310 10 * 1mm Bakin karfe naɗaɗɗen nau'in sinadarai na tubing, N-terminal domains na spidroin samar da hydrogels dangane da amyloid fibrils da samar da wani dandamali ga gina jiki immobilization.

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Ƙayyadaddun bayanai

310 10*1mm Bakin Karfe nada bututu masu kaya

Haɗin Sinadari

Kayayyakin Injini

Recombinant gizo-gizo sunadaran siliki (protein siliki na gizo-gizo) suna da yuwuwar aikace-aikace masu yawa a cikin haɓaka sabbin abubuwan halitta, amma yanayinsu na multimodal da haɗuwa-sauƙi yana sa su wahala a samu da sauƙin amfani.Anan mun ba da rahoton cewa ƙananan sunadaran spidroin suna sake haɗawa da, mahimmanci, yankin N-terminal (NT) da kansa yana yin saurin samar da kai da madaidaicin hydrogels a 37 ° C.sunadaran fusion wanda ya ƙunshi NT da koren furotin mai kyalli ko purine nucleoside phosphorylase suna samar da cikakkiyar sunadaran fusion ɗin aiki.Hydrogels.Sakamakonmu ya nuna cewa recombinant NT da fusion sunadaran suna ba da babban adadin furci kuma suna ba da hydrogels tare da kyawawan kaddarorin kamar nuna gaskiya, gelation ba tare da haɗin kai ba, da kuma hana su sunadaran da ke aiki kai tsaye a babban yawa.
Spiders suna da nau'ikan siliki daban-daban har guda bakwai, kowannensu yana samar da takamaiman nau'in siliki.Dukkan nau'ikan siliki guda bakwai sun ƙunshi sunadaran siliki na gizo-gizo (spidroins) kusan ragowar 6000 tsayi kuma suna ɗauke da babban yanki mai maimaitawa na tsakiya kewaye da yankuna N- da C-terminal (NT da CT) 1,2.Nau'in siliki da aka fi yin nazari a kai, farkon ampulla, ana samar da shi ne ta glandan ampulla na farko.A cikin wannan gland, monolayer na sel epithelial yana haɗa sunadaran spidroin kuma ya ɓoye su a cikin lumen na gland, inda suke a cikin nau'i mai narkewa (doping) a cikin adadi mai yawa (30-50% w / v) 3,4.An yi muhawara kan tsari da daidaituwa na manyan sunadaran spidroin na ampullar a cikin gland, amma yawancin shaidun gwaji sun nuna kasancewar wani nau'i mai mahimmanci da / ko bazuwar helical da kuma tsarin micellar ko lamellar5,6,7,8,9,10.Yayin da yankuna masu maimaitawa ke tsara kaddarorin inji na filayen siliki, suna samar da β-sheet nanocrystals da tsarin amorphous11,12,13,14,15, yankuna na ƙarshe suna daidaita filayen siliki don amsa yanayin canza yanayin tare da siliki na siliki16,17,18.Ta hanyar sarrafa samuwar siliki, 19. Ƙimar yanki ana kiyaye su ta hanyar juyin halitta kuma aikinsu na iya zama gama gari ga duk sunadaran spidroin 2,20,21.Lokacin wucewa ta cikin gland, pH na spidroin yana raguwa daga kusan 7.6 zuwa <5.716 kuma yana ƙaruwa tare da ƙarfi da shimfidawa ta hanyar motsi ta hanyar raguwa a hankali.A cikin bayani, CT shine α-helical constitutive parallel dimer17, amma a mayar da martani ga ƙananan pH da karfi, CT ya bayyana kuma ya canza β-layers16, 17, mai yiwuwa ya haifar da β-layers a cikin yankuna masu maimaitawa na Maimaita 16. NT sune monomeric a ƙarƙashin yanayin da ke nuna yanayi a cikin lumen na gland kuma yana yin sulhu da solubility na spidroin, amma a rage pH, protonation na adadin carboxylic acid gefe sassan yana kaiwa zuwa dimerization na NT tare da pKa na kusan 6.5, game da shi stabilizing NT da kayyade spidroin a cikin manyan. yawa.hanyoyin sadarwa 16,18.Don haka, NT yana taka muhimmiyar rawa wajen samar da filament, yana canzawa daga monomer a cikin sutura zuwa dimer a cikin fiber23,24,25.NT ya kasance mai narkewa sosai kuma yana da ƙarfi a ƙarƙashin duk yanayin da aka yi nazari har zuwa yau16, 18, 19, 20, 26, 27, 28, 29, wanda ya ƙarfafa haɓakarsa a matsayin lakabin haɓaka mai narkewa don samar da sunadaran ƙwayoyin cuta.
Recombinant mini gizo-gizo furotin siliki, wanda ya ƙunshi NT guda ɗaya, ɗan gajeren yanki guda ɗaya, CT ɗaya, da alamar His6 (His-NT2RepCT) don tsarkakewa, yana narkewa a cikin buffer mai ruwa kamar yadda furotin siliki na gizo-gizo na asali kuma yana kwaikwayi mahimman halaye na siliki gizo-gizo. .rufe 25.31.Ana iya jujjuya shi-NT2RepCT cikin fibers masu ci gaba ta amfani da injin biomimetic wanda aka fitar da murfin pH 8 mai narkewa a cikin pH 525,32,33,34,35 wanka ruwa.Bioreactor fermentation na E. coli yana bayyana His-NT2RepCT da kuma bayan jiyya ya haifar da> 14 g/L yawan amfanin ƙasa bayan tsarkakewa.Babban yawan amfanin ƙasa, babban solubility, da isassun amsa na His-NT2RepCT zuwa yanayin acidic duk ana danganta su ga NT23, 25, 34.
Anan mun ba da rahoton saurin samuwar hydrogels masu gaskiya daga sunadaran spidroin recombinant, gami da NT kadai, ta hanyar haifar da maganin furotin a 37 ° C.Yin amfani da thioflavin T fluorescence (ThT), Fourier yana canza infrared spectroscopy (FTIR), nukiliyar magnetic resonance spectroscopy (NMR) da microscope na lantarki (TEM), mun gano cewa sunadaran NT da microspider suna jurewa tsarin canji zuwa β-sheets da amyloid-kamar fibrils. lokacin da aka kafa gels.Bugu da ƙari, sunadaran fusion na NT da koren furotin mai kyalli (GFP) ko purine nucleoside phosphorylase (PNP) suna samar da hydrogels tare da gutsuttsuran fusion ɗin aiki cikakke.Babban magana mai girma a cikin runduna masu ban sha'awa, tare da saurin haɓakar hydrogels a ƙarƙashin yanayin ilimin lissafi, yana buɗe yuwuwar samar da ingantaccen farashi na hydrogels tare da ayyukan injiniya.
