310 10 * 1mm tšepe e sa hloekang e kopantsoeng ka motsoako oa lik'hemik'hale, Likarolo tsa N-terminal tsa spidroin li theha li-hydrogel tse thehiloeng ho li-amyloid fibrils le ho fana ka sethala sa ho thibela protheine.

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Tlhaloso

310 10 * 1mm Bafani ba li-tubing tsa tšepe tse sa hloekang tse kopantsoeng

Sebopeho sa Lik'hemik'hale

Thepa ea Mechini

Liprotheine tsa silika tsa sekho (liprotheine tsa silika tsa sekho) li na le ts'ebeliso e ngata ho nts'etsopele ea li-biomaterials tse ncha, empa tlhaho ea tsona ea multimodal le aggregation e etsa hore ho be thata ho li fumana le ho li sebelisa habonolo.Mona re tlaleha hore liprotheine tse nyane tsa spidroin, 'me, habohlokoa, sebaka sa N-terminal (NT) ka boeona se theha li-hydrogel tse itšebetsang le tse bonaletsang ho 37 ° C.liprotheine tsa fusion tse nang le NT le protheine e tala ea fluorescent kapa purine nucleoside phosphorylase e etsa liprotheine tse sebetsang ka botlalo.Li-Hydrogels.Liphetho tsa rona li bonts'a hore liprotheine tse kopaneng tsa NT le fusion li fana ka lihlahisoa tse phahameng tsa polelo le ho fa li-hydrogel tse nang le thepa e khahlang joalo ka ponaletso, gelation ntle le ho kopanya, le ho emisa ka kotloloho liprotheine tse sebetsang ka bongata bo boholo.
Likho li na le lihlopha tse ka bang supileng tse fapaneng tsa litšoelesa tsa silika, tse hlahisang mofuta o itseng oa silika.Mefuta eohle e supileng ea silika e entsoe ka liprotheine tsa silika tsa sekho (spidroins) tse ka bang 6000 mesaletsa ka bolelele 'me li na le sebaka se seholo se pheta-phetoang bohareng se pota-potiloeng ke libaka tse chitja tsa N- le C-terminal (NT le CT)1,2.Mofuta oa silika o ithutoang ka ho fetisisa, e leng ampulla e ka sehloohong, e hlahisoa ke tšoelesa e ka sehloohong ea ampulla.Ho tšoelesa ena, monolayer ea lisele tsa epithelial e kopanya liprotheine tsa spidroin ebe li li kenya ka har'a lumen ea tšoelesa, moo li leng teng ka mokhoa o qhibilihang (doping) maemong a phahameng haholo (30-50% w/v)3,4.Ho hlophisoa le ho lumellana ha liprotheine tse ka sehloohong tsa ampullar spidroin ka har'a tšoelesa ho 'nile ha phehisana khang, empa bopaki bo bongata ba liteko bo bontša ho ba teng ha sebopeho se tloaelehileng sa helical le / kapa se sa tloaelehang le mehaho ea micellar kapa lamellar5,6,7,8,9,10.Le hoja libaka tse pheta-phetoang li laola mekhoa ea mechine ea likhoele tsa silika, ho etsa li-nanocrystals tsa β-sheet le mehaho ea amorphous11,12,13,14,15, libaka tsa ho qetela li laola likhoele tsa silika ho arabela maemo a fetohang hammoho le silika ea silika16,17,18.Ka ho laola sebopeho sa silika, 19. Libaka tsa li-terminal li bolokiloe ka mokhoa oa ho iphetola ha lintho 'me mosebetsi oa tsona o ka ba o tloaelehileng ho liprotheine tsohle tsa spidroin 2,20,21.Nakong ea ho pholletsa le tšoelesa, pH ea spidroin e fokotseha ho tloha hoo e ka bang 7.6 ho ea ho <5.716 'me e eketseha ka ho kuta le ho otlolla ho kenella ka ho sisinyeha ka mokhoa o fokolang butle-butle.Ka tharollo, CT ke α-helical constitutive parallel dimer17, empa ho arabela pH e tlaase le matla a ho kuta, CT e hlahisa le ho fetola β-layers16, 17, mohlomong e bakang β-layers libakeng tse pheta-phetoang tsa Convert 16. NT ke monomeric tlas'a maemo a le mong tlas'a maemo a le mong. maemo a bonts'ang maemo a lumen ea tšoelesa le ho kopanya ho qhibiliha ha spidroin, empa ha pH e fokotsehile, tlhahiso ea liketane tse ngata tsa mahlakoreng a carboxylic acid e lebisa ho fokotseha ha NT ka pKa ea hoo e ka bang 6.5, kahoo e tsitsisa NT le ho lokisa spidroin ka bongata. bongata.marang-rang16,18.Ka hona, NT e phetha karolo ea bohlokoa ho thehoeng ha filament, ho fetoha ho tloha monoma ka ho roala ho ea ho dimer ho fiber23,24,25.NT e lula e qhibiliha haholo ebile e le helical tlas'a maemo ohle a ithutoang ho fihlela joale16, 18, 19, 20, 26, 27, 28, 29, e ileng ea susumetsa tsoelo-pele ea eona e le leibole e matlafatsang ho qhibiliha bakeng sa tlhahiso ea liprotheine tsa heterologous.
Protheine ea silika ea sekho e nyane, e nang le NT e le 'ngoe, sebaka se le seng se iphetang, CT e le' ngoe, le tag ea His6 (His-NT2RepCT) bakeng sa tlhoekiso, e ka qhibiliha ka har'a metsi joaloka protheine ea silika ea sekho 'me e etsisa litšobotsi tsa bohlokoa tsa sekho sa silika. .Setšoantšo sa 25.31.His-NT2RepCT e ka ohloa ka har'a likhoele tse tsoelang pele ho sebelisoa mochini oa biomimetic oo ho ona pH 8 e qhibilihang e kenngoa ka har'a bate ea metsi ea pH 525,32,33,34,35.Bioreactor fermentation ea E. coli e hlalosang His-NT2RepCT le phekolo e ileng ea latela ka mor'a phekolo e ile ea fella ka > 14 g/L chai ka mor'a tlhoekiso.Kotulo e phahameng, ho qhibiliha ho holimo, le karabelo e lekaneng ea His-NT2RepCT maemong a asiti kaofela li hlahisoa ke NT23, 25, 34.
Mona re tlaleha popeho e potlakileng ea li-hydrogel tse bonaletsang ho tsoa ho liprotheine tsa spidroin tse kopaneng, ho kenyeletsoa NT feela, ka ho kenya tharollo ea protheine ho 37 °C.Re sebelisa thioflavin T fluorescence (ThT), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) le transmission electron microscopy (TEM), re fumane hore NT le microspider proteins li kena phetohong ea sebopeho ho ba β-sheets le fibrils tse kang amyloid. ha disele di bopeha.Ho phaella moo, liprotheine tsa fusion tsa NT le protheine e tala ea fluorescent (GFP) kapa purine nucleoside phosphorylase (PNP) li etsa li-hydrogel tse nang le likaroloana tse sebetsang ka botlalo.Polelo e phahameng ka ho fetisisa ho lihlopha tsa heterologous, hammoho le ho thehoa ka potlako ha li-hydrogel tlas'a maemo a 'mele, ho bula monyetla oa ho hlahisa li-hydrogel tse nang le mesebetsi ea boenjiniere e theko e tlaase.
