozi bwa NT bwo guhinduka β -urupapuro rwubaka no gukora hydrogels bigaragara ko ikoreshwa kuri bamwe ariko siko bose ba NT domaine ya spidroin.NTs ziva mubwoko butandukanye bwa silike nubwoko bwigitagangurirwa, clavipes Trichonephila (NTFlSp), zakoze geles nubwo zifite methionine nkeya kandi zifite ubushyuhe bwinshi (Igicapo 5e, f hamwe nimbonerahamwe 2).Ibinyuranye na byo, NT yo muri poroteyine ntoya ya ampullar yo muri Araneus ventricosus (NTMiSp) ifite ubushyuhe buke buke hamwe na methionine nyinshi ntabwo yakoze hydrogels (Imbonerahamwe yinyongera 2 nishusho 5e, f).Iheruka irashobora kuba ifitanye isano no kuba intambamyi ya disulfide ihuza 29,47.Burigihe, mugihe imiyoboro ya disulfide ya NTMiSp yagabanutse, yakoze hydrogel nyuma yubushuhe kuri 37 ° C muminota 10 (Ishusho 5e).Mu gusoza, twakagombye kumenya ko guhuza imiterere ningirakamaro, ariko sibyo byonyine, igipimo cyo gukora gel kuva muri NT.Ikindi kintu gishobora kuba ingirakamaro ni ugushaka gukora fibrile ya amyloide, kandi isesengura hamwe na base ya zipper hamwe na algorithm ya Waltz ryerekanye isano iri hagati yubushobozi bwo gukora gele no kuba hari uturere twa amyloidogene, ndetse nubunini bwakarere kahanuwe Kuri Gushiraho.Hariho ihuriro (Imbonerahamwe yinyongera 2 nishusho yinyongera 9).
Ubushobozi bwa NT bwo gukora fibrile no gukora gele mubihe byiza byaduteye gushidikanya ko NT guhuza hamwe nibindi bice bya poroteyine bishobora gukomeza gukora gele ifite imikorere yuzuye yabafatanyabikorwa.Kugirango tugerageze ibi, twashyizeho proteine ​​yicyatsi kibisi (GFP) na purine nucleoside fosifora (PNP) kuri C-terminus ya NT.Intungamubiri za poroteyine zavutse zagaragaye muri E. coli n'umusaruro mwinshi cyane (150 mg / L na 256 mg / L shake flask imico ya His-NT-GFP na His-NT-PNP), bihuye nibyerekanwe kubindi poroteyine zahujwe na NT Ref.30. Poroteyine ye-NT-GFP (300mg / mL) hamwe na poroteyine ye-NT-PNP (100mg / mL) ikora geles nyuma yamasaha 2 namasaha 6.5 kuri 37 ° C, kandi icy'ingenzi, igice cya GFP nticyahindutse.byagaragaye nyuma yo kuzunguruka, hamwe na> 70% byimbaraga za fluorescence zasigaye nyuma yo kuzunguruka (Ishusho 6a).Kugirango dusuzume ibikorwa bya PNP mubisubizo bye-NT-PNP hamwe na geles, twagombaga kuvanga poroteyine ya fusion hamwe na NT kuko ibikorwa byimisemburo yo kwitegura neza byari hanze yubushakashatsi bwakorewe kuri gelling.Gele yakozwe nuruvange rurimo 0.01 mg / mL His-NT-PNP na 100 mg / mL NT yagumanye 65% yibikorwa bya enzymatique yambere yintangarugero (Ishusho 6b).Gele yagumye idahwitse mugihe cyo gupima (Ishusho yinyongera).
ubukana bwa fluorescence mbere na nyuma yo kuzunguruka-NT-GFP (300 mg / mL) hamwe na vial ihindagurika irimo hydrogel ye-NT-GFP (300 mg / mL) munsi yumucyo ugaragara na UV.Ingingo zerekana ibipimo byihariye (n = 3), utubari twibeshya twerekana gutandukana bisanzwe.Impuzandengo yagaciro irerekanwa hagati yamakosa.b Igikorwa cya PNP cyabonetse hakoreshejwe isesengura rya fluorometric ukoresheje ibisubizo na geles bigizwe na NT (100 mg / ml) hamwe nuruvange rurimo 0.01 mg / ml ye-NT-PNP na 100 mg / ml Amadolari ya Tayiwani.Inet yerekana vial ihindagurika irimo hydrogel irimo His-NT-PNP (umurongo wa mm 5).