Ba kamar yawancin sunadaran sunadaran spidroin36 da aka ruwaito ba, His-NT2RepCT yana da ƙarfi a cikin buffer Tris-HCl a pH 8 kuma ana iya tattara shi har zuwa 500 mg/mL ba tare da hazo25 ba.Sabili da haka, mun yi mamakin ganin cewa wannan sunadaran da sauri ya samar da haske mai haske, hydrogels masu goyon bayan kai lokacin da aka sanya su a 37 ° C (Fig. 1b-d).Ƙarin karatu ya nuna cewa Gelation na His-NT2RepCT ya faru a kan nau'in furotin mai yawa (10-300 mg / mL) kuma wannan ƙaddamarwa ya bambanta da lokacin gelation (Fig. 1c da Ƙarin Fig. 1).Don gano waɗanne sassa na His-NT2RepCT ne ke daidaita tsarin samar da hydrogel, sai mu bincika kowane yanki daban-daban kuma a cikin haɗuwa daban-daban ta amfani da gwajin juzu'i na flask (Hoto 1a,b).Duk ɓangarorin da aka gwada na recombinant spidroin da aka kafa gels (a adadin furotin na 300 mg/mL) a ƙasa da 1 h, sai dai 2Rep da aka haɓaka (Fig. 1b).Wannan yana nuna cewa NT da CT kadai, a hade, ko hade da maimaitawa, na iya yin gel a 37 ° C kuma alamar His6 ba ta shafar wannan tsari har zuwa wani matsayi mai mahimmanci.Idan aka ba da ra'ayi na gama gari cewa NT furotin ne mai narkewa kuma barga, kuma rahotannin da suka gabata na recombinant spidroin hydrogels sun danganta tasirin gelation zuwa canje-canje masu daidaituwa a cikin maimaita yankuna da / ko CTs, NT da kanta na iya.Gano gelation ya kasance ba zato ba tsammani.Ƙarin Teburin 1) 37, 38, 39. Abin mamaki, NT an riga an yi gelled a cikin minti 10 a taro na ≥ 300 mg/mL (Fig. 1c).Gwaje-gwajen jujjuyawar Vial tare da nau'o'in NT daban-daban sun nuna cewa a> 50 mg / mL maganin NT ya yi sauri fiye da His-NT2RepCT a daidaitaccen taro (w / v, Hoto 1c).
Tsarin tsari na ginin spidroin daban-daban da aka yi nazari a cikin wannan aikin.b Gel lokacin a 37 ° C don nau'ikan sunadaran spidroin recombinant (300 mg/mL) an tabbatar ta hanyar jujjuya vial.CT gel nan da nan ba tare da shiryawa ba (<300 mg / ml), 2Rep precipitates (300 mg / ml, 5 mm sikelin).c lokacin Gel na His-NT2RepCT da NT a adadin furotin da aka nuna a 37 ° C.d Hotunan His-NT2RepCT da NT hydrogels tare da gizo-gizo da harafin "NT" da aka buga a ƙasa, bi da bi (duka 200 mg/ml, sikelin 5 mm).
Hydrogels da aka kirkira ta daban-daban sunadaran sunadaran spidroin recombinant suna da ɗan launi daban-daban, kuma kallon ido tsirara yana nuna mabanbantan matakan bayyanawa (Fig. 1b).Gel na NT sun fito fili a sarari yayin da sauran gels suka zama mara kyau.Gel ɗin sa-NT2RepCT da NT da aka jefa a cikin bututun silinda za a iya cire su daga cikin mold (Fig. 1d).
Don gwada ko gel na siliki na gizo-gizo gizo-gizo a ƙarƙashin yanayin da aka gano yanzu yana haifar da gelation na furotin spidroin recombinant, an tattara sutura daga babban glandar ampulla na gizo-gizo gada na Sweden (Larinioides sclopetarius).An adana sutura a cikin 20 mM Tris-HCl buffer a 50 MG / mL (dangane da ma'aunin busassun ma'auni), amma ba a lura da gelation ba a cikin kwanakin 21 a cikin 37 ° C (Ƙarin Hoto 2a).
Don ƙididdige waɗannan gels, ana iya amfani da ma'auni na rheological don nazarin tsarin gelation da kuma ƙayyade duk kayan aikin injiniya.Musamman ma, kula da ma'auni na ajiya (elasticity) a yanayin zafi mai girma zai iya ba da bayani game da zafin jiki na gelling da kuma abubuwan viscoelastic na sutura.Gwajin zafin zafin jiki (ta yin amfani da 1 ° C / min a 25-45 ° C, dangane da binciken da aka yi a baya ta yin amfani da hanyoyin siliki na siliki na halitta) 40,41 ya nuna cewa tsarin ajiya na His-NT2RepCT da NT mafita ya karu tare da yawan zafin jiki.an ƙara (Fig. 2 da Ƙarin Hoto 3).Musamman ma, tsarin NT ya fara girma a ƙananan zafin jiki idan aka kwatanta da His-NT2RepCT, daidai da lokacin gel mai sauri da aka lura lokacin da NT ya kasance kai tsaye tare da His-NT2RepCT a 37 ° C (Hoto 1).Bayan faɗuwar zafin jiki na gaba, ma'aunin ajiya bai dawo zuwa ƙananan ƙima ba kuma ya kasance sama da ma'aunin asara (duba Ƙarin Hoto 3), yana nuni da kwanciyar hankali da ba za a iya juyawa ba.Bayan gelation, na ƙarshe na roba modulus ya tashi daga 15 zuwa 330 kPa don His-NT2RepCT hydrogels a wani taro na 100-500 mg / ml, da kuma na karshe na roba modulus ga NT hydrogels (100-500 mg / ml) jeri daga 2 zuwa 1400. kPa (Hoto, 2 da cikakkun bayanan ramp) duba Ƙarin Hoto 3).
Canjin zafin jiki yayin ma'aunin His-NT2RepCT (300 mg/mL) da b NT (300 mg/mL) tare da girgiza.Kibiyoyin suna nuna yanayin zafin jiki, kuma ƙaramin inuwa na bayanan tsarin ajiya yana nuna gwaji a ƙananan ƙimar ƙarfi don kayan aiki fiye da ƙayyadaddun da masana'anta suka kayyade, wanda shine dalilin ƙarar amo.c Ƙarshen-module tara na His-NT2RepCT da NT bayan haɓakar zafin jiki (100, 300, da 500 mg/mL).Ana ɗaukar duk karatun module a mitar 0.1 Hz.
A matsayin hanya mai yuwuwa don bincika sauye-sauyen yanayi masu alaƙa da gelation, mun yi rikodin sifofin FTIR na His-NT2RepCT da NT kafin da bayan gelation a 37 ° C (Hoto 3a,b).Kamar yadda aka zata, bakan na His-NT2RepCT da NT mafita sun dace da sunadaran da ke nuna tsarin na biyu na α-helix / bazuwar coil, tare da madaidaicin band a 1645 cm-1.Ga duka hydrogels, gelation ya haifar da samuwar makamai biyu a tsakiyar I band a kusan 1617 cm-1 da 1695 cm-1 (Fig. 3a, b), yana nuna samuwar sifofin β-sheet na antiparallel.Ana iya ganin waɗannan canje-canje a fili a cikin nau'ikan abubuwan da suka samo asali na biyu da bambancin gelation spectra (Ƙarin Hoton 4b).Ƙungiyoyin biyu na NT β-Layer sun fi na His-NT2RepCT, suna nuna cewa jimillar abun ciki na β-Layer bands a cikin NT hydrogel ya fi na NT2RepCT hydrogel.
wani nau'in sha na FTIR na His-NT2RepCT da b NT (duka 500 mg/mL) kafin (mafifi) da kuma bayan (gel) shiryawa a 37 ° C.c Hotunan TEM na 50 mg/ml NT2RepCT gels da d NT.Matsakaicin girman 200 nm.e Fiber diamita na His-NT2RepCT da NT hydrogels.n = 100 da aka auna fibrils, p <0.0001.Sandunan kurakurai suna nuna daidaitaccen karkatacciyar hanya.Tsakiyar sandunan kuskure shine ma'ana.An yi amfani da t-gwajin mara guda (mai-wutsiya biyu) don nazarin ƙididdiga.Hasken haske na ThT na furotin spidroin daban-daban (100 mg/mL) a 37 ° C ba tare da girgiza ba.g NT (100 mg/mL) gwaje-gwajen inoculation daga 100 mg/mL NT gel tare da 0%, 5%, 10%, da 20% tsaba.