Ho fapana le liprotheine tse ngata tse tlalehiloeng tsa spidroin36, His-NT2RepCT e tsitsitse ho Tris-HCl buffer ka pH 8 mme e ka tsepamisoa ho fihla ho 500 mg/mL ntle le pula25.Ka hona, re ile ra makala ha re fumana hore protheine ena e etsa ka potlako li-hydrogel tse hlakileng, tse ikemetseng ha li kenngoa ka 37 ° C (Fig. 1b-d).Liphuputso tse ling li bontšitse hore gelation ea Hae-NT2RepCT e etsahetse ho feta mefuta e mengata ea liprotheine (10-300 mg / mL) le hore mahloriso ana a ne a amana ka tsela e fapaneng le nako ea gelation (setšoantšo sa 1c le Supplementary Fig. 1).Ho fumana hore na ke likarolo life tsa His-NT2RepCT mediate hydrogel formation, re ile ra hlahloba sebaka se seng le se seng ka bonngoe le ka metsoako e fapaneng re sebelisa tlhahlobo ea inversion ea flask (Setšoantšo sa 1a,b).Likarolo tsohle tse lekiloeng tsa recombinant spidroin li thehile li-gel (ka motsoako oa protheine ea 300 mg / mL) ka nako e ka tlase ho 1 h, ntle le 2Rep (Fig. 1b).Sena se fana ka maikutlo a hore NT le CT feela, hammoho, kapa tse amanang le ho pheta-pheta, li ka gel ho 37 ° C le hore tag ea His6 ha e ame ts'ebetso ena ho isa tekanyong leha e le efe ea bohlokoa.Ha ho nahanoa ka maikutlo a tloaelehileng a hore NT ke protheine e qhibilihang haholo ebile e tsitsitse, le hore litlaleho tse fetileng tsa recombinant spidroin hydrogels li bakile litlamorao tsa gelation ho liphetoho tse amanang le libaka tse pheta-phetoang le/kapa CTs, NT ka boeona e ka khona.Ho sibolloa ha gelation ho ne ho sa lebelloa.Tlatsetso Letlapa 1) 37, 38, 39. Ka mokhoa o tsotehang, NT e se e ntse e kenngoa ka hare ho metsotso ea 10 sebakeng sa mahloriso a ≥ 300 mg / mL (setšoantšo sa 1c).Liteko tsa inversion tsa vial tse nang le likhakanyo tse fapaneng tsa NT li bonts'itse hore ho> 50 mg/mL tharollo ea NT e ile ea phalla ka potlako ho feta His-NT2RepCT ka mahloriso a tšoanang (w/v, Figure 1c).
Boemeli ba moralo oa mefuta e fapaneng ea li-spidroin tse ithutoang mosebetsing ona.b Nako ea gel ho 37 °C bakeng sa liprotheine tse fapaneng tsa spidroin (300 mg/mL) e netefalitsoe ka ho khelosa vial.CT gel hang-hang ntle le ho qhoqhoa (<300 mg/mL), 2Rep precipitates (300 mg/mL, 5 mm scale).c Nako ea Gel ea His-NT2RepCT le NT maemong a protheine a bontšitsoeng ho 37°C.d Lifoto tsa His-NT2RepCT le NT hydrogels tse nang le sekho le tlhaku "NT" e hatisitsoeng ka tlase, ka ho latellana (ka bobeli 200 mg/mL, sekala sa bar 5 mm).
Li-Hydrogel tse entsoeng ke liprotheine tse fapaneng tsa spidroin li na le mebala e fapaneng hanyenyane, 'me ho shebella mahlo a hlobotse ho bontša likarolo tse sa tšoaneng tsa pepenene (setšoantšo sa 1b).Li-gel tsa NT li hlakile ka mokhoa o ikhethang ha li-gel tse ling li fetoha opaque.Li-gel tsa hae-NT2RepCT le NT tse lahleloang ka har'a li-cylindrical tubes li ne li ka tlosoa hlobo e sa tsitsang (setšoantšo sa 1d).
Ho lekola hore na litlolo tsa silika tsa tlhaho tsa sekho li gel tlas'a maemo a teng hona joale a bakang ho ruruha ha liprotheine tsa spidroin, ho ile ha bokelloa liphahlo ho tsoa karolong e kholo ea ampulla ea sekho sa borokho sa Sweden (Larinioides sclopetarius).Liaparo li ne li bolokoa ka har'a 20 mM Tris-HCl buffer ho 50 mg / mL (ho itšetlehile ka boima bo lekantsoeng bo omileng), empa ha ho gelation e ileng ea hlokomeloa nakong ea matsatsi a 21 a incubation ho 37 ° C (Supplementary Figure 2a).
Ho lekanya li-gel tsena, litekanyo tsa rheological li ka sebelisoa ho ithuta ts'ebetso ea gelation le ho fumana thepa ea mochini ka kakaretso.Haholo-holo, ho shebella modulus ea polokelo (elasticity) ka mocheso o phahameng ho ka fana ka tlhahisoleseding mabapi le mocheso oa gelling hammoho le thepa ea viscoelastic ea ho roala.Liteko tsa ho phahama ha mocheso (ho sebelisa 1 ° C / min ho 25-45 ° C, ho itšetlehile ka lithuto tse fetileng tse sebelisang litharollo tsa lihlahisoa tsa silika tsa tlhaho) 40,41 e bontšitse hore moduli ea polokelo ea tharollo ea His-NT2RepCT le NT e eketsehile ka mocheso o ntseng o eketseha .e ile ea eketseha (setšoantšo sa 2 le sa Tlatsetso sa 3).Ka ho hlakileng, mochine oa NT o ile oa qala ho hōla ka mocheso o tlaase ha o bapisoa le His-NT2RepCT, e lumellanang le nako ea gel e potlakileng e hlokometsoeng ha NT e ne e kenngoa ka ho toba le His-NT2RepCT ho 37 ° C (Setšoantšo sa 1).Ka mor'a ho theoha ha mocheso ka mor'a moo, modulus ea polokelo ha ea ka ea khutlela ho litekanyetso tse tlaase 'me ea lula ka holim'a modulus ea tahlehelo (sheba Supplementary Fig. 3), e bontšang gelation e tsitsitseng e ke keng ea fetoloa.Ka mor'a gelation, modulus ea ho qetela ea elastic e ne e tloha ho 15 ho ea ho 330 kPa bakeng sa li-hydrogel tsa His-NT2RepCT sebakeng sa 100-500 mg / mL, 'me modulus ea ho qetela ea elastic bakeng sa NT hydrogels (100-500 mg / mL) e ne e tloha ho 2 ho ea ho 1400. kPa (setšoantšo, 2 le data e feletseng ea ramp) bona Supplementary Fig. 3).
Phetoho ea mocheso nakong ea litekanyo tsa His-NT2RepCT (300 mg/mL) le b NT (300 mg/mL) ka ho thothomela.Metsu e bonts'a mokhoa oa mocheso, 'me moriti o bobebe oa data ea mojule oa polokelo o bonts'a tlhahlobo ka boleng bo tlase ba torque bakeng sa sesebelisoa ho feta se boletsoeng ke moetsi, e leng sesosa sa lerata le ntseng le eketseha.c Ho bokella mojule oa ho qetela oa His-NT2RepCT le NT ka mor'a mocheso o phahameng (100, 300, le 500 mg/mL).Lipalo tsohle tsa mojule li nkuoa ka sekhahla sa 0.1 Hz.
Joalo ka mokhoa o ka bang teng oa ho etsa lipatlisiso ka liphetoho tsa conformational tse amanang le gelation, re rekotile ponahalo ea FTIR ea His-NT2RepCT le NT pele le ka morao ho gelation ho 37 ° C (Setšoantšo sa 3a,b).Joalokaha ho lebeletsoe, ponahalo ea litharollo tsa His-NT2RepCT le NT li ne li lumellana le liprotheine tse bontšang sebopeho sa bobeli sa α-helix / random coil, se nang le sehlopha se phatlalalitsoeng ho 1645 cm-1.Bakeng sa li-hydrogel ka bobeli, gelation e ile ea etsa hore ho thehoe matsoho a mabeli bohareng ba I band hoo e ka bang 1617 cm-1 le 1695 cm-1 (setšoantšo sa 3a, b), se bontšang ho thehoa ha li-antiparallel β-sheet structures.Liphetoho tsena li ka boela tsa bonoa ka ho hlaka karolong e fapaneng ea bobeli e nkiloeng le phapang ea gelation spectra (Supplementary Fig. 4b).Lihlopha tse peli tsa NT β-layer li ne li tsebahala ho feta tsa His-NT2RepCT, e leng se bontšang hore kakaretso ea lihlopha tsa β-layer ho NT hydrogel e ne e phahame ho feta ea NT2RepCT hydrogel.
a FTIR absorption spectra ea His-NT2RepCT le b NT (bobeli 500 mg/mL) pele (tharollo) le ka mor'a (gel) ho qhoqhoa ka 37°C.c litšoantšo tsa TEM tsa li-gel tsa 50 mg/ml tsa NT2RepCT tse emisitsoeng hape le d NT.Scale bar 200 nm.e Fiber diameters tsa His-NT2RepCT le NT hydrogels.n = 100 li-fibrils tse lekantsoeng, p <0.0001.Lits'oants'o tsa liphoso li bonts'a ho kheloha ho tloaelehileng.Bohareng ba mekoallo ea liphoso ke moelelo.Teko ea t e sa sebetseng (ea mehatla e 'meli) e ile ea sebelisoa bakeng sa tlhahlobo ea lipalo.f ThT fluorescence ea mefuta e fapaneng ea liprotheine tsa spidroin (100 mg/mL) ka 37 °C ntle le ho sisinyeha.g NT (100 mg/mL) liteko tsa ho enta ho tloha ho 100 mg/mL gel ea NT e nang le 0%, 5%, 10%, le 20% ea peo.