Hano, turatanga raporo yibikorwa bya hydrogel biva muri NT hamwe nizindi poroteyine za spombroin recombinant dushyiramo proteine ​​kuri 37 ° C (Ishusho 1).Twerekana ko gelation ifitanye isano no guhindura α-helices mubice β no gushiraho fibrile isa na amyloide (Ishusho 3 na 4).Ubu bushakashatsi buratangaje kuko NT zashizwe hamwe nisi yose ya helix eshanu zizwiho gukomera cyane hamwe no guhagarara neza kuri concentration> 200 mg / mL kuri 4 ° C muminsi myinshi27.Mubyongeyeho, NTs byoroshye guhinduka nyuma yubushyuhe bwa protein nkeya muri µM.Dukurikije ibisubizo byacu, kwibumbira kwa fibril bisaba guhuza> 10 mg / mL ya protein hamwe nubushyuhe bwo hejuru gato (Ishusho 1).Ibi bihuye nigitekerezo kivuga ko fibrile ya amyloide ishobora guturuka kuri poroteyine zifunitse ku isi ziri mu buryo butagaragara kubera ihindagurika ry’ubushyuhe mu bihe bya physiologique 48.Ingero za poroteyine zigenda zihinduka harimo insuline 49,50, β2-microglobuline, transthyretin na lysozyme 51,5,5,53.Nubwo NT ari α-helix mu gihugu cyayo kavukire, hafi 65% by'urunigi rwa polypeptide bihujwe no gukora zipper steric (Ishusho 4e) 45.Kubera ko monomer igenda igendanwa 46, irashobora kwerekana uturere dushobora kuba amyloidogeneque ku bushyuhe bwo hejuru kandi ku bwinshi bwa poroteyine zose zishobora kugera ku kintu gikomeye cyo kwibumbira muri amiloide fibril 54.Dukurikije iyi mitekerereze, twasanze isano iri hagati yibitekerezo bya spidroin nigihe cyo guterana (Igicapo 1c), kandi niba ihinduka rya monomeric NT rihindagurika haba mubihinduka (NT *, His-NT-L6) cyangwa no kongera umunyu, birashobora gukumira u gukora hydrogels (Ishusho 5).
Kenshi na kenshi, fibrile ya amyloide ibura igisubizo nkigicucu, ariko mubihe bimwe na bimwe birashobora gukora hydrogels 55,56,57.Hydrogel ikora fibrile mubisanzwe ifite igipimo kinini kandi igakora imiyoboro ihamye yibice bitatu ikoresheje molekulari, 55,58 ihuye nibisubizo byacu.Kugirango hydrogel ikorwe muri vitro, poroteyine akenshi ziba zuzuye cyangwa igice, urugero, muguhura n'umuti ukomoka ku bimera, ubushyuhe bwo hejuru (70-90 ° C) na / cyangwa pH nkeya (1.5-3.0) 59,60,61,62.Hydrogel ya spidroin yasobanuwe hano ntabwo isaba gutunganywa bikaze, kandi ntisaba guhuza imiyoboro ihuza hydrogels.
Byaravuzwe mbere ko spidroin isubiramo na QDs, bigaragara ko ihinduranya β -impapuro mugihe cyo kuzunguruka imyenda, ikora hydrogels.Ugereranije nibyo twabonye, ​​ibihe byubushakashatsi hamwe na / cyangwa ubushyuhe bwubushyuhe byari birebire cyangwa birebire, kandi hydrogène yavuyemo yakunze kuba idahwitse (Igicapo 7 nimbonerahamwe yinyongera 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. ) na kolagen.(0,6%) (Igicapo 7 hamwe n'imbonerahamwe y'inyongera 1 na 3) 37,39,66,67,68,69,70,71,72,73,74.