Binciken gel ta amfani da microscopy na watsawa (TEM) ya nuna cewa hydrogel ya ƙunshi fibrils amyloid (Figs. 3c, 3d).Fibrils na NT da aka kafa sun kasance masu tsayi (5-12 nm a diamita) kuma ba tare da reshe ba, yayin da His-NT2RepCT fibrils sun fi guntu tsayi kuma sun fi girma a diamita (7-16 nm) (Fig. 3e).Wadannan sakamakon sun ba mu damar bin tsarin motsa jiki na fibrosis ta amfani da gwajin thioflavin T (ThT).Don duk sunadaran spidroin recombinant, siginar mai kyalli ya karu lokacin da aka ƙaddamar da samfurori a 37 ° C (Fig. 3f, Ƙarin Hoto 5a).Daidai da wannan binciken, binciken da aka yi na NT da His-NT2RepCT a ƙarƙashin yanayin gelling ya nuna haɓakar haɓakar haɓakar haske na ThT ba tare da annashuwa na gida na abubuwan da suka dace ba (Ƙarin Hoto 5b,c).Samar da fibrils mai kyau na ThT ba a tare da karuwa a cikin NT da NTCT turbidity (Ƙarin Hoton 5d), wanda ke nufin cewa hanyar sadarwa na fibrils a cikin gel na iya samuwa ba tare da lalata tsabtar gel ba.Shuka ta hanyar ƙara ƙananan fibrils waɗanda aka riga aka kafa na iya haɓaka haɓakar fibril na wasu amyloid42,43,44 amma ƙara 5%, 10% ko 20% (w/w) NT zuwa maganin NT hydrocoagulants.sakamakon shuka (Fig. 3g).Wataƙila wannan shi ne saboda gaskiyar cewa fibrils a cikin hydrogel suna da ƙayyadaddun ƙayyadaddun abubuwa kuma ba za a iya amfani da su azaman tsaba ba.
Halin da ba zato ba tsammani na sunadaran spidroin recombinant a yanayin zafi mai zafi ya haifar da ƙarin nazarin yanayin maganadisu na nukiliya (NMR) don gano sauye-sauyen yanayi da ke da alaƙa da samuwar gel.NMR spectra na His-NT2RepCT mafita da aka rubuta akan lokaci a 37 ° C ya nuna cewa CT har yanzu yana naɗe-kaɗe, yayin da siginar NT da 2Rep suka ɓace (Fig. 4a), yana nuna cewa galibi NT da 2Rep ne ke sarrafa samuwar His- Saukewa: NT2RepCT.Hakanan an rage siginar CT zuwa kashi 20% na ƙarfinsa na asali, yana nuna cewa CT shima an daidaita shi kuma an haɗa shi cikin tsarin hydrogel.Don ƙaramin yanki na CT, wanda yake da wayar hannu kamar yadda yake a cikin samfurin da aka riga aka ƙaddamar kuma don haka ana lura da shi ta hanyar NMR mafita, bakan bakan ba su da sigina don ragowar 10 na farko da aka tsara, mai yiwuwa saboda rashin motsi na ɓangaren da aka haɗe na His-NT2Rep.Siffar NMR na -state of hydrogels -NT2RepCT ya bayyana babban kasancewar α-helices da β-layers kuma, zuwa ƙarami, haɓakar coil bazuwar (Fig. 4b).Binciken canjin sinadarai na ragowar methionine da ke cikin NT kawai ya nuna cewa an canza wannan yanki zuwa tsarin β-sheet.Abubuwan da suka dogara da lokaci na NT a cikin bayani sun nuna raguwar daidaituwa a cikin ƙarfin sigina (Fig. 4c), kuma NMR mai ƙarfi na NT hydrogels ya nuna cewa yawancin ragowar NT sun canza zuwa tsarin β-sheet (Fig. 4d).Ba za a iya tantance daidaiton 2Rep daban ba saboda halinsa na tarawa.Koyaya, ingantaccen yanayin NMR na NTCT da His-NT2RepCT hydrogels yayi kama da kamanni (Fig. 4b; Ƙarin Hoto 6b), yana nuna cewa 2Rep ya ba da gudummawa kaɗan ga tsarin tsarin His-NT2RepCT hydrogel.Don CT hydrogels, α-helices, β-sheets, da bazuwar sifofi na biyu na helical an samo su (Ƙarin Hoton 6d).Wannan yana nuna cewa wasu sassan CT sun kasance α-helices yayin da wasu suka zama β-sheets.Don haka, sakamakon NMR spectroscopy yana ba da shawarar cewa NT yana da mahimmanci ga samuwar hydrogel kuma yana canzawa zuwa ɓangarorin β-sheet akan haɗuwa tare da 2Rep da CT.Daidai da wannan, kwanan nan mun gano cewa amyloid spatial zippers na iya samuwa a cikin dukkanin helices biyar na yankin NT, kuma Waltz algorithm ya annabta yankin amyloidogenic a cikin helix 1 (Fig. 4e).
2D bakan na 15N-HSQC 10 mg/mL Maganin His-NT2RepCT kafin (blue) da 19 hours bayan shiryawa (ja) a 37°C.Kololuwar giciye ɗaya a cikin bakan ja da F24, G136, polyA a cikin bakan shuɗi ana nuna su ta alamomin amino acid harafi ɗaya da ragowar lambobi.Abubuwan da aka shigar suna nuna dogaro da ƙarfin siginar akan lokaci don zaɓaɓɓun rago daga yankunan NT, 2Rep, da CT.b Siffar mitar rediyo mai ƙarfi (RFDR) na His-NT2RepCT hydrogels.Abubuwan da suka dace na ragowar Cα/Cβ da aka gani a cikin sifofin RFDR an ƙaddara su ta hanyar kwatanta da juzu'in sinadarai na peptide da ƙimar da aka samo daga ƙididdiga82,83 da tsarinsu na biyu.SSB – jujjuyawar gefe.c Bakan mai girma ɗaya na 15N-HSQC 10 mg/mL NT bayani yayin shiryawa a 37 ° C na awanni 36.Wurin shigarwa yana nuna ƙarfin juzu'i tare da lokaci.d Takaitaccen yanayin yanayin RFDR na NT hydrogels.An nuna alaƙar ragowar Cα/Cβ da tsarinsu na biyu da aka gani a cikin sifofin RFDR.e Bisa ga bayanin martabar fibrillation na NT45.79 daga bayanan Zipper (https://services.mbi.ucla.edu/zipperdb/).Ƙarfin Rosetta na taga motsi na walƙiya na hexapeptide yana nunawa a cikin kcal/mol.Sandunan ja suna nuna hexapeptides tare da haɓakar fibrosis mai girma (makamashi na Rosetta a ƙasa -23 kcal / mol; ƙasa da layin dige).Koren sanduna suna nuna gutsuttsura tare da kuzarin Rosetta sama da bakin kofa don haka ƙasa da yuwuwar samar da zippers masu siffa.An cire sassan da ke dauke da proline daga bincike (ba tare da ginshiƙai ba).Maɗaukaki suna nuna wuraren amyloidosis da Waltz algorithm81 (https://waltz.switchlab.org) ya annabta.Jerin ragowar amino acid na NT yana saman, kuma nau'ikan ragowar da aka samo a cikin tsarin na biyu (wanda aka ƙaddara ta hanyar ingantaccen yanayin NMR spectroscopy) ana nuna su da ja.Matsayin biyar NT α-helices an tsara su azaman (H1-H5)28.