Tlhahlobo ea gel e sebelisang microscopy electron microscopy (TEM) e bontšitse hore hydrogel e na le li-fibrils tse kang amyloid (Figs. 3c, 3d).Li-fibrils tse entsoeng ka NT li ne li lelefalitsoe (5-12 nm bophara) 'me li se na lekala, ha li-fibrils tsa His-NT2RepCT li ne li le khutšoanyane ka bolelele le bophara bo boholo ka bophara (7-16 nm) (Setšoantšo sa 3e).Liphetho tsena li re lumelletse ho latela kinetics ea fibrosis re sebelisa tlhahlobo ea thioflavin T (ThT).Bakeng sa liprotheine tsohle tsa spidroin tse tsosolosang, lets'oao la fluorescent le ile la eketseha ha disampole li kenngoa ka 37 ° C (Fig. 3f, Supplementary Fig. 5a).Tumellanong le se fumanoeng, tlhahlobo ea microscopic ea NT le His-NT2RepCT tlas'a maemo a gelling e senotse keketseho e ts'oanang ea ThT fluorescence ntle le ho bokellana ha sebaka sa ThT-positive aggregates (Supplementary Fig. 5b, c).Ho thehoa ha li-fibrils tsa ThT-positive ha hoa ka ha tsamaea le ho eketseha ha NT le His-NTCT turbidity (Supplementary Fig. 5d), e bolelang hore marang-rang a li-fibrils ka gel a ka theha ntle le ho sekisetsa ho hlaka ha gel.Ho jala peo ka ho eketsa likhahla tse nyenyane tse entsoeng pele ho nako ho ka potlakisa haholo ho thehoa ha fibril ea amyloids42,43,44 empa ho eketsa 5%, 10% kapa 20% (w/w) NT ho tharollo ea NT hydrocoagulants.seeding phello (sa feiga. 3g).Mohlomong sena se bakoa ke taba ea hore li-fibrils tsa hydrogel li batla li tsitsitse 'me li ke ke tsa sebelisoa e le peo.
Boitšoaro bo sa lebelloang ba liprotheine tsa spidroin tse nang le lithempereichara tse phahameng li ile tsa susumetsa lithuto tse ling tsa spectroscopy tsa nuclear magnetic resonance (NMR) ho hlwaya liphetoho tse amanang le sebopeho sa lijeli.NMR spectra ea His-NT2RepCT tharollo e tlalehiloeng ka nako ho 37 ° C e bontšitse hore CT e ne e ntse e phuthoa ka mokhoa o fokolang, athe lipontšo tsa NT le 2Rep li ne li nyametse (Fig. 4a), e fana ka maikutlo a hore e ne e le haholo-holo NT le 2Rep tse neng li laoloa ka mokhoa o itseng oa His- NT2RepCT hydrogel.Letšoao la CT le lona le ile la fokotsoa ho 20% ea matla a lona a pele, a fana ka maikutlo a hore CT e boetse e tsitsitse haholo 'me e kenngoa mohahong oa hydrogel.Bakeng sa karolo e nyane ea CT, e tsamaeang joalo ka sampole e kentsoeng pele 'me ka hona e bonoa ke tharollo ea NMR, ponahalo ha e na matšoao bakeng sa masala a 10 a hlophisitsoeng a pele, mohlomong ka lebaka la ho se sebetse hantle ha karolo e kentsoeng ea His-NT2Rep.Sebopeho sa NMR sa -state of hydrogels -NT2RepCT se senoletse boteng bo boholo ba α-helices le β-layers 'me, ho isa tekanyong e fokolang, ho kopana ha li-coil ka mokhoa o sa tloaelehang (setšoantšo sa 4b).Tlhahlobo ea phetoho ea lik'hemik'hale ea masala a methionine a teng feela ho NT e bontšitse hore sebaka sena se fetotsoe ho sebopeho sa β-sheet.Tlhaloso e itšetlehileng ka nako ea NT ka tharollo e bontšitse ho fokotseha ha junifomo ka matla a pontšo (setšoantšo sa 4c), le NMR e tiileng ea NT hydrogels e bontšitse hore boholo ba masala a NT a fetotsoe ho mehaho ea β-sheet (Fig. 4d).Khokahano ea 2Rep ha e khone ho khethoa ka thoko ka lebaka la tloaelo ea ho kopanya.Leha ho le joalo, boemo bo tiileng ba NMR spectra ea NTCT le His-NT2RepCT hydrogels e ne e shebahala e tšoana haholo (Fig. 4b; Supplementary Fig. 6b), e fana ka maikutlo a hore 2Rep e ile ea kenya letsoho hanyenyane karolong ea mohaho oa His-NT2RepCT hydrogel.Bakeng sa li-hydrogel tsa CT, α-helices, β-sheets, le mehaho ea bobeli ea li-helical tse sa tloaelehang li fumanoe li le teng (Supplementary Fig. 6d).Sena se fana ka maikutlo a hore likarolo tse ling tsa CT li sala e le α-helices ha tse ling li fetoha β-sheets.Ka hona, liphetho tsa NMR spectroscopy li fana ka maikutlo a hore NT ke ea bohlokoa bakeng sa ho thehoa ha hydrogel hape e fetoha sebopeho sa β-sheet holim'a fusion le 2Rep le CT.Tumellanong le sena, re sa tsoa fumana hore li-zippers tsa sebaka sa amyloid li ka 'na tsa theha li-helices tse hlano tsa sebaka sa NT,' me algorithm ea Waltz e boletse esale pele sebaka sa amyloidogenic ho helix 1 (setšoantšo sa 4e).
2D spectra ea 15N-HSQC 10 mg/mL Tharollo ea His-NT2RepCT pele (e putsoa) le lihora tse 19 ka mor'a ho qhoqhoa (e khubelu) ho 37°C.Litlhōrō tsa batho ka bomong liphatlalatsong tse khubelu le F24, G136, polyA ka har'a spectrum e putsoa li bontšoa ke matšoao a tlhaku e le 'ngoe ea amino acid le linomoro tse setseng.Li-insets li bonts'a ho its'etleha ha matla a lets'oao ka nako bakeng sa masala a khethiloeng ho tsoa libakeng tsa NT, 2Rep, le CT.b Pontšo e matla ea seea-le-moea (RFDR) ea li-hydrogel tsa His-NT2RepCT.Likamano tsa masala a Cα / Cβ tse hlokometsoeng ho RFDR spectra li ile tsa khethoa ka ho bapisoa le liphetoho tsa lik'hemik'hale tsa peptide ea mohlala le litekanyetso tse nkiloeng ho lipalo-palo82,83 le mehaho ea bona ea bobeli.SSB - letlapa le ka lehlakoreng le potolohang.c Pontšo ea lehlakore le le leng la 15N-HSQC 10 mg/mL NT tharollo nakong ea ho qhoqhoa ka 37 °C bakeng sa lihora tse 36.Inset e bonts'a matla a volumetric khahlano le nako.d Boemo bo tiileng ba RFDR spectra ea li-hydrogel tsa NT.Likamano tsa masala a Cα / Cβ le mehaho ea bona ea bobeli e hlokometsoeng ho RFDR spectra e bontšoa.e Thehiloe holima boemo ba NT45.79 fibrillation propensity ho tsoa polokelong ea Zipper (https://services.mbi.ucla.edu/zipperdb/).Matla a Rosetta a fensetere ea sebaka sa sebaka sa lehalima la hexapeptide a bonts'oa ka kcal/mol.Libaka tse khubelu li bolela li-hexapeptide tse nang le tšekamelo e phahameng ea fibrosis (Rosetta eneji e ka tlase ho -23 kcal/mol; ka tlas'a mola o nang le matheba).Lits'oants'o tse tala li bonts'a likhechana tse nang le matla a Rosetta ka holim'a monyako, ka hona, ha ho na monyetla oa ho etsa lizipi tse hlabang.Likaroloana tse nang le proline li ne li sa kenyelletsoa tlhahlobong (ntle le litšiea).Masekwere a bontša libaka tsa amyloidosis tse boletsoeng esale pele ke Waltz algorithm81 (https://waltz.switchlab.org).Tatelano ea masala a amino acid ea NT e ka holimo, 'me mefuta ea masalla a fumanoang mohahong oa bobeli oa β (o khethiloeng ke boemo bo tiileng ba NMR spectroscopy) bo bontšoa ka bofubelu.Maemo a li-helice tse hlano tsa NT α a khethiloe e le (H1-H5)28.