Igihe cya gel hamwe na moderi ya elastike ya hydrogels muri ubu bushakashatsi yagereranijwe nizindi hydrogène ishingiye kuri spidroin hamwe na hydrogels karemano yatoranijwe.Ibisobanuro byatanzwe hamwe nibisobanuro byimiterere ya gelation.APS Amonium perulfate, ubushyuhe bwicyumba.Amakuru 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
Igitagangurirwa gisa nkicyateye imbere uburyo bwo kwirinda spidroin guterwa mugihe cyo kubika.Nubwo intungamubiri nyinshi za poroteyine ziri muri glande ya silk, akarere kanini gasubiramo kajyanye na domaine ya terefone bivuze ko kugaragara kwa NT na CT muri glande bihwanye na mg / ml 10-20, kumupaka wubu bushakashatsi.bisabwa muri vitro yitegereje hydrogel.Byongeye kandi, imyunyu isa niyumunyu 16 ituje NT, nko muri glande ya silike (Ishusho 5b).Guhindura NT byakorewe ubushakashatsi muri cytosol ya E. coli ugasanga bikubye cyane kuruta iyo bisuzumwe muri vitro, bikerekana ko umunyu cyangwa ibindi bintu bibuza kwegeranya muri vivo.Ariko, ubushobozi bwa NTs bwo guhinduka β-urupapuro rwa fibrile birashobora kuba ingenzi mugukora filament kandi bigomba gukorwaho ubushakashatsi mubushakashatsi buzaza.
Usibye ibice bishya byerekana NT-amyloide isa na fibril na hydrogel igaragara muri ubu bushakashatsi, twerekana kandi ko iki kintu gishobora kuba gifite ibinyabuzima na biologiya (Ishusho 8).Nkikimenyetso cyigitekerezo, twahujije NT na GFP cyangwa PNP hanyuma twerekana ko proteine ​​ya fusion nayo ikora hydrogène iyo yashyizwe kuri 37 ° C kandi ko uduce twa GFP na PNP ahanini tugumana ibikorwa byabwo nyuma yo kuzunguruka (Ishusho 6).Nucleoside fosifora ningirakamaro ya catalizike ya synthesis ya nucleoside igereranya75, ibyo bigatuma ibyo tuvumbura bifitanye isano ninganda zikomoka ku binyabuzima.Igitekerezo cyo kwerekana poroteyine zo mu bwoko bwa fusion zigizwe na hydrogène ibonerana mu bihe byiza bituma habaho gukora hydrogène ikora ifite imitungo myiza yo gukoresha ibintu byinshi nka enzyme immobilisation, kurekura ibiyobyabwenge no gukora inganda.Byongeye kandi, NT na NT * ni ibimenyetso byerekana neza30, bivuze ko NT hamwe nibihinduka byayo bishobora gukoreshwa mugutanga umusaruro mwinshi wa poroteyine zishushe hamwe no gukora poroteyine zidafite intego muri hydrogels ya 3D.
NT irashonga, α-ihindagurika kandi itajegajega (µM) na 37 ° C.Ku bushyuhe bumwe, ariko mugihe cyo kwiyongera (> 10 mg / ml), NT ikora geles igizwe na fibrile isa na amyloide.Intungamubiri za NT fusion nazo zikora fibrillar geles hamwe nibice byuzuye bya fusion bikora, bigatuma poroteyine zitandukanye zidahinduka muri hydrogel ya 3D ukoresheje NT.Hasi: NT (PDB: 4FBS) n'amashusho y'urusobe rwa fibre hamwe na poroteyine bifitanye isano (byafashwe kandi ntibishushanyije ku gipimo, GFP PDB: 2B3Q, 10.2210 / pdb2B3Q / pdb; PNP PDB: 4RJ2, 10.2210 / pdb4RJ2 / pdb).