A pH <6.5, HT dimerizes, kasancewa juriya ga zafi- ko urea-jawo denaturation18.Don bayyana yadda NT dimerization da kwanciyar hankali ke shafar gelation, an sarrafa maganin da ke dauke da 100 mg / ml NT a pH 8, 7, da 6 ta amfani da gwajin inversion vial.Samfurin NT da aka haɗa a pH 8 da 7 gelled bayan 30 min a 37 ° C, amma pH 8 gel ya kasance a sarari, yayin da pH 7 gel ya nuna hazo mai gani (Fig. 5a).Sabanin haka, maganin da ke dauke da HT a pH 6 bai samar da gel ba, kuma ana iya ganin babban hazo bayan 20 min a 37 ° C.Wannan yana nuna cewa dimers da kansu da / ko kwanciyar hankali mafi girma idan aka kwatanta da monomers suna hana gelation.Samuwar hazo don NT a pH 7 da 6 ba a sa ran ba, tunda an bayar da rahoton cewa NT yana narkewa a 200 mg / ml27, cikin sauƙi yana juyawa bayan zafin zafi, kuma yana riƙe da α-helix a ƙananan ƙimar. pH 18. Bayani mai yiwuwa ga waɗannan bambance-bambancen shine cewa an gudanar da gwaje-gwajen da aka ruwaito a baya a dakin da zafin jiki ko ƙasa, ko kuma a ƙananan ƙananan furotin16,18,19.
Gwajin inversion NT vial (100 mg/mL) a pH 8, 7, 6 da 154 mM NaCl (pH 8) bayan shiryawa a 37°C.b NT CD bakan tare da kuma ba tare da 154 mM NaF da 154 mM NaCl, bi da bi.Molar ellipticity a 222 nm ana canza shi zuwa rabon folds na halitta.c NT inversion assay (100 mg/mL) NT* (37 °C da 60 °C), NTA72R (37 °C), da His-NT-L6 (37 °C da 60 °C).d bakan CD na NT mutants NT*, NTA72R, da His-NT-L6.Molar ellipticity a 222 nm ana canza shi zuwa rabon folds na halitta.e Gwajin jujjuyawar NTFlSp, NTMiSp da rage NTMiSp (100 mg/mL).Matsakaicin girman 5 mm.f Siffar CD na NT, NTFLSp, NTMiSp da rage NTMiSp.Molar ellipticity a 222 nm ana canza shi zuwa rabon folds na halitta.Ana nuna cikakkun sifofin NT a 25 °C da 95 °C a Ƙarin Hoto 8.
Matsakaicin gishiri na jiki yana ƙayyade hulɗar electrostatic tsakanin NT subunits da dimerization na NT canja wuri zuwa ƙananan pH18.Mun gano cewa kasancewar 154 mM NaCl da NaF sun hana gelation, bi da bi (Fig. 5a, b; Ƙarin Fig. 2b) da kuma cewa waɗannan salts sun kara yawan kwanciyar hankali na NT monomers (Fig. 5b, Ƙarin Hoto 8). .Har ila yau, yana nuna cewa haɓakar kwanciyar hankali, maimakon dimerization, yana hana samuwar gel.
Don ci gaba da bincika rawar da ke tattare da lalata furotin da kwanciyar hankali a cikin gelation, mun yi amfani da mutants guda biyu, NT * da NTA72R, waɗanda kuma sun kasance monomeric a ƙananan pH28.30.NT* shine mutantan juzu'i biyu na caji wanda a cikinsa aka daidaita rarraba cajin dipolar na monomer, wanda ke hana dimerization kuma yana ƙaruwa da kwanciyar hankali na monomer.NTA72R da aka caje dipole ne, amma Arg-musanya Ala yana kan iyakar dimer, don haka maye gurbi yana tsoma baki tare da mu'amalar subunit da ake buƙata don daidaitawa.Bayan shigar da shi a 37 ° C, NT * bai samar da hydrogel ba, yayin da NTA72R ya kafa gel ɗin opaque don 15 min (Fig. 5c).Tun da duka NT* da NTA72R ba za su iya raguwa ba amma sun bambanta a cikin kwanciyar hankali na monomer (Fig. 5d), waɗannan sakamakon suna nuna ƙarfi sosai cewa kwanciyar hankali mai ƙarfi yana hana NT daga gelling.Wannan kuma yana goyan bayan gaskiyar cewa HT * yana samar da gel lokacin da ba shi da kwanciyar hankali a babban zafin jiki (bayan 8 min a 60 ° C; Fig. 5c).An nuna a baya cewa babban abun ciki na methionine a cikin NT yana haifar da nadawa na halitta kuma cewa shida Met zuwa Leu maye gurbin (wanda ake magana a nan a matsayin sa-NT-L6) yana ƙarfafa NT46 monomer.Dangane da zato cewa ana buƙatar sassaucin tsari don samuwar NT gel, mun gano cewa ƙwanƙolin ɗan adam na His-NT-L6 bai wuce 37 ° C ba (Hoto 5c, d).Duk da haka, His-NT-L6 kuma ya samar da gel akan shiryawa a 60 ° C na 60 min (Fig. 5c).
Ƙarfin NT don canzawa zuwa tsarin β-sheet da samar da hydrogels ya bayyana yana aiki ga wasu amma ba duk wuraren NT na spidroin ba.NTs daga nau'ikan siliki daban-daban da nau'in gizo-gizo, Trichonephila clavipes (NTFlSp), sun kafa gels duk da ƙarancin abun ciki na methionine da ƙarancin kwanciyar hankali na thermal (Fig. 5e, f da Ƙarin Table 2).Sabanin haka, NT daga ƙananan furotin ampullar spidroin daga Araneus ventricosus (NTMiSp) tare da ƙananan kwanciyar hankali na thermal da babban abun ciki na methionine bai samar da hydrogels (Ƙarin Table 2 da Fig. 5e, f).Ƙarshen na iya haɗawa da kasancewar intramolecular disulfide bonds29,47.Kullum, lokacin da aka rage haɗin disulfide na NTMiSp, ya kafa hydrogel bayan shiryawa a 37 ° C na 10 min (Fig. 5e).A ƙarshe, ya kamata a lura cewa sassaucin tsari yana da mahimmanci, amma ba kawai, ma'auni na samuwar gel daga NT ba.Wani abin da zai iya zama mai dacewa shi ne yiwuwar samar da fibrils amyloid, da bincike tare da bayanan zipper da Waltz algorithm ya nuna dangantaka tsakanin ikon samar da gels da kasancewar yankunan amyloidogenic, da kuma girman yankunan da aka annabta. don samar da zippers masu siffa.Akwai alaƙa (Ƙarin Teburin 2 da Ƙarin Hoto 9).