Ho pH <6.5, HT e fokotseha, e hanyetsana le mocheso-kapa denaturation e bakoang ke urea18.Ho hlakisa hore na NT dimerization le botsitso li ama jelation joang, litharollo tse nang le 100 mg/ml NT li ne li laoloa ho pH 8, 7, le 6 ho sebelisoa tlhahlobo ea vial inversion.NT disampole incubated ka pH 8 le 7 gelled ka mor'a metsotso e 30 ka 37 ° C, empa pH 8 gel o ile a lula e hlakile, ha pH 7 gel e bontša precipitate bonahalang (sa feiga. 5a).Ka lehlakoreng le leng, tharollo e nang le HT ho pH 6 ha ea ka ea theha gel, 'me pula e kholo e ne e ka bonoa ka mor'a metsotso e 20 ho 37 ° C.Sena se fana ka maikutlo a hore dimers ka botsona le / kapa botsitso ba bona bo phahameng ha bo bapisoa le li-monomers li thibela gelation.Ho theoa ha pula ea NT ho pH 7 le 6 ho ne ho sa lebelloa, kaha ho tlalehiloe hore NT e qhibiliha ho 200 mg/ml27, e ipheta habonolo ka mor'a ho fifala ha mocheso, hape e boloka α-helix maemong a tlase. pH 18. Tlhaloso e ka bang teng bakeng sa liphapang tsena ke hore liteko tse tlalehiloeng pele li entsoe ka mocheso oa kamore kapa ka tlase, kapa ka liprotheine tse batlang li le tlaase16,18,19.
Teko ea NT vial inversion (100 mg/mL) ho pH 8, 7, 6 le 154 mM NaCl (pH 8) ka mor'a ho qhoqhoa ka 37°C.b NT CD spectra e nang le le ntle le 154 mM NaF le 154 mM NaCl, ka ho latellana.Molar ellipticity ho 222 nm e fetoloa ho karolo ea mameno a tlhaho.c NT inversion assay (100 mg/mL) NT* (37 °C le 60 °C), NTA72R (37 °C), le His-NT-L6 (37 °C le 60 °C).d CD spectra ea NT mutants NT*, NTA72R, le His-NT-L6.Molar ellipticity ho 222 nm e fetoloa ho karolo ea mameno a tlhaho.e Teko ea ho fetola NTFlSp, NTMiSp le ho fokotsa NTMiSp (100 mg/mL).Sekhahla sa 5 mm.f CD spectra ea NT, NTFlSp, NTMiSp le NTMiSp e fokolitsoeng.Molar ellipticity ho 222 nm e fetoloa ho karolo ea mameno a tlhaho.Ponahalo e felletseng ea NT ho 25 °C le 95 °C e bonts'oa ho Setšoantšo sa Tlatsetso 8.
Letsoai la fisioloji le etsa qeto ea ho sebelisana ha electrostatic pakeng tsa li-subunits tsa NT le dimerization ea phetisetso ea NT ho ea tlase pH18.Re fumane hore ho ba teng ha 154 mM NaCl le NaF ho hlile ho thibela gelation, ka ho latellana (setšoantšo sa 5a, b; Fig. 2b ea Tlatsetso) le hore letsoai lena le ile la eketsa botsitso ba mocheso oa li-monomers tsa NT (setšoantšo sa 5b, Supplementary Fig. 8) .E boetse e fana ka maikutlo a hore ntlafatso ea botsitso, ho e-na le dimerization, e thibela ho thehoa ha li-gel.
Ho tsoela pele ho hlahloba karolo ea protheine dimerization le botsitso ho gelation, re sebelisitse liphetoho tse peli, NT * le NTA72R, tse lulang li le monomeric ka pH28.30 e tlaase.NT* ke phetoho e feto-fetohang ea litefello tse peli eo ho eona ho bonahalang eka kabo ea tefiso ea dipolar ea monoma e batalatsoa, ​​​​e thibelang dimerization le ho eketsa botsitso ba monoma haholo.NTA72R ke dipole tse lefelloang, empa Arg-substituted Ala e moeling oa dimer, ka hona liphetoho li kena-kenana le tšebelisano ea subunit e hlokahalang bakeng sa dimerization.Ka mor'a ho kenngoa ha 37 ° C, NT * ha ea ka ea etsa hydrogel, ha NTA72R e theha gel e opaque bakeng sa 15 min (setšoantšo sa 5c).Kaha ka bobeli NT * le NTA72R ha li khone ho fokotseha empa li fapane ka botsitso ba monomer (setšoantšo sa 5d), liphetho tsena li fana ka maikutlo a matla a hore botsitso bo phahameng ba thermodynamic bo thibela NT ho tloha gelling.Sena se boetse se tšehetsoa ke taba ea hore HT * e etsa gel ha e sa tsitsa ka mocheso o phahameng (ka mor'a metsotso ea 8 ho 60 ° C; Setšoantšo sa 5c).Ho 'nile ha bontšoa hore litaba tse phahameng tsa methionine ho NT li qhibilihisa ho phuthoa ha eona ka tlhaho le hore li-substitutes tse tšeletseng tsa Met to Leu (tseo ho buuoang ka tsona mona e le His-NT-L6) li tsitsisa monoma oa NT46 ka matla.Ho itšetlehile ka maikutlo a hore ho feto-fetoha ha sebopeho hoa hlokahala bakeng sa ho etsoa ha li-gel tsa NT, re fumane hore phetoho e tsitsitseng ea His-NT-L6 ha ea ka ea e-ba 37 ° C (Setšoantšo sa 5c, d).Leha ho le joalo, His-NT-L6 e boetse e thehile gel ha e kenngoa ka 60 ° С bakeng sa 60 min (setšoantšo sa 5c).
Bokhoni ba NT ba ho fetoha meaho ea β-sheet le ho etsa li-hydrogel bo bonahala bo sebetsa ho tse ling empa eseng kaofela ha libaka tsa NT tsa spidroin.NTs ho tsoa mefuteng e fapaneng ea silika le mefuta ea likho, Trichonephila clavipes (NTFlSp), e ile ea theha li-gel ho sa tsotellehe hore na li na le methionine e fokolang le botsitso bo phahameng ba mocheso (Setšoantšo sa 5e, f le Tlatsetso Letlapa la 2).Ka lehlakoreng le leng, NT e tsoang ho protheine e nyenyane ea ampullar spidroin e tsoang ho Araneus ventricosus (NTMiSp) e nang le botsitso bo tlaase ba mocheso le methionine e phahameng ha ea ka ea etsa li-hydrogel (Supplementary Table 2 le Fig. 5e, f).E ka 'na ea amahanngoa le ho ba teng ha intramolecular disulfide bonds29,47.Ka mokhoa o ts'oanang, ha litlamo tsa disulfide tsa NTMiSp li fokotsehile, li ile tsa theha hydrogel ka mor'a ho kenngoa ka 37 ° C bakeng sa 10 min (setšoantšo sa 5e).Qetellong, ho lokela ho hlokomeloa hore ho feto-fetoha ha sebopeho ke ntho ea bohlokoa, empa eseng eona feela, mokhoa oa ho thehoa ha gel ho tloha NT.Ntho e 'ngoe e ka' nang ea e-ba ea bohlokoa ke tšekamelo ea ho theha li-amyloid fibrils, 'me tlhahlobo e nang le database ea zipper le algorithm ea Waltz e bontšitse kamano pakeng tsa bokhoni ba ho etsa li-gel le boteng ba libaka tsa amyloidogenic, hammoho le boholo ba libaka tse boletsoeng esale pele. ho etsa li-zippers tsa steric.Ho ne ho e-na le kamano (Tlaletso ea Tlatsetso 2 le Tlatsetso ea setšoantšo sa 9).