Inyubako (reba Imbonerahamwe ya 4 yinyongera kurutonde rwuzuye harimo na aside amine ikurikirana) yashyizwe muri plasmid pT7 ihinduka E coli BL21 (DE3).E. coli irimo plasima yakozwe na injeniyeri yatewe mu muhogo wa Luria hiyongeraho kanamycine (70 mg / l) hanyuma ikura ijoro ryose kuri 30 ° C na 250 rpm.Umuco waje guterwa 1/100 muburyo bwa LB burimo kanamycine kandi butera imbere kuri 30 ° C na 110 rpm kugeza OD600 igeze kuri 0.8.Kubushakashatsi bwa NMR, bagiteri zahinzwe muri M9 ntoya irimo g 2 za D-glucose 13C (Aldrich) na g 1 ya chloride ya amonium 15N (Cambridge Isotope Laboratories, Inc.) kugirango proteine ​​yanditseho isotopi.Gabanya ubushyuhe kugera kuri dogere selisiyusi 20 hanyuma utere proteine ​​imvugo hamwe na 0,15 mM isopropylthiogalactopyranoside (concentration ya nyuma).Nyuma yo kwerekana poroteyine ijoro ryose, selile zasaruwe kuri 7278 × g, 4 ° C kuri min 20.Pelletes selile zahagaritswe muri mM 20 Tris-HCl, pH 8, hanyuma zirahagarikwa kugeza igihe zizakoreshwa.Utugingo ngengabuzima twajugunywe twakoresheje lisitori (imashini ya TS, imashini ya Constant Systems Limited, Ubwongereza) kuri 30 kPa.Hanyuma lysates yashyizwe kuri 25.000 g muminota 30 kuri 4 ° C.Kuri NTMiSp, pellet yahise ihagarikwa muri 2 M urea, 20 mM Tris-HCl, pH 8, hanyuma ihindurwamo iminota 2 (2 s kuri / kuzimya, 65%), hanyuma yongera gushyirwamo 25,000 xg, 4 ° C. imbere 30 min.Supernatant yapakiwe ku nkingi ya Ni-NTA, yogejwe na mM 20 Tris-HCl, mM imidazole 2 mM, pH 8, hanyuma poroteyine isimburwa na mM 20 Tris-HCl, 200 mM imidazole, pH 8. Kubyara NT2RepCT na NTCT, igogorwa rya trombin ryerekana urubuga (ThrCleav) hagati ye na NT.Imbuga za Thrombin ziraboneka no muri His-NT-ThrCleav-2Rep (itanga 2Rep), His-thioredoxin-ThrCleav-NT (itanga NT), His-thioredoxin-ThrCleav-CT (itanga CT), His-Thioredoxin-ThrCleav-NT .*.Inyubako zashizwemo na trombine (1: 1000) hanyuma zandikirwa ijoro ryose kuri 4 ° C. hamwe na mM 20 Tris-HCl, pH 8, ukoresheje membrane ya Spectra / Por dialyse ifite uburemere bwa molekile ya 6-8 kDa.Nyuma ya dialyse, igisubizo gishyirwa kumurongo wa Ni-NTA hanyuma imyanda irimo proteine ​​yinyungu ikusanywa.Intungamubiri za poroteyine zagenwe no gupima iyinjizwa rya UV kuri 280 nm ukoresheje coefficente de lisansi ya buri poroteyine, usibye NTF1Sp, yakoresheje ubushakashatsi bwa Bradford ukurikije protocole yuwabikoze.Isuku yagenwe na SDS polyacrylamide (4-20%) gel electrophorei na Coomassie nziza cyane yubururu.Poroteyine zegeranijwe hifashishijwe akayunguruzo ka centrifuge (VivaSpin 20, GE Healthcare) kuri 4000 xg hamwe na 10 kDa ya molekile yagabanutse mu minota 20.