Ƙarfin NT don samar da fibrils da samar da gels a ƙarƙashin yanayi masu kyau ya sa mu yi tunanin cewa NT fusions tare da sauran gutsuttsura furotin na iya zama gels tare da cikakken aikin abokan haɗin gwiwa.Don gwada wannan, mun gabatar da furotin mai kyalli (GFP) da purine nucleoside phosphorylase (PNP) a C-terminus na NT, bi da bi.Sakamakon sunadaran fusion an bayyana su a cikin E. coli tare da yawan amfanin ƙarshe na ƙarshe (150 MG / L da 256 mg / L al'adun flask ɗin girgiza don His-NT-GFP da His-NT-PNP, bi da bi), daidai da abin da aka nuna. don Wasu sunadaran da aka haɗa zuwa NT Ref.30. His-NT-GFP (300mg / mL) da His-NT-PNP (100mg / mL) sunadaran fusion sun kafa gels bayan 2 hours da 6.5 hours a 37 ° C kuma, mahimmanci, ɓangaren GFP ya kasance ba canzawa ba.ana lura da shi bayan gelation, tare da> 70% na farkon ƙarfin haske ya rage bayan gelation (Fig. 6a).Don auna ayyukan PNP a cikin maganin sa-NT-PNP da gels, dole ne mu tsarma furotin fusion tare da NT saboda aikin enzymatic na shiri mai tsabta ya kasance a waje da kewayon ganowa na ƙididdigar gelling.Gel da aka kafa tare da cakuda mai dauke da 0.01 mg / mL His-NT-PNP da 100 mg / mL NT ya riƙe 65% na aikin enzymatic na farko na samfurori da aka riga aka yi (Fig. 6b).Gel ɗin ya kasance cikakke yayin aunawa (Ƙarin Hoto 10).
Ƙarfin haske mai alaƙa kafin da bayan gelation na His-NT-GFP (300 mg/mL) da jujjuyawar vial mai ɗauke da His-NT-GFP hydrogel (300 mg/mL) ƙarƙashin haske da haske UV.Makiyoyi suna nuna ma'auni ɗaya (n = 3), sandunan kuskure suna nuna daidaitattun sabani.Ana nuna matsakaicin ƙimar a tsakiyar sandunan kuskure.b PNP aiki da aka samu ta hanyar bincike na fluorometric ta amfani da mafita da gels da ke kunshe da NT (100 mg / ml) da cakuda mai dauke da 0.01 mg / ml his-NT-PNP da 100 mg / ml New Taiwan dollars.Inset ɗin yana nuna jujjuyawar vial ɗin da ke ɗauke da hydrogel mai ɗauke da His-NT-PNP (masanin sikelin mm 5).
Anan, mun bayar da rahoton samuwar hydrogels daga NT da sauran sunadaran spidroin recombinant ta hanyar haifar da maganin furotin a 37 ° C (Hoto 1).Mun nuna cewa gelation yana hade da canzawar α-helices zuwa β-layers da kuma samar da fibrils amyloid (Figs. 3 da 4).Wannan binciken yana da ban mamaki yayin da NTs aka naɗe globular globular-helix bundles sanannen su matuƙar solubility da babban kwanciyar hankali a taro>200 mg/mL a 4°C na da yawa kwanaki27.Bugu da kari, NTs suna sake ninkawa da sauri bayan ƙarancin zafi a ƙananan adadin furotin a cikin µM.Dangane da sakamakon mu, samuwar fibril yana buƙatar haɗuwa da> 10 MG / ml sunadarin sunadaran sunadaran da ƙananan zafin jiki (Fig. 1).Wannan ya dace da ra'ayin cewa fibrils amyloid na iya samuwa daga sunadaran sunadaran globularly wanda ke cikin yanayin da ba a bayyana ba saboda yanayin zafi a ƙarƙashin yanayin ilimin lissafi 48.Misalan sunadaran da ke fuskantar wannan juzu'i sun haɗa da insulin49,50, β2-microglobulin, transthyretin da lysozyme51,52,53.Ko da yake NT shine α-helix a cikin mahaifarsa, kusan 65% na sarkar polypeptide ya dace da steric zipper samuwar (Fig. 4e) 45.Tunda monomer ɗin yana da ƙarfi ta hannu46, yana iya fallasa waɗannan yankuna masu yuwuwar amyloidogenic a matsakaicin matsakaicin yanayin zafi kuma a babban adadin furotin duka na iya kaiwa ga ƙima mai mahimmanci don samuwar amyloid fibril54.Bayan wannan dalili, mun sami wani mummunan dangantaka tsakanin spidroin maida hankali da kuma lokacin gelation (Fig. 1c), kuma idan monomeric NT conformation aka daidaita ko dai ta maye gurbi (NT *, His-NT-L6) ko ta gishiri Bugu da kari, zai iya hana. samuwar hydrogels (Fig. 5).
A mafi yawan lokuta, amyloid fibrils bace daga bayani a matsayin hazo, amma a karkashin wasu yanayi za su iya samar da hydrogels55,56,57.Fibrils masu samar da hydrogel yawanci suna da babban al'amari rabo kuma suna samar da tsayayyen hanyoyin sadarwa mai girma uku ta hanyar haɗin kwayoyin halitta,55,58 daidai da sakamakonmu.Don samar da hydrogel a cikin vitro, sunadaran suna sau da yawa cikakke ko kuma an buɗe su, alal misali, ta hanyar bayyanar da abubuwan kaushi, babban zafin jiki (70-90 ° C) da / ko ƙananan pH (1.5-3.0) 59,60,61,62.Spidroin hydrogels da aka kwatanta a nan ba sa buƙatar aiki mai tsauri, kuma ba sa buƙatar masu haɗin kai don daidaita hydrogels.
An riga an ba da rahoton cewa spidroin yana maimaitawa da QDs, waɗanda ke bayyana suna jujjuyawar β-sheet yayin jujjuyawar siliki, suna samar da hydrogels.Idan aka kwatanta da bincikenmu, lokutan shiryawa da/ko yanayin zafin jiki sun fi tsayi ko sama da haka, kuma sakamakon hydrogels yawanci ba su da kyau (Hoto na 7 da Ƙarin Tebu 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. Baya ga saurin gel sau, NT hydrogels> 300 mg / mL (30%) ya fi duk sauran abubuwan da aka kwatanta recombinant gizo-gizo siliki protein hydrogels, da na halitta hydrogels irin su gelatin, alginate (2%), agar (0.5 %). ) da collagen.(0.6%) (Hoto na 7 da Ƙarin Tables 1 da 3)37,39,66,67,68,69,70,71,72,73,74.
An kwatanta lokacin gel da na'urorin roba na hydrogels a cikin wannan binciken tare da sauran hydrogels na tushen spidroin da zaɓaɓɓun hydrogels na halitta.Ana ba da nassoshi tare da bayanin yanayin gelation.APS Ammonium persulfate, zafin jiki.Bayanai 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
Spiders sun bayyana sun haɓaka hanyoyin da za su hana spidroin daga gelling yayin ajiya.Duk da yawan adadin furotin a cikin siliki na siliki, babban yanki mai maimaitawa da ke hade da yanki na ƙarshe yana nufin cewa bayyanar NT da CT a cikin gland shine daidai da kusan 10-20 mg / ml, a iyakar wannan binciken.da ake bukata don in vitro lura da samuwar hydrogel.Bugu da kari, irin wannan taro na salts 16 stabilized NT, kamar yadda a cikin siliki gland (Fig. 5b).An yi nazarin NT conformation a cikin E. coli cytosol kuma an gano ya fi nannade sosai fiye da lokacin da aka duba shi a cikin vitro, yana ƙara nuna cewa gishiri ko wasu abubuwan suna hana haɗuwa a cikin vivo.Duk da haka, ikon NTs don canzawa zuwa β-sheet fibrils na iya zama mahimmanci don samuwar filament kuma ya kamata a bincika a cikin binciken gaba.