Bokhoni ba NT ba ho etsa li-fibrils le ho etsa li-gel tlas'a maemo a matle bo ile ba etsa hore re nahane hore ho kopana ha NT le likaroloana tse ling tsa protheine ho ntse ho ka etsa li-gel tse nang le ts'ebetso e feletseng ea balekane ba fusion.E le ho hlahloba sena, re hlahisitse protheine e tala ea fluorescent (GFP) le purine nucleoside phosphorylase (PNP) ho C-terminus ea NT, ka ho latellana.Liprotheine tsa fusion tse hlahisitsoeng li hlahisitsoe ho E. coli ka lihlahisoa tse phahameng haholo tsa ho qetela (150 mg/L le 256 mg/L shake flask cultures bakeng sa His-NT-GFP le His-NT-PNP, ka ho latellana), ho lumellana le se bontšitsoeng. bakeng sa liprotheine tse ling tse kopantsoeng le NT Ref.30. His-NT-GFP (300mg / mL) le His-NT-PNP (100mg / mL) liprotheine tsa fusion li ile tsa theha li-gel ka mor'a lihora tsa 2 le lihora tsa 6.5 ho 37 ° C 'me, habohlokoa, karoloana ea GFP e ile ea lula e sa fetohe.e hlokometsoe ka mor'a gelation, e nang le > 70% ea matla a pele a fluorescence a setseng ka mor'a gelation (setšoantšo sa 6a).Ho lekanya ts'ebetso ea PNP ho litharollo le li-gel tsa hae-NT-PNP, re ile ra tlameha ho hlapolla protheine ea fusion le NT hobane ts'ebetso ea enzymatic ea tokiso e hloekileng e ne e le kantle ho sebaka sa tlhahlobo ea tlhahlobo ea likhakanyo tsa gelling.Gel e entsoeng ka motsoako o nang le 0.01 mg / mL His-NT-PNP le 100 mg / mL NT e bolokile 65% ea ts'ebetso ea pele ea enzymatic ea li-sampuli tse entsoeng pele ho nako (setšoantšo sa 6b).Gel e ile ea lula e tiile nakong ea tekanyo (Fig. 10 ea Tlatsetso).
A Relative fluorescence intensity pele le ka morao ho gelation ea His-NT-GFP (300 mg/mL) le vial inverted e nang le His-NT-GFP hydrogel (300 mg/mL) tlas'a khanya e bonahalang le UV.Lintlha li bontša litekanyo tsa motho ka mong (n = 3), mekoallo ea liphoso e bonts'a ho kheloha ho tloaelehileng.Theko e tloaelehileng e bonts'oa bohareng ba li-barbar tsa liphoso.b PNP e ile ea fumanoa ka tlhahlobo ea fluorometric ho sebelisa tharollo le li-gel tse nang le NT (100 mg / ml) le motsoako o nang le 0.01 mg / ml ea hae-NT-PNP le 100 mg / ml Lidolara tse ncha tsa Taiwan.Sets'oants'o se bonts'a vial e sothehileng e nang le hydrogel e nang le His-NT-PNP (5 mm scale bar).
Mona, re tlaleha ho thehoa ha li-hydrogel tse tsoang NT le liprotheine tse ling tsa spidroin ka ho kenya motsoako oa protheine ho 37 ° C (Setšoantšo sa 1).Re bontša hore gelation e amahanngoa le phetoho ea α-helices ho β-layers le ho thehoa ha li-fibrils tse kang amyloid (Fig. 3 le 4).Se fumaneng sena sea makatsa kaha NTs ke li-globular tse kopantsoeng li-helix tse hlano tse tsejoang ka ho qhibiliha ha tsona ho phahameng ka ho fetisisa le botsitso bo phahameng ka ho fetisisa > 200 mg / mL ho 4 ° C ka matsatsi a 'maloa27.Ho feta moo, NTs e ipheta habonolo ka mor'a hore mocheso o fokotsehe maemong a tlaase a protheine ho µM.Ho ea ka liphello tsa rona, ho thehoa ha fibril ho hloka motsoako oa > 10 mg / mL ea protheine ea mahloriso le mocheso o phahameng hanyenyane (setšoantšo sa 1).Sena se lumellana le maikutlo a hore li-amyloid fibrils li ka theha ho tloha ho liprotheine tse nang le globularly folded tse boemong bo sa phutholohang ka lebaka la ho fetoha ha mocheso tlas'a maemo a physiological 48.Mehlala ea liprotheine tse kenang phetohong ena li kenyelletsa insulin49,50, β2-microglobulin, transthyretin le lysozyme51,52,53.Le hoja NT e le α-helix sebakeng sa eona sa tlhaho, hoo e ka bang 65% ​​ea ketane ea polypeptide e lumellana le sebopeho sa zipper ea steric (Setšoantšo sa 4e) 45.Kaha monoma e na le dynamically mobile46, e ka pepesa libaka tsena tse ka bang teng tsa amyloidogenic ka mocheso o itekanetseng o phahameng 'me maemong a phahameng a protheine e feletseng e ka finyella mahloriso a bohlokoa bakeng sa amyloid fibril formation54.Ka mor'a mabaka ana, re fumane kamano e mpe pakeng tsa mahloriso a spidroin le nako ea gelation (Setšoantšo sa 1c), 'me haeba conformation ea monomeric NT e tsitsisoa ka liphetoho (NT *, His-NT-L6) kapa ka ho eketsa letsoai, e ka thibela sebopeho sa hydrogels (setšoantšo sa 5).
Maemong a mangata, li-fibrils tsa amyloid li nyamela ho tloha tharollo e le precipitate, empa tlas'a maemo a itseng li ka etsa hydrogels55,56,57.Hydrogel-forming fibrils hangata e na le tekanyo e phahameng ea likarolo 'me e theha marang-rang a tsitsitseng a mahlakore a mararo ka ho kenngoa ha molek'hule,55,58 e lumellanang le liphetho tsa rona.Bakeng sa tlhahiso ea hydrogel in vitro, liprotheine li atisa ho senoloa ka botlalo kapa ka mokhoa o fokolang, ka mohlala, ka ho pepesehela lihlahisoa tsa tlhaho, mocheso o phahameng (70-90 ° C) le / kapa pH e tlaase (1.5-3.0) 59,60,61,62.Li-spidroin hydrogel tse hlalositsoeng mona ha li hloke ts'ebetso e matla, ebile ha li hloke li-agent tse hokahanyang ho tsitsisa li-hydrogel.
Ho kile ha tlalehoa hore spidroin e pheta-pheta le li-QD, tse bonahalang li fetoha β-sheet switching nakong ea ho ohla silika, li etsa li-hydrogel.Ha ho bapisoa le seo re se fumaneng, linako tsa ho qhoqhoa le/kapa mocheso oa ho qhoqhoa li ne li le telele haholo kapa li phahame, ka ho latellana, 'me li-hydrogel tse hlahisoang hangata li ne li le opaque (Figure 7 le Supplementary Table 1) 37, 38, 63, 64, 65, 66, 67, 68 , 69. Ho phaella linakong tsa li-gel tse potlakileng, li-hydrogel tsa NT>300 mg/mL (30%) li ile tsa feta tse ling tsohle tse hlalositsoeng tse amanang le liprotheine tsa silika tsa sekho, hammoho le li-hydrogel tsa tlhaho tse kang gelatin, alginate (2%), agar (0.5 %). ) le collagen.(0.6%) (Setšoantšo sa 7 le Litafole tsa Tlatsetso 1 le 3) 37,39,66,67,68,69,70,71,72,73,74.
Nako ea gel le modulus elastic ea hydrogels thutong ena li ne li bapisoa le li-hydrogel tse ling tse thehiloeng ho spidroin le li-hydrogel tse khethiloeng tsa tlhaho.Litšupiso li fanoa hammoho le tlhaloso ea maemo a gelation.APS Ammonium sulfate, mocheso oa kamore.Lintlha 37, 38, 39, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74.
Ho bonahala likho li na le mekhoa ea ho thibela spidroin hore e se ke ea tsoa nakong ea polokelo.Ho sa tsotellehe boemo bo phahameng ba protheine ka har'a tšoelesa ea silika, sebaka se seholo sa ho pheta-pheta se amanang le sebaka sa ho qetela se bolela hore motsoako o bonahalang oa NT le CT ka tšoelesa o lumellana le hoo e ka bang 10-20 mg / ml, moeling oa thuto ena.e hlokahalang bakeng sa in vitro e hlokometseng sebopeho sa hydrogel.Ho phaella moo, mahloriso a tšoanang a letsoai 16 a tsitsitse NT, joalo ka litšoelesa tsa silika (setšoantšo sa 5b).NT conformation e ithutoe ho E. coli cytosol mme e fumanoe e mennoe ka thata ho feta ha e hlahlojoa in vitro, ho bontša hape hore letsoai kapa lintlha tse ling li thibela ho kopana ha eona ka har'a vivo.Leha ho le joalo, bokhoni ba NTs ho fetoha β-sheet fibrils bo ka ba bohlokoa bakeng sa ho thehoa ha likhoele 'me bo lokela ho batlisisoa lithutong tse tlang.