Kuramo poroteyine hanyuma witondere witonze 150 µl muri ml 1 isukuye ya septum (8 x 40 mm Thermo Scientific).Imiyoboro yafunzwe kandi ifunzwe na parafilm kugirango birinde guhumeka.Ingero (n = 3) zashyizwe kuri 37 ° C cyangwa 60 ° C kandi zagiye zihindagurika kugirango harebwe gelation.Ingero zidaje zashizwemo byibura icyumweru.Mugabanye NTMiSp disulfide ihuza hamwe na 10 mM DTT kuri proteine ​​10 µM.Kugira ngo dusesengure imiterere y’igitagangurirwa cy’igitagangurirwa gisanzwe, igitagangurirwa cy’ikiraro cya Suwede cyaraciwe, glande ebyiri nyamukuru zaciwe zashyizwe muri 200 μl za 20 mM ya Tris-HCl buffer pH 8 hanyuma iracibwa kugira ngo igifuniko gitandukane na glande..Ibiri muri glande byashongeshejwe muri buffer, 50 µl kugirango hamenyekane uburemere bwumye (ukoresheje inkingi zifunguye kuri 60 ° C kugeza uburemere burigihe) na 150 µl kugirango zive kuri 37 ° C.
Gupima geometrie / igikoresho gikozwe mubyuma bidafite ingese ukoresheje isahani ibangikanye na diameter yo hejuru ya mm 20 hamwe nicyuho cya mm 0.5.Shyushya icyitegererezo kuva kuri 25 ° C kugeza kuri 45 ° C hanyuma usubire kuri 25 ° C ku gipimo cya 1 ° C ku munota ukoresheje icyuma cyo hasi cya Peltier.Ibipimo byo kunyeganyega byakorewe ku nshuro ya 0.1 Hz no mu karere ka viscoelastic umurongo ugizwe n'ibikoresho ku gipimo cya 5% na 0.5% ku ngero za 100 mg / mL na 300-500 mg / mL.Koresha icyumba cyabugenewe kugirango wirinde guhumeka.Amakuru yasesenguwe hakoreshejwe Prism 9.
Mugukusanya infrarafarike (IR) mubushyuhe bwicyumba kuva kuri 800 kugeza 3900 cm - 1.Igikoresho cya ATR, kimwe n'inzira yumucyo unyuze kuri spekrometero, isukurwa n'umwuka wayungurujwe mbere no mugihe cy'igerageza.Umuti (500 mg / mL kugirango ugabanye impanuka zo kwinjiza amazi muri spekiteri) washyizwe kuri kristu, hanyuma geles (500 mg / mL) zakozwe mbere yo gupimwa hanyuma zijyanwa muri kristu (n = 3).Gusikana 1000 byafashwe amajwi bifite ibyemezo bya cm 2-1 na zeru yumusoro wa 2. Inkomoko ya kabiri yabazwe ikoresheje OPUS (Bruker) ikoresheje uburyo bworoshye bw'amanota icyenda.Ibyerekanwe byari bisanzwe mukarere kamwe ko guhuza hagati ya 1720 na 1580 cm-1 ukoresheje F. Menges “Spectragryph - Optical Spectroscopy Software”.Muri ATR-IR spectroscopy, ubujyakuzimu bwinjira mumurambararo wa infragre mucyitegererezo biterwa na wavenumber, bikaviramo kwinjirira cyane kumashanyarazi yo hepfo kuruta kuri wavenumbers.Izi ngaruka ntizakosowe kuri ecran yerekanwe mubishushanyo.3 kubera ko ari nto cyane (Ishusho yinyongera. 4).Ikosorwa ryakosowe kuriyi shusho ryabazwe ukoresheje software ya Bruker OPUS.
Ihame, ingano yuzuye ya poroteyine irashoboka nyuma yo kwizerwa kwizerwa ryibigize muri amide I mpinga.Ariko, inzitizi zimwe zivuka mubikorwa.Urusaku muri specran rushobora kugaragara nkimpinga (ibinyoma) mugihe cya deconvolution.Byongeye kandi, impinga kubera kunama kwamazi ihura nu mwanya wa amide I mpinga kandi irashobora kugira ubunini busa kuburugero burimo amazi menshi, nka gel y'amazi yize hano.Ntabwo rero, twagerageje kubora burundu hagati yimpinga ya I, kandi ibyo twabonye bigomba gutekerezwa gusa mugushigikira ubundi buryo nka NMR spectroscopy.