Baya ga al'amuran labari na NT-amyloid-kamar fibril da haɓakar hydrogel da aka lura a cikin wannan binciken, mun kuma nuna cewa wannan sabon abu na iya samun aikace-aikacen ilimin halittu da ilimin halittu (Fig. 8).A matsayin hujja na ra'ayi, mun haɗu da NT tare da GFP ko PNP kuma mun nuna cewa furotin fusion shima yana samar da hydrogels lokacin da aka sanya shi a 37 ° C kuma cewa GFP da ɓangarorin PNP suna riƙe da ayyukan su bayan gelation (Hoto 6).Nucleoside phosphorylases sune mahimman abubuwan haɓaka haɗin haɗin nucleoside analogues75, wanda ya sa bincikenmu ya dace da masana'antar biopharmaceutical.Manufar bayyana sunadaran haɗin gwiwa waɗanda ke samar da ingantaccen hydrogels a ƙarƙashin yanayi masu kyau suna ba da damar ƙirƙirar hydrogels masu aiki tare da kyawawan kaddarorin don aikace-aikacen da yawa kamar rashin motsin enzyme, sakin magunguna da sarrafa nama.Bugu da kari, NT da NT* su ne ingantattun alamomin magana30, wanda ke nufin ana iya amfani da NT da bambance-bambancensa don samar da furotin mai narkewa mai narkewa da kuma ƙirƙirar sunadaran da ba a iya motsi a cikin 3D hydrogels.
NT mai narkewa ne, α-helical kuma barga a ƙananan ƙira (µM) da 37°C.A daidai wannan zafin jiki, amma a karuwa mai yawa (> 10 mg / ml), NT yana samar da gels wanda ya ƙunshi fibrils amyloid.NT fusion proteins suma suna samar da gels na fibrillar tare da gutsuttsuran fusion ɗin aiki cikakke, suna ba da damar sunadaran sunadaran da ba su iya motsi a cikin 3D hydrogels ta amfani da NT.Kasa: NT (PDB: 4FBS) da kuma misalai na hanyoyin sadarwa na fiber da tsarin gina jiki masu alaƙa (wanda aka ɗauka kuma ba a zana su zuwa sikelin ba, GFP PDB: 2B3Q, 10.2210/pdb2B3Q/pdb; PNP PDB: 4RJ2, 10.2210/2/pd4).
Gine-ginen (duba Ƙarin Tebu 4 don cikakken jerin abubuwan da suka haɗa da jerin amino acid) an haɗa su zuwa plasmid pT7 kuma an canza su zuwa E. coli BL21 (DE3).E. coli dauke da plasmids na injiniya an yi amfani da su a cikin Luria broth wanda aka kara da kanamycin (70 mg / l) kuma ya girma cikin dare a 30 ° C da 250 rpm.Daga nan aka sanya al'adar 1/100 cikin matsakaicin LB mai ɗauke da kanamycin kuma an yi al'adar a 30 ° C da 110 rpm har OD600 ya kai 0.8.Don nazarin NMR, an haɓaka ƙwayoyin cuta a cikin M9 ƙaramin matsakaici mai ɗauke da 2 g na D-glucose 13C (Aldrich) da 1 g na ammonium chloride 15N (Cambridge Isotope Laboratories, Inc.) don alamar furotin tare da isotopes.Rage yawan zafin jiki zuwa digiri 20 na ma'aunin celcius kuma haifar da furcin furotin tare da 0.15 mM isopropylthiogalactopyranoside (haɗuwar ƙarshe).Bayan bayyanar furotin na dare, an girbe sel a 7278 × g, 4 ° C na 20 min.An sake dakatar da pellets na salula a cikin 20 mM Tris-HCl, pH 8, da daskararre har sai an ƙara amfani.Kwayoyin da aka nannade an lalata su ta hanyar amfani da mai rushewa tantanin halitta (injunan jerin TS, Constant Systems Limited, Ingila) a 30 kPa.Sa'an nan kuma lysates an centrifuged a 25,000 g na minti 30 a 4 ° C.Don NTMiSp, an sake dakatar da pellet ɗin a cikin 2M urea, 20mM Tris-HCl, pH 8, kuma ana sonicated na 2 min (2 s kunnawa/kashe, 65%), sannan a sake sanyawa a 25,000 xg, 4° C. a ciki. 30 min.An ɗora ma'auni a kan ginshiƙi na Ni-NTA, an wanke shi tare da 20 mM Tris-HCl, 2 mM imidazole, pH 8, kuma a ƙarshe an cire furotin tare da 20 mM Tris-HCl, 200 mM imidazole, pH 8. Don samar da NT2RepCT da NTCT, thrombin narkewa yana gabatar da shafin (ThrCleav) tsakanin sa da NT.Shafukan cleavage na Thrombin kuma suna cikin His-NT-ThrCleav-2Rep (yana samar da 2Rep), His-thioredoxin-ThrCleav-NT (yana samar da NT), His-thioredoxin-ThrCleav-CT (yana samar da CT), His-Thioredoxin-ThrCleav-NT. .* (yana samar da NT *), His-Thioredoxin-ThrCleav-NTA72R (yana samar da NTA72R), His-Thioredoxin-ThrCleav-NTFlSp (yana samar da NTF1Sp), da His-sulfur Redoxin-ThrCleav-NTMiSp (yana samar da NTMiSp).An narkar da abubuwan da aka gina tare da thrombin (1: 1000) kuma an yi su a cikin dare a 4 ° C. tare da 20 mM Tris-HCl, pH 8, ta yin amfani da membrane na Spectra / Por dialysis tare da ma'aunin nauyin kwayoyin halitta na 6-8 kDa.Bayan dialysis, ana ɗora maganin a kan ginshiƙi na Ni-NTA kuma ana tattara magudanar da ke ɗauke da furotin mai sha'awa.An ƙaddara yawan adadin furotin ta hanyar auna ɗaukar nauyin UV a 280 nm ta amfani da ƙimar ƙarancin kowane furotin, ban da NTF1Sp, wanda yayi amfani da gwajin Bradford bisa ga ka'idar masana'anta.An ƙaddara tsarki ta SDS polyacrylamide (4-20%) gel electrophoresis da Coomassie shuɗi mai haske.Sunadaran an tattara su ta amfani da matattarar centrifuge (VivaSpin 20, GE Healthcare) a 4000 xg tare da yanke nauyin kwayoyin halitta 10 kDa a cikin zagayowar mintuna 20.
Narke maganin furotin kuma a hankali pipet 150 µl cikin 1 ml bayyananne vial septum (8 x 40 mm Thermo Scientific).An lullube bututun kuma an rufe su da parafilm don hana fitar da iska.Samfurori (n = 3) an haɗa su a 37°C ko 60°C kuma ana juyar da su lokaci-lokaci don lura da gelation.Samfurori waɗanda ba su da gel an shirya su na akalla mako guda.Rage NTMiSp disulfide bond tare da 10 mM DTT a kowace furotin 10 µM.Don nazarin gelation na halitta gizo-gizo siliki coatings, da Sweden gada gizogizo da aka yanke, da biyu main yanke gland an sanya a cikin 200 μl na 20 mM Tris-HCl buffer pH 8 da kuma yanke don ba da damar da shafi ya rabu da gland..Abubuwan da ke cikin gland suna narkar da su a cikin buffer, 50 µl don ƙayyade busassun nauyi (ta hanyar shirya buɗaɗɗen vials a 60 ° C zuwa nauyi akai-akai) da 150 µl don gelation a 37 ° C.