Ntle le likarolo tse ncha tsa NT-amyloid-like fibril le hydrogel sebopeho se hlokometsoeng thutong ena, re boetse re bontša hore ketsahalo ena e ka 'na ea e-ba le lisebelisoa tsa biotechnological le biomedical (setšoan. 8).E le bopaki ba khopolo, re ile ra kopanya NT le GFP kapa PNP 'me ra bontša hore protheine ea fusion e boetse e etsa li-hydrogel ha li kenngoa ka 37 ° C le hore likaroloana tsa GFP le PNP li boloka haholo mosebetsi oa tsona ka mor'a gelation (Setšoantšo sa 6).Nucleoside phosphorylases ke li-catalysts tsa bohlokoa tsa motsoako oa nucleoside analogues75, e leng se etsang hore ho sibolloa ha rona ho sebetse indastering ea biopharmaceutical.Mohopolo oa ho hlahisa liprotheine tsa fusion tse etsang li-hydrogel tse pepeneneng tlas'a maemo a matle li lumella ho theoa ha li-hydrogel tse sebetsang tse nang le thepa e ntle bakeng sa lits'ebetso tse fapaneng tse kang immobilization ea enzyme, tokollo ea lithethefatsi e laoloang le boenjiniere ba lisele.Ho feta moo, NT le NT* ke matshwao a sebetsang hantle a polelo30, ho bolelang hore NT le mefuta e fapaneng ya yona e ka sebediswa bakeng sa tlhahiso e phahameng ya diprotheine tsa fusion e qhibidihang le tlhahiso e latelang ya diprotheine tse hlahiswang tse sa sebetseng ho 3D hydrogel.
NT e qhibiliha, α-helical ebile e tsitsitse maemong a tlase (µM) le 37°C.Ka mocheso o ts'oanang, empa ha ho ntse ho eketseha (> 10 mg / ml), NT e etsa li-gel tse nang le li-fibrils tse kang amyloid.Liprotheine tsa fusion tsa NT li boetse li theha li-gel tsa fibrillar tse nang le likaroloana tse sebetsang ka botlalo, tse lumellang liprotheine tse fapaneng hore li se ke tsa tsamaea ka har'a li-hydrogel tsa 3D tse sebelisang NT.Ka tlase: NT (PDB: 4FBS) le lipapiso tsa marang-rang a fiber le libopeho tsa protheine tse amanang le tsona (tse nkoang ebile li sa huleloe ho sekaleng, GFP PDB: 2B3Q, 10.2210/pdb2B3Q/pdb; PNP PDB: 4RJ2, 10.2210/pdb4RJ2).
The constructs (bona Supplementary Table 4 bakeng sa lenane le feletseng ho kenyeletsa le tatellano ea amino acid) e ile ea etsoa ka plasmid pT7 'me ea fetoloa E. coli BL21 (DE3).E. coli e nang le li-plasmids tse entsoeng ka boenjiniere li ile tsa kenngoa ka har'a moro oa Luria o tlatsitsoeng ka kanamycin (70 mg / l) 'me oa hōla bosiu bo le bong ka 30 ° C le 250 rpm.Ka nako eo setso se ile sa kenngoa 1 / 100 ho LB medium e nang le kanamycin 'me ea lengoa ka 30 ° C le 110 rpm ho fihlela OD600 e fihla ho 0.8.Bakeng sa lithuto tsa NMR, libaktheria li ne li lengoa ka M9 e nyenyane e nang le 2 g ea D-glucose 13C (Aldrich) le 1 g ea ammonium chloride 15N (Cambridge Isotope Laboratories, Inc.) bakeng sa ho ngola liprotheine tse nang le isotopes.Fokotsa mocheso ho likhato tse 20 tsa Celsius 'me u khothalletse polelo ea protheine ka 0.15 mM isopropylthiogalactopyranoside (concentration ea ho qetela).Ka mor'a polelo ea protheine ea bosiu bo le bong, lisele li ile tsa kotuloa ho 7278 × g, 4 ° C bakeng sa 20 min.Li-cell pellets li ile tsa emisoa hape ka 20 mM Tris-HCl, pH 8, 'me tsa hoamisoa ho fihlela li sebelisoa hape.Lisele tse qhibilihisitsoeng li ne li sebelisoa ho sebelisa mochine o senyang lisele (mechini ea TS series, Constant Systems Limited, Engelane) ka 30 kPa.Ebe li-lysates li ne li le centrifuged ho 25,000 g bakeng sa metsotso e 30 ho 4 ° C.Bakeng sa NTMiSp, pellet e ile ea tsosolosoa ho 2 M urea, 20 mM Tris-HCl, pH 8, 'me ea sonicated bakeng sa 2 min (2 s on / off, 65%), ebe e kenngoa centrifuged hape ho 25,000 xg, 4 ° C. ka hare ho eona. 30 mets.Supernatant e ile ea laeloa holim'a kholomo ea Ni-NTA, ea hlatsuoa ka 20 mM Tris-HCl, 2 mM imidazole, pH 8, 'me qetellong protheine e ile ea hlakoloa ka 20 mM Tris-HCl, 200 mM imidazole, pH 8. Ho hlahisa NT2RepCT le NTCT, tšilo ea thrombin e hlahisa sebaka (ThrCleav) lipakeng tsa Hae le NT.Liwebsaete tsa Thrombin cleavage le tsona li teng ho His-NT-ThrCleav-2Rep (e hlahisa 2Rep), His-thioredoxin-ThrCleav-NT (e hlahisa NT), His-thioredoxin-ThrCleav-CT (e hlahisa CT), His-Thioredoxin-ThrCleav-NT .* (e hlahisa NT*), His-Thioredoxin-ThrCleav-NTA72R (e hlahisa NTA72R), His-Thioredoxin-ThrCleav-NTFlSp (e hlahisa NTF1Sp), le His-Sulphur Redoxin-ThrCleav-NTMiSp (e hlahisa NTMiSp).Lihahi li ne li chekoa ka thrombin (1: 1000) 'me tsa dialyzed ka bosiu bo le bong ho 4 ° C. le 20 mM Tris-HCl, pH 8, ho sebelisa lesela la Spectra / Por dialysis le molek'hule oa boima ba 6-8 kDa.Ka mor'a dialysis, tharollo e laeloa kholomong ea Ni-NTA 'me litšila tse nang le protheine ea thahasello lia bokelloa.Maemo a protheine a ile a khethoa ka ho lekanya monyetla oa UV ho 280 nm ho sebelisoa coefficient ea ho timela ea protheine ka 'ngoe, ntle le NTF1Sp, e sebelisitseng tlhahlobo ea Bradford ho latela protocol ea moetsi.Bohloeki bo ile ba khethoa ke SDS polyacrylamide (4–20%) gel electrophoresis le Coomassie brilliant blue staining.Liprotheine li ne li tsepamisitsoe ho sebelisoa li-filters tsa centrifuge (VivaSpin 20, GE Healthcare) ho 4000 xg e nang le 10 kDa molecular weight cutoff ka metsotsoana ea 20.
Qhibiliha motsoako oa protheine 'me ka hloko o pete 150 µl ka har'a vial ea 1 ml e hlakileng ea septum (8 x 40 mm Thermo Scientific).Li-tubes li ne li koaheloa 'me li tiisitsoe ka parafilm ho thibela ho fetoha mouoane.Mehlala (n = 3) e ne e kenngoa ka 37 ° C kapa 60 ° C 'me nako le nako e ne e fetoloa ho shebella gelation.Lisampole tse sa kang tsa hloekisoa ka gel li ne li eketsoa bonyane beke e le 'ngoe.Fokotsa libonto tsa disulfide tsa NTMiSp ka 10 mM DTT ka protheine ea 10 µM.Ho hlahlobisisa ho hlajoa ha liphahlo tsa silika tsa sekho, ho ile ha khaoloa sekho sa borokho sa Sweden, litšoelesa tse peli tse kholo tse khaotsoeng li ile tsa beoa ka har'a 200 μl ea 20 mM Tris-HCl buffer pH 8 le ho seha ho lumella ho roala ho arohana le litšoelesa..Likahare tsa litšoelesa li qhibiliha ka har'a buffer, 50 µl bakeng sa qeto ea boima bo omileng (ka ho kenya linkho tse bulehileng ka 60 °C ho ea boima bo sa feleng) le 150 μl bakeng sa gelation ho 37 °C.