Umuti wa 50 mg / ml NT na His-NT2RepCT watanzwe ijoro ryose kuri 37 ° C.Hydrogel yahise ivangwa na mM 20 Tris-HCl (pH 8) kugeza kuri 12,5 mg / ml, ihungabana neza hanyuma irasukurwa kugirango ivunike gel.Ubukurikira, hydrogel yavomwe inshuro 10 hamwe na 20 mM Tris-HCl (pH 8), 5 μl z'icyitegererezo zashyizwe kuri gride y'umuringa wasizwe na formvar, hanyuma icyitegererezo kirenga gikurwaho n'impapuro.Ingero zogejwe kabiri hamwe na 5 µl y'amazi ya MilliQ hanyuma zisiga irangi 1% ya uranyl muminota 5.Kuraho irangi ryinshi hamwe nimpapuro zinjira, hanyuma umwuka wumishe mesh.Kwerekana amashusho byakorewe kuri gride ukoresheje FEI Tecnai 12 Spirit BioTWIN ikora kuri 100 kV.Amashusho yafashwe kuri x 26.500 na x 43.000 akoresheje kamera ya Veleta 2k × 2k CCD (Olympus Soft Imaging Solutions, GmbH, Münster, Ubudage).Kuri buri cyitegererezo (n = 1), amashusho 10-15 yarafashwe.ImageJ (https://imagej.nih.gov/) yakoreshejwe mu gusesengura amashusho no gupima ibipimo bya fibre (n = 100, fibre zitandukanye).Prism 9 yakoreshejwe mugukora t-ibizamini bidakorewe (umurizo ibiri).Hagati ya fibrile ye-NT2RepCT na NT yari 11.43 (SD 2.035) na 7.67 (SD 1.389) nm.Intera yicyizere (95%) ni -4.246 kugeza -3.275.impamyabumenyi z'ubwisanzure = 198, p <0.0001.
80 µl by'icyitegererezo cy'amazi arimo 10 µM thioflavin T (ThT) yapimwe muri bitatu (n = 3) mubihe bihagaze hifashishijwe Corning 96-iriba ryirabura ryirabura risobanutse neza (Corning Glass 3881, USA).Itandukaniro rya Fluorescence ryanditswe hifashishijwe akayunguruzo ka 440 nm hamwe n’iyungurura rya 480 nm (FLUOStar Galaxy yo muri BMG Labtech, Offenburg, mu Budage).Ikimenyetso cya ThT nticyari cyuzuye cyangwa ngo kizimye, kuko ubushakashatsi hamwe nubushakashatsi butandukanye bwa ThT bwakozwe nta guhindura ubukana bwikimenyetso.Andika kwinjiza kuri 360 nm yo gupima igihu.Kubushakashatsi bwimbuto, geles 100 mg / mL zakozwe kuri 37 ° C., zirahagarikwa, kandi zikoreshwa mu gutera imbuto ku gipimo cya 5%, 10%, na 20%.Amakuru yasesenguwe hakoreshejwe Prism 9.
Thaw ububiko bwa His-NT2RepCT na NT> 100 mg / mL kurubura hanyuma uyungurure unyuze kuri 0.22 µm.Ihuriro ryabazwe mukupima kwinjiza kuri 280 nm ukoresheje Nanodrop.Mu mariba ya 96-iriba ryirabura ridahambiriye (Corning) rifite epfo na ruguru, ingero zavomwe kuri mg / ml 20 muri 20 mM Tris-HCl pH 8 hanyuma zivangwa na 5 μM ThT (kwibanda kwanyuma), hamwe hamwe Ingano ya 50 μl.Icyitegererezo cyashushanijwe buri minota 10 kuri 37 ° C kuri microscope ya CellObserver (Zeiss) hamwe numuyoboro wogukwirakwiza hamwe na FITC ishimishije hamwe nayungurura ibyuka byerekana amashusho ya ThT.Lens ya 20x / 0.4 ikoreshwa mugushushanya.Zen Ubururu (Zeiss) na ImageJ (https://imagej.nih.gov/) byakoreshejwe mu gusesengura amashusho.Gels nayo yateguwe kuva NT na His-NT2RepCT ibisubizo byibanze kuri 50 mg / mL irimo 20 mM Tris pH 8 na 5 µM ThT kandi yashyizwe kuri 37 ° C muminota 90.Ibice bya gel byimuriwe ku iriba rishya ririmo 20 mM Tris, pH 8, na 5 μM ThT mu gikara kidahambiriye 96 iriba risobanutse neza.Shaka icyatsi kibisi fluorescence hamwe namashusho yumurima kuri 20x / 0.4.ImageJ yakoreshejwe mu gusesengura amashusho.