Auna ma'auni / kayan aiki an yi shi da bakin karfe ta amfani da farantin layi daya tare da saman diamita na 20 mm da rata na 0.5 mm.Zafin samfurin daga 25 °C zuwa 45 °C kuma baya zuwa 25 ° C a cikin adadin 1 °C a minti daya ta amfani da farantin karfe na kasa Peltier.An gudanar da ma'auni na vibration a mita na 0.1 Hz kuma a cikin yanki na viscoelastic na layi na kayan aiki a wani nau'i na 5% da 0.5% don samfurori na 100 mg / mL da 300-500 mg / ml, bi da bi.Yi amfani da ɗakin zafi na al'ada don hana ƙawancewar.An yi nazarin bayanai ta amfani da Prism 9.
Don tattara bakan infrared (IR) a dakin da zafin jiki daga 800 zuwa 3900 cm-1.Na'urar ATR, da kuma hanyar haske ta cikin na'urar gani, ana wanke ta da busasshiyar iskar da aka tace kafin da kuma lokacin gwajin.Magani (500 MG / mL don rage girman kololuwar ruwa a cikin bakan) an yi amfani da su a kan lu'ulu'u, kuma an kafa gels (500 mg / mL) kafin aunawa sannan kuma a canza su zuwa lu'ulu'u (n = 3).An yi rikodin sikanin 1000 tare da ƙuduri na 2 cm-1 da sifili na aikin sake zagayowar 2. An ƙididdige ƙididdiga ta biyu ta amfani da OPUS (Bruker) ta amfani da kewayon smoothing na maki tara.An daidaita siginar zuwa yankin haɗin kai ɗaya tsakanin 1720 da 1580 cm-1 ta amfani da F. Menges "Spectragryph - Software Spectroscopy Optical".A cikin ATR-IR spectroscopy, zurfin shigar da katakon infrared a cikin samfurin ya dogara da adadin raƙuman ruwa, wanda ke haifar da ɗaukar ƙarfi a ƙananan raƙuman raƙuman ruwa fiye da mafi girman raƙuman ruwa.Ba a gyara waɗannan tasirin ba don bakan da aka nuna a cikin Fig.3 domin suna da ƙanƙanta (Ƙarin Hoto na 4).An ƙididdige abubuwan da aka gyara don wannan adadi ta amfani da software na Bruker OPUS.
A ka'ida, ƙididdige ƙididdige ƙididdigar furotin yana yiwuwa bayan ingantaccen juzu'i na abubuwan da ke cikin kololuwar amide I.Duk da haka, wasu cikas suna tasowa a aikace.Hayaniya a cikin bakan na iya fitowa azaman (ƙarya) kololuwa yayin ƙaddamar da juyin juya hali.Bugu da ƙari, kololuwa saboda lankwasa ruwa ya dace da matsayi na amide I peak kuma yana iya samun irin wannan girman don samfurori da ke dauke da ruwa mai yawa, irin su gel na ruwa da aka yi nazari a nan.Sabili da haka, ba mu yi ƙoƙari mu lalata kololuwar amide I gaba ɗaya ba, kuma abubuwan da muka lura ya kamata a yi la’akari da su ne kawai don tallafawa wasu hanyoyin kamar NMR spectroscopy.
Maganin 50 mg/ml NT da His-NT2RepCT an yi gelled na dare a 37°C.Bayan haka, an diluted hydrogel tare da 20 mM Tris-HCl (pH 8) zuwa maida hankali na 12.5 mg / ml, girgiza da kyau kuma an cire shi don karya gel.Bayan haka, an narkar da hydrogel sau 10 tare da 20 mM Tris-HCl (pH 8), 5 μl na samfurin an yi amfani da shi a kan grid na tagulla wanda aka lullube shi da formvar, kuma an cire samfurin da ya wuce tare da takarda.An wanke samfurori sau biyu tare da 5 µl na ruwan MilliQ kuma an lalata su da 1% uranyl formate na minti 5.Cire tabo mai yawa tare da takarda mai shayarwa, sannan iska bushe ragar.Anyi hoto akan waɗannan grid ta amfani da FEI Tecnai 12 Spirit BioTWIN aiki a 100 kV.Hotunan an yi rikodin su a x 26,500 da x 43,000 girma ta amfani da kyamarar Veleta 2k × 2k CCD (Olympus Soft Imaging Solutions, GmbH, Münster, Jamus).Ga kowane samfurin (n = 1), an yi rikodin hotuna 10-15.An yi amfani da ImageJ (https://imagej.nih.gov/) don nazarin hoto da auna diamita na fiber (n = 100, filaye daban-daban).An yi amfani da Prism 9 don yin gwaje-gwajen t-t-tes marasa guda biyu (mai-wutsiya biyu).Ma'anar His-NT2RepCT da NT fibrils sun kasance 11.43 (SD 2.035) da 7.67 (SD 1.389) nm, bi da bi.Tazarar amincewa (95%) shine -4.246 zuwa -3.275.digiri na 'yanci = 198, p <0.0001.
80 µl na samfuran ruwa mai ɗauke da 10 µM thioflavin T (ThT) an auna su a cikin sau uku (n = 3) a ƙarƙashin yanayi na tsaye ta amfani da Corning 96- rijiyar baƙar fata bayyanan faranti na ƙasa (Corning Glass 3881, USA).An yi rikodin bambance-bambancen fluorescence ta amfani da matatar tashin hankali na 440nm da kuma tacewa na 480 nm (FLUOStar Galaxy daga BMG Labtech, Offenburg, Jamus).Siginar ThT ba ta cika ko kashewa ba, saboda an yi gwaje-gwaje tare da mabambantan abubuwan ThT ba tare da canza ƙarfin siginar ba.Yi rikodin sha a 360 nm don auna haze.Don gwaje-gwajen iri, an kafa gels 100 mg/mL a 37 ° C., an sake dakatar da su, kuma an yi amfani da su don shuka a ƙimar molar na 5%, 10%, da 20%.An yi nazarin bayanai ta amfani da Prism 9.
Narke hannun jari na His-NT2RepCT da NT>100 mg/mL akan kankara kuma tace ta hanyar tacewa 0.22µm.An ƙididdige abubuwan tattarawa ta hanyar auna abin sha a 280 nm ta amfani da Nandrop.A cikin rijiyoyin 96-riji baƙar fata ba tare da ɗauri ba (Corning) tare da bayyananniyar ƙasa, samfuran an diluted zuwa 20 MG / ml a cikin 20 mM Tris-HCl pH 8 kuma an haɗe su da 5 μM ThT (ƙarshen taro), jimlar ƙaddamarwar samfurin. 50 μl girma.An zana samfurori kowane minti 10 a 37 ° C akan na'urar duban dan tayi na CellObserver (Zeiss) tare da tashar haske da aka watsa da FITC tashin hankali da saitin tacewa don hoton ThT.Ana amfani da ruwan tabarau 20x/0.4 don yin hoto.An yi amfani da Zen Blue (Zeiss) da ImageJ (https://imagej.nih.gov/) don nazarin hoto.An kuma shirya gels daga mafita na NT da His-NT2RepCT a maida hankali na 50 mg/mL dauke da 20 mM Tris pH 8 da 5 µM ThT kuma an sanya shi a 37 ° C na 90 min.An canza guntun gel ɗin zuwa sabuwar rijiya mai ɗauke da 20 mM Tris, pH 8, da 5 μM ThT a cikin baƙar fata 96 baƙar fata mara kyau.Nemi koren kyalli da hotunan fili mai haske a girman 20x/0.4.An yi amfani da ImageJ don nazarin hoto.