Tekanyo ea geometry / sesebelisoa e entsoe ka tšepe e sa hloekang e sebelisa poleiti e bapileng e nang le bophara ba 20 mm le lekhalo la 0.5 mm.Chesa sampole ho tloha ho 25 °C ho ea ho 45 °C 'me u khutlele ho 25 °C ka sekhahla sa 1 °C ka motsotso u sebelisa poleiti ea Peltier ea tšepe e katlase.Litekanyo tsa ho sisinyeha li ne li etsoa ka makhetlo a 0.1 Hz le sebakeng se nang le li-viscoelastic tsa lisebelisoa ka khatello ea 5% le 0.5% bakeng sa disampole tsa 100 mg / mL le 300-500 mg / mL, ka ho latellana.Sebelisa kamore ea mongobo e tloaelehileng ho thibela mouoane.Lintlha li ile tsa hlahlojoa ho sebelisoa Prism 9.
Bakeng sa ho bokella spectra infrared (IR) mocheso oa kamore ho tloha ho 800 ho isa ho 3900 cm-1.Sesebelisoa sa ATR, hammoho le tsela e khanyang ka spectrometer, e hloekisoa ka moea o omileng o sefiloeng pele le nakong ea teko.Litharollo (500 mg / mL ho fokotsa litlhōrō tsa ho monya metsi ka pono) li ile tsa kenngoa ka liphaephe holim'a likristale, 'me li-gel (500 mg / mL) li ile tsa thehoa pele ho tekanyo ebe li fetisetsoa ho likristale (n = 3).Li-scans tsa 1000 li ngotsoe ka qeto ea 2 cm-1 le potoloho ea mosebetsi oa zero ea 2. Motsoako oa bobeli o ne o baloa ho sebelisoa OPUS (Bruker) ho sebelisa mefuta e bonolo ea lintlha tse robong.Setšoantšo se ne se tloaelehile sebakeng se le seng sa ho kopanya pakeng tsa 1720 le 1580 cm-1 ho sebelisa F. Menges "Spectragryph - Optical Spectroscopy Software".Ho spectroscopy ea ATR-IR, botebo ba ho kenella ha lebone la infrared ho sampole bo ipapisitse le palo ea maqhubu, e bakang ho monya ho matla ho linomoro tse tlase ho feta linomoro tse phahameng tsa maqhubu.Liphello tsena ha lia lokisoa bakeng sa ponahalo e bontšitsoeng ho Feiga.3 hobane li nyane haholo (Supplementary Fig. 4).Pono e nepahetseng ea palo ena e baloa ho sebelisoa software ea Bruker OPUS.
Ha e le hantle, palo e pharaletseng ea li-protein conformations e ka khoneha ka mor'a hore ho be le phetoho e ka tšeptjoang ea likarolo tse ka hare ho tlhōrō ea amide I.Leha ho le joalo, litšitiso tse ling lia hlaha ha li sebetsa.Lerata ka har'a spectrum le ka hlaha e le litlhōrō tsa (mashano) nakong ea deconvolution.Ho feta moo, tlhōrō ka lebaka la ho kobeha ha metsi e lumellana le boemo ba tlhōrō ea amide I mme e ka 'na ea e-ba le boholo bo tšoanang bakeng sa lisampole tse nang le metsi a mangata, joalo ka gel e nang le metsi e ithutoang mona.Ka hona, ha rea ​​ka ra leka ho senya ka ho feletseng tlhōrō ea amide I, 'me maikutlo a rona a lokela ho nkoa feela ho tšehetsa mekhoa e meng e kang NMR spectroscopy.
Litharollo tsa 50 mg/ml NT le His-NT2RepCT li ile tsa kenngoa bosiu bo le bong ka 37°C.Joale hydrogel e ile ea hlapolloa ka 20 mM Tris-HCl (pH 8) ho ea ho motsoako oa 12.5 mg/ml, ea sisinyeha hantle le ho tšeloa ka liphaephe ho senya gel.Ka mor'a moo, hydrogel e ile ea hlapolloa ka makhetlo a 10 ka 20 mM Tris-HCl (pH 8), 5 μl ea sampuli e ile ea sebelisoa ho grid ea koporo e koahetsoeng ka formvar, 'me sampuli e feteletseng e ile ea tlosoa ka pampiri e senyang.Mehlala e ile ea hlatsuoa habeli ka 5 µl ea metsi a MilliQ mme e silafalitsoe ka 1% ea formate ea uranyl bakeng sa metsotso e 5.Tlosa letheba le feteletseng ka pampiri e monyang, ebe moea o omisa letlooeng.Ho etsa litšoantšo ho li-grids tsena ho sebelisoa FEI Tecnai 12 Spirit BioTWIN e sebetsang ka 100 kV.Litšoantšo li ngotsoe ho x 26,500 le x 43,000 magnifications ho sebelisa Veleta 2k × 2k CCD khamera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).Bakeng sa sampole ka 'ngoe (n = 1), litšoantšo tsa 10-15 li ngotsoe.ImageJ (https://imagej.nih.gov/) e ne e sebelisetsoa ho hlahloba litšoantšo le ho lekanya li-diameter tsa fiber (n = 100, likhoele tse fapaneng).Prism 9 e ne e sebelisetsoa ho etsa liteko tsa t tse sa sebetseng (tse nang le mehatla e 'meli).Li-fibrils tsa His-NT2RepCT le NT li ne li le 11.43 (SD 2.035) le 7.67 (SD 1.389) nm, ka ho latellana.Nako ea kholiseho (95%) ke -4.246 ho -3.275.likhato tsa tokoloho = 198, p <0.0001.
80 µl ea disampole tsa mokelikeli tse nang le 10 µM thioflavin T (ThT) li lekantsoe ka makhetlo a mararo (n = 3) tlas'a maemo a tsitsitseng ho sebelisoa lipoleiti tse tlase tse hlakileng tsa Corning 96-botšo bo botšo (Corning Glass 3881, USA).Phapano ya Fluorescence e ile ya rekotwa ho sebediswa sefene se hlasimollang sa 440 nm le sefene sa 480 nm emission (FLUOStar Galaxy ho tswa ho BMG Labtech, Offenburg, Germany).Letšoao la ThT ha lea ka la tlala kapa la tima, kaha liteko tse nang le likarolo tse fapaneng tsa ThT li entsoe ntle le ho fetola matla a lets'oao.Rekota ho monya ho 360 nm bakeng sa ho metha haze.Bakeng sa liteko tsa peo, li-gel tsa 100 mg/mL li entsoe ka 37 ° C., tsa emisoa hape, 'me tsa sebelisoa bakeng sa ho jala peo ka tekanyo ea molar ea 5%, 10%, le 20%.Lintlha li ile tsa hlahlojoa ho sebelisoa Prism 9.
Thaw stocks of His-NT2RepCT le NT >100 mg/mL holim'a leqhoa ebe u sefa ka sefe ea 0.22 µm.Likhakanyo li ne li baloa ka ho lekanya ho monya ho 280 nm ho sebelisa Nanodrop.Ka lilibeng tsa poleiti e 96 e ntšo e sa tlamang (Corning) e nang le botlaaseng bo hlakileng, lisampole li ile tsa hlapolloa ho 20 mg/ml ho 20 mM Tris-HCl pH 8 mme li kopantsoe le 5 μM ThT (concentration ea ho qetela), kakaretso ea sampuli ea mahloriso. 50 μl boima.Mehlala e ne e nkuoa metsotso e meng le e meng ea 10 ho 37 ° C ka microscope ea CellObserver (Zeiss) e nang le kanale ea leseli le fetisoang ea FITC le li-filters tsa emission bakeng sa litšoantšo tsa ThT.Ho sebelisoa lense ea 20x/0.4 bakeng sa ho nka litšoantšo.Zen Blue (Zeiss) le ImageJ (https://imagej.nih.gov/) li ile tsa sebelisoa bakeng sa tlhahlobo ea litšoantšo.Li-gel li ne li boetse li lokiselitsoe ho tloha NT le His-NT2RepCT tharollo ka har'a motsoako oa 50 mg / mL e nang le 20 mM Tris pH 8 le 5 µM ThT 'me e kenngoa ka 37 ° C bakeng sa 90 min.Likotoana tsa gel li ile tsa fetisetsoa selibeng se secha se nang le 20 mM Tris, pH 8, le 5 μM ThT ka poleiti e ntšo e sa tlameng e 96 e hlakileng hantle.Fumana fluorescence e tala le litšoantšo tsa lebala tse khanyang ka 20x/0.4 magnification.ImageJ e ne e sebelisetsoa tlhahlobo ea litšoantšo.