Igisubizo cya NMR cyabonetse kuri 310 K kuri 600 MHz Bruker Avance Neo spectrometer ifite ibikoresho bya QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN).Ingero za NMR zirimo proteine ​​10 mg / mL za poroteyine zanditseho 13C, 15N, zishonga muri mM 20 Tris-HCl (pH 8), 0.02% (w / v) NaN3, 5% DO (v / v), (n = 1) .Guhindura imiti ya NT2RepCT kuri pH 6.7 byakoreshejwe mugutanga impinga 23 muri 2D ya 15N-HSQC.
Magic angle izunguruka ikomeye ya NMR (MAS) ya 13C, 15N yanditseho hydrogel yanditse kuri ecran ya Bruker Avance III HD kuri 800 MHz ifite 3.2 mm 13C / 15N {1H} iperereza ridafite electron.Ubushyuhe bw'icyitegererezo bwagenzuwe hifashishijwe ubushyuhe bwa gazi ihindagurika kuri 277 K. Ikigereranyo cya dipole ebyiri zuzunguruka (DARR) 76 hamwe na radiyo yo guhuza imirongo (RFDR) 77 yabonetse kuri MAS yumurongo wa 12.5 kHz na 20 kHz.Kwambukiranya umusaraba (CP) kuva 1H kugeza 13C byakozwe hifashishijwe umurongo uva kuri 60.0 kugeza 48.0 kHz kuri 1H, 61.3 / 71,6 kHz kuri 13C (kuri 12.5 / 20 kHz MAS) nigihe cyo kuvugana na 0.5-1 ms.Spinal6478 decoupling kuri 73.5 kHz yakoreshejwe mugihe cyo gukusanya amakuru.Igihe cyo kugura cyari milisegonda 10 naho gutinda kwinzira byari amasegonda 2.5.Ihuriro rimwe rya Cα / Cβ ryagaragaye muri spekure ya RFDR ryahawe hashingiwe ku miterere iranga ubwoko bw’ibisigisigi by’imiti ihindagurika no guhuza byinshi bifitanye isano na DARR.
Ububiko bwa Zipper79 (https://services.mbi.ucla.edu/zipperdb/) bwakoreshejwe mugusuzuma ibyifuzo bya flutter hamwe ningufu za Rosetta kuri NT, NTFlSp, na NTMiSp.Ububiko bwa Zipper bugereranya Rosetta Energy80, ihuza ibikorwa byinshi byingufu zubusa kugirango bigaragaze kandi bisesengure imiterere ya poroteyine.Urwego rwingufu -23 kcal / mol cyangwa munsi yerekana impengamiro yo hejuru ya fibrillate.Ingufu zo hasi zisobanura gutuza kwinshi β -imigozi muburyo bwa zipper.Byongeye kandi, algorithm ya Waltz yakoreshejwe mu guhanura uturere twa amyloidogenic muri NT, NTFlSp na NTMiSp Ref.81. (https://waltz.switchlab.org/).
Umuti wa poroteyine NT wavanze na 2- (N-morpholino) etanesulfonike aside (MES) kuri pH 5.5 na 6.0 kugirango umanure pH kuri pH 6 na 7.Intungamubiri za poroteyine zanyuma zari 100 mg / ml.