An sami mafita na NMR a 310 K akan 600 MHz Bruker Avance Neo spectrometer sanye take da QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN).Samfuran NMR da ke ɗauke da 10 mg/mL sunadaran sunadaran da aka yi wa lakabi da 13C, 15N, narkar da su a cikin 20 mM Tris-HCl (pH 8), 0.02% (w/v) NaN3, 5% DO (v/v), (n = 1) .An yi amfani da sauye-sauyen sinadarai na NT2RepCT a pH 6.7 don sanya kololuwar 23 a cikin bakan 2D na 15N-HSQC.
An yi rikodin siginar kusurwar sihiri mai ƙarfi na NMR (MAS) na 13C, 15N mai alamar hydrogels akan na'urar hangen nesa na Bruker Avance III HD a 800 MHz sanye take da 3.2 mm 13C/15N{1H} binciken lantarki.An sarrafa samfurin zafin jiki ta amfani da madaidaicin iskar gas mai zafi a 277 K. Dimensional dipole rotational resonance (DARR) 76 da sake haɗawa da mitar rediyo (RFDR) 77 spectra an samo su a mitocin MAS na 12.5 kHz da 20 kHz, bi da bi.Cross polarization (CP) daga 1H zuwa 13C an yi ta ta amfani da ramin layi daga 60.0 zuwa 48.0 kHz a 1H, 61.3/71.6 kHz a 13C (a 12.5/20 kHz MAS) da lokacin lamba 0.5-1 ms.Spinal6478 decoupling a 73.5 kHz an yi amfani dashi yayin tattara bayanai.Lokacin sayan ya kasance millise seconds 10 kuma jinkirin sake zagayowar ya kasance 2.5 seconds.Abubuwan haɗin gwiwar Cα/Cβ masu alaƙa guda ɗaya da aka lura a cikin sifofin RFDR an sanya su bisa ga halayen saura-nau'in sinadarai da sauye-sauye masu alaƙa da yawa a cikin bakan DARR.
An yi amfani da bayanan Zipper79 (https://services.mbi.ucla.edu/zipperdb/) don kimanta abubuwan da ake so da kuma kuzarin Rosetta don NT, NTFLSp, da NTMiSp.Rukunin bayanai na Zipper yana ƙididdige Rosetta Energy80, wanda ke haɗa ayyukan makamashi kyauta da yawa don ƙira da kuma nazarin tsarin furotin.Matsayin makamashi na -23 kcal / mol ko ƙananan yana nuna babban hali na fibrillate.Ƙarƙashin makamashi yana nufin ƙarin kwanciyar hankali na β-strands guda biyu a cikin daidaitattun zik din.Bugu da ƙari, an yi amfani da algorithm na Waltz don tsinkaya yankunan amyloidogenic a cikin NT, NTFLSp da NTMiSp Ref.81. (https://waltz.switchlab.org/).
An gauraya maganin furotin na NT tare da 2- (N-morpholino) ethanesulfonic acid (MES) buffer a pH 5.5 da 6.0 don rage pH zuwa pH 6 da 7, bi da bi.Matsakaicin furotin na ƙarshe shine 100 mg / ml.
An yi ma'auni a kan J-1500 CD spectrometer (JASCO, USA) ta amfani da 300 μL cuvette tare da hanyar gani na 0.1 cm.An diluted sunadaran zuwa 10 μM (n = 1) a cikin 20 mM phosphate buffer (pH 8).Don nazarin kwanciyar hankali na gina jiki a gaban gishiri, an yi nazarin sunadaran a daidai wannan taro (n = 1) a cikin 20 mM phosphate buffer (pH 8) dauke da 154 mM NaF ko NaCl, bi da bi.An yi rikodin yanayin zafin jiki a 222 nm daga 25 ° C zuwa 95 ° C tare da ƙimar dumama na 1 ° C / min.An ƙididdige adadin sunadaran da aka naɗe su ta hanyar amfani da dabara (KDmeasure - KDfinal)/(KDstart - KDfinal).Bugu da kari, an rubuta bakan biyar ga kowane samfurin daga 260 nm zuwa 190 nm a 25 ° C kuma bayan dumama zuwa 95 ° C.An daidaita nau'ikan bakan gizo guda biyar, an daidaita su kuma an juyar da su zuwa madaidaicin molar.An yi nazarin bayanai ta amfani da Prism 9.
An auna ƙarfin haske na His-NT-GFP (300 mg/mL, 80 µL) a cikin sau uku (n = 3) a cikin 96-rijiya Corning faranti tare da ƙasa mai haske (Corning Glass 3881, Amurka) a ƙarƙashin yanayi na tsaye.Auna samfurori tare da mai karanta faranti na tushen haske tare da tsayin motsin rai na 395 nm kuma rikodin fitarwa a 509 nm kafin gelation da sa'o'i 2 daga baya a 37 ° C.An yi nazarin bayanai tare da Prism 9.
Purine nucleoside phosphorylase kit kit (hanyar fluorometric, Sigma Aldrich) an yi amfani da shi bisa ga umarnin masana'anta.Don auna aiki a cikin gels da mafita waɗanda ke ɗauke da His-NT-PNP, haɗa 10 ng na His-NT-PNP tare da 100 mg/mL NT zuwa jimlar ƙarar 2 µL saboda gel ɗin ya ba da sigina sama da tazarar gano saitin.An haɗa sarrafawa don gels da mafita ba tare da His-NT-PNP ba.An gudanar da ma'aunin sau biyu (n = 2).Bayan an auna aikin, an cire cakudar da aka yi kuma an dauki hoton gel don tabbatar da cewa gel ɗin ya kasance daidai lokacin aunawa.An yi nazarin bayanai ta amfani da Prism 9.
Don ƙarin bayani kan ƙirar binciken, duba ƙayyadaddun binciken Nature mai alaƙa da wannan labarin.
Figures 1 da 2 suna gabatar da bayanan farko.1c, 2a–c, 3a, b, e–g, 4, 5b, d, f, da 6, Karin Fig.3, karin fig.5a, d, ƙarin fig.6 da ƙarin fig.8. Bayanai daga wannan binciken an shirya su a cikin Zenodo database https://doi.org/10.5281/zenodo.6683653.Bayanan NMR da aka samu a cikin wannan binciken an buga su zuwa ma'ajiyar BMRBig a ƙarƙashin ID na shigarwa bmrbig36.An ɗauki tsarin GFP da PNP daga PDB (GFP 2B3Q, PNP 4RJ2).
Tashi, A. da Johansson, J. Kadin siliki na gizo-gizo na wucin gadi.Chemical National.ilmin halitta.11, 309-315 (2015).
Babb, PL et al.Nephila clavipes genome yana ba da haske game da bambancin kwayoyin siliki na gizo-gizo da hadaddun maganganunsu.National Genette.49, 895-903 (2017).