Tharollo NMR spectra e ile ea fumanoa 310 K ka 600 MHz Bruker Avance Neo spectrometer e nang le QCI Quadrupole Resonance Pulsed Gradient Field Cryoprobe (HFCN).Mehlala ea NMR e nang le 10 mg/mL protheine ea homogeneous e ngotsoeng 13C, 15N, e qhibilihisitsoeng ka 20 mM Tris-HCl (pH 8), 0.02% (w/v) NaN3, 5% DO (v/v), (n = 1) .Liphetoho tsa lik'hemik'hale tsa NT2RepCT ho pH 6.7 li ile tsa sebelisoa ho fana ka peak 23 ho 2D spectrum ea 15N-HSQC.
Magic angle spinning solid NMR (MAS) spectra ea 13C, 15N-lebeled hydrogels e rekotiloe ho Bruker Avance III HD spectrometer ka 800 MHz e nang le 3.2 mm 13C/15N{1H} probe e se nang mohala.Mocheso oa sampuli o ne o laoloa ho sebelisoa mocheso o fapaneng oa mocheso oa khase ho 277 K. Li-dimensional dipole rotational resonance (DARR) 76 le radio frequency reconnection (RFDR) 77 spectra li ile tsa fumanoa ka maqhubu a MAS a 12.5 kHz le 20 kHz, ka ho latellana.Cross polarization (CP) ho tloha 1H ho ea ho 13C e ne e etsoa ka ho sebelisa marang-rang a marang-rang ho tloha 60.0 ho ea ho 48.0 kHz ho 1H, 61.3 / 71.6 kHz ho 13C (ho 12.5 / 20 kHz MAS) le nako ea ho kopana 0.5-1 ms.Spinal6478 decoupling ho 73.5 kHz e sebelisitsoe nakong ea pokello ea data.Nako ea ho reka e ne e le 10 milliseconds 'me tieho e ne e le metsotsoana e 2.5.Likamano tse le 'ngoe tsa Cα / Cβ tse hlokometsoeng ho RFDR spectra li ne li abeloa ho ipapisitsoe le mekhoa ea ho fetoha ha lik'hemik'hale tsa mofuta oa masala le likamano tse ngata-ngata tse amanang le ponahalo ea DARR.
Setsi sa polokelo ea Zipper79 (https://services.mbi.ucla.edu/zipperdb/) se ile sa sebelisoa ho hlahloba mekhoa ea flutter le matla a Rosetta bakeng sa NT, NTFlSp, le NTMiSp.Setsi sa polokelo ea Zipper se kopanya Rosetta Energy80, e kopanyang mesebetsi e mengata ea mahala ea matla ho etsa mohlala le ho sekaseka sebopeho sa protheine.Matla a matla a -23 kcal / mol kapa a tlase a bontša tšekamelo e phahameng ea fibrillate.Matla a tlase a bolela botsitso bo eketsehileng ba likhoele tse peli tsa β ho conformation ea zipper.Ho feta moo, algorithm ea Waltz e ne e sebelisetsoa ho bolela esale pele libaka tsa amyloidogenic ho NT, NTFlSp le NTMiSp Ref.81. (https://waltz.switchlab.org/).
Tharollo ea protheine ea NT e kopantsoe le 2-(N-morpholino) ethanesulfonic acid (MES) buffer ho pH 5.5 le 6.0 ho theola pH ho pH 6 le 7, ka ho latellana.Motsoako oa ho qetela oa protheine e ne e le 100 mg / ml.
Litekanyo li entsoe ka J-1500 CD spectrometer (JASCO, USA) ho sebelisa 300 μL cuvette e nang le tsela ea optical ea 0.1 cm.Liprotheine li ile tsa hlapolloa ho 10 μM (n = 1) ho 20 mM phosphate buffer (pH 8).Ho hlahloba botsitso ba liprotheine boteng ba letsoai, liprotheine li ile tsa hlahlojoa ka mokhoa o ts'oanang (n = 1) ho 20 mM phosphate buffer (pH 8) e nang le 154 mM NaF kapa NaCl, ka ho latellana.Lisebelisoa tsa mocheso li ngotsoe ho 222 nm ho tloha 25 ° C ho ea ho 95 ° C ka tekanyo ea mocheso oa 1 ° C / min.Palo ea liprotheine tse phuthetsoeng ka tlhaho e ile ea baloa ho sebelisoa foromo (KDmeasure – KDfinal)/(KDstart – KDfinal).Ho feta moo, ho ile ha tlalehoa li-spectra tse hlano bakeng sa sampole ka 'ngoe ho tloha ho 260 nm ho ea ho 190 nm ho 25 ° C le ka mor'a ho futhumatsa ho 95 ° C.Lipontšo tse hlano li ile tsa lekanngoa, tsa theoleloa 'me tsa fetoloa ho molar ellipticity.Lintlha li ile tsa hlahlojoa ho sebelisoa Prism 9.
Matla a fluorescence a His-NT-GFP (300 mg/mL, 80 µL) a lekantsoe ka makhetlo a mararo (n = 3) ka lipoleiti tsa Corning tse 96-well tse tlase botšo bo bonaletsang (Corning Glass 3881, USA) tlas'a maemo a tsitsitseng.Lekanya disampole ka sebali sa poleiti se thehiloeng ho fluorescence se nang le maqhubu a hlasimollang a 395 nm 'me u tlalehe tlhahiso ho 509 nm pele ho gelation le lihora tse 2 hamorao ho 37°C.Lintlha li ile tsa hlahlojoa ka Prism 9.
Purine nucleoside phosphorylase assay kit (mokhoa oa fluorometric, Sigma Aldrich) o sebelisitsoe ho latela litaelo tsa moetsi.Ho lekanya ts'ebetso ho li-gel le litharollo tse nang le His-NT-PNP, kopanya 10 ng ea His-NT-PNP le 100 mg / mL NT ho kakaretso ea 2 µL hobane gel e fane ka letšoao ka holimo ho nako ea ho lemoha ea sete.Litaolo tsa li-gel le tharollo ntle le His-NT-PNP li ne li kenyelelitsoe.Litekanyo li entsoe habeli (n = 2).Ka mor'a hore ketsahalo e lekanngoe, motsoako oa karabelo o ile oa tlosoa 'me gel e nkoa foto ho etsa bonnete ba hore gel e lula e tiile nakong ea tekanyo.Lintlha li ile tsa hlahlojoa ho sebelisoa Prism 9.
Bakeng sa tlhaiso-leseling e batsi mabapi le moralo oa thuto, bona bukana ea thuto ea Tlhaho e hokahaneng le sengoloa sena.
Lipalo 1 le 2 li hlahisa lintlha tsa pele.1c, 2a–c, 3a, b, e–g, 4, 5b, d, f, le 6, Feiga tsa Tlatsetso.3, feiga ea tlatsetso.5a, d, feiga ea tlatsetso.6 le feiga ea tlatsetso.8. Lintlha tse tsoang phuputsong ena li bolokiloe polokelong ea boitsebiso ea Zenodo https://doi.org/10.5281/zenodo.6683653.Lintlha tsa NMR tse fumanoeng thutong ena li rometsoe sebakeng sa polokelo ea BMRBig tlasa ID ea ho kena bmrbig36.Mehaho ea GFP le PNP e nkiloe ho PDB (GFP 2B3Q, PNP 4RJ2).
Rising, A. le Johansson, J. Ho ohla silika ea maiketsetso ea sekho.Lik'hemik'hale tsa Sechaba.baeloji.11, 309–315 (2015).
Babb, PL le ba bang.Genome ea Nephila clavipes e totobatsa mefuta-futa ea liphatsa tsa lefutso tsa silika ea sekho le ponahalo ea tsona e rarahaneng.Genette ea Sechaba.49, 895–903 (2017).