Ibipimo byakorewe kuri CD ya J-1500 (JASCO, USA) hakoreshejwe cuvette 300 μL ifite inzira nziza ya cm 0.1.Poroteyine zavomwe kuri 10 μM (n = 1) muri 20 mM ya fosifate (pH 8).Kugira ngo dusesengure ituze rya poroteyine imbere y’umunyu, poroteyine zasesenguwe ku kintu kimwe (n = 1) muri 20 mM ya fosifate (pH 8) irimo 154 mM NaF cyangwa NaCl.Isuzuma ry'ubushyuhe ryanditswe kuri 222 nm kuva kuri 25 ° C kugeza kuri 95 ° C hamwe n'ubushyuhe bwa 1 ° C / min.Umubare wa poroteyine zavutse kavukire wabazwe ukoresheje formula (KDmeasure - KDfinal) / (KDstart - KDfinal).Hiyongereyeho, ibintu bitanu byanditswe kuri buri cyitegererezo kuva kuri 260 nm kugeza kuri 190 nm kuri 25 ° C na nyuma yo gushyuha kugeza kuri 95 ° C.Ibice bitanu byagereranijwe, byoroheje kandi bihinduka kuri elliptike.Amakuru yasesenguwe hakoreshejwe Prism 9.
Ubwinshi bwa fluorescence ya His-NT-GFP (300 mg / mL, 80 µL) yapimwe muri bitatu (n = 3) muri 96-iriba rya plaque Corning ifite hepfo yumukara (Corning Glass 3881, USA) mubihe bihamye.Gupima icyitegererezo hamwe na florescence ishingiye ku isahani yerekana isahani ifite uburebure bwa 395 nm hanyuma wandike ibyuka bihumanya kuri 509 nm mbere yo gusohora na nyuma yamasaha 2 kuri 37 ° C.Amakuru yasesenguwe hamwe na Prism 9.
Purine nucleoside fosifora yibikorwa byibikoresho (uburyo bwa fluorometric, Sigma Aldrich) yakoreshejwe ukurikije amabwiriza yabakozwe.Kugirango upime ibikorwa muri geles nibisubizo birimo-NT-PNP, vanga 10 ng ya His-NT-PNP na 100 mg / mL NT kugeza kuri 2 µL yose kuko gel yatanze ikimenyetso hejuru yintera yo kumenya.Igenzura rya geles nigisubizo nta-NT-PNP yarimo.Ibipimo byakozwe kabiri (n = 2).Igikorwa kimaze gupimwa, ivangwa rya reaction ryakuweho hanyuma gel ifotorwa kugirango barebe ko geli yagumye idahwitse mugihe cyo gupima.Amakuru yasesenguwe hakoreshejwe Prism 9.
Kubindi bisobanuro kubijyanye nigishushanyo mbonera cyo kwiga, reba ubushakashatsi bwibidukikije bujyanye niyi ngingo.
Igishushanyo cya 1 nicya 2 byerekana amakuru yambere.1c, 2a - c, 3a, b, e - g, 4, 5b, d, f, na 6, Imitini yinyongera.3, umutini winyongera.5a, d, umutini winyongera.6 n'umutini winyongera.8. Amakuru yamakuru yavuye muri ubu bushakashatsi yakiriwe muri base ya Zenodo https://doi.org/10.5281/zenodo.6683653.Amakuru ya NMR yabonetse muri ubu bushakashatsi yashyizwe ku bubiko bwa BMRBig munsi y'irangamuntu bmrbig36.Imiterere ya GFP na PNP yakuwe muri PDB (GFP 2B3Q, PNP 4RJ2).
Kuzamuka, A. na Johansson, J. Kuzunguruka igitagangurirwa cyigitagangurirwa.Imiti yigihugu.ibinyabuzima.11, 309–315 (2015).
Babb, PL n'abandi.Nephila clavipes genome yerekana ubudasa bwubwoko bwigitagangurirwa nigitekerezo cyacyo.Genette y'igihugu.49, 895–903 (2